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The emergence of Epizootic Hemorrhagic Disease in mainland France in 2023 led to thousands of clinical outbreaks in cattle herds and likely led to the natural immunization of a large number of animals
uncertainties persist regarding the extent of this immunity
both within herds and across affected regions
This study therefore aimed at investigating the variability of within-herd seroprevalence in clinically affected and non-affected herds across geographical areas with differing levels of disease incidence.A study was launched in February 2024 to assess the variability of within-herd seroprevalence in three geographical areas with varying EHDV-8 clinical incidence
A total of 2,763 serums samples from cattle over 24 months in 30 herds with clinical outbreaks and 31 herds without reported clinical case were analyzed using a commercial competitive ELISA.Results: A strong south-north seroprevalence gradient was observed
with the highest animal-level seroprevalence evidenced in the southernmost zone (Pyrenean Piémont) (82.6 %
which also experienced the highest incidence of clinical outbreaks
significantly lower seroprevalence levels were found in the more northern areas: (zone 2: 11.6%
where clinical outbreaks were less frequent
The within-herd seroprevalence varied widely among herds but was significantly higher in those located in the southernmost zone
no significant differences in seroprevalence were observed between clinical outbreak herds and non-outbreak herds.Discussion: this study highlights significant geographic and between herd variability in seroprevalence against EHDV-8 after the major virus circulation experienced in 2023
and provides critical insights into regional risks and the potential impact of future EHDV-8 circulation
Epizootic Hemorrhagic Disease Virus type 8
Received: 18 Jan 2025; Accepted: 30 Apr 2025
Copyright: © 2025 Anthonioz, Abadie, Reversat, Lafargue, Delalande, Renaudineau, Delobel, Verdeille, Ngwa Mbot, Gache, GARIN and Corbiere. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY)
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a key epigenetic mark involved in gene regulation
is commonly analyzed using Whole-Genome Bisulfite Sequencing (WGBS)
bisulfite treatment causes significant DNA degradation
Enzymatic Methyl-seq (EM-seq) offers a short-read alternative that preserves DNA integrity but requires conversion steps
limiting its compatibility with downstream analyses
such as Oxford Nanopore Technologies (ONT) and PacBio
enable direct detection of DNA modifications without altering the DNA
providing simultaneous genome and epigenome information
This work presents a comprehensive dataset combining long- and short-read sequencing data
for two agronomically relevant species: pig (Sus scrofa) and quail (Coturnix japonica)
Data quality evaluation reveals high nucleotide quality scores for PacBio and short reads
and inter-method correlations in CpG methylation calling ranging from 0.76 to 0.99
This dataset is a valuable resource for training methylation callers and represents the first combined methylation dataset for these species
providing an essential benchmark for assessing emerging sequencing technologies
Because these methods alter native DNA integrity and consequently modify bases
the resulting reads are more difficult to use for variant calling and genome assembly
have made it possible to detect DNA modifications without chemical or enzymatic alteration
Both methods rely on machine learning models trained to detect DNA methylation from raw or intermediate signal fluctuations during sequencing
The raw electrical signal from the nanopore is used to call modifications during DNA basecalling
nucleotide incorporation kinetics during sequencing are used to detect epigenetic modifications
Overview of study design and dataset produced
The quail and pig samples were sequenced using EM-Seq and WGBS methods on Illumina HiSeq 3000 or NovaSeq 6000
as well as Oxford Nanopore technology on PromethION
the quail sample was sequenced using PacBio on Sequel II
The dataset was then processed by 1) analyzing the format of raw reads produced by each method
2) applying specific CpG methylation calling pipelines
3) compiling fractions of 5mC methylation and depth of coverage per CpG position and strand (one file per run)
and 4) performing R analyses with the result data to evaluate and compare the technologies
with approval from the local ethical committee for animal experimentation (Val de Loire) and the French Ministry of Higher Education and Scientific Research (authorization n 4609-2016032115196879)
The pig blood sample was harvested from an adult Large White boar as part of the national selection program for the French LargeWhite breed
conducted by the three French breeding companies of the Alliance R&D association (composed of Axiom
The procedure was performed in compliance with the guidelines of the French Ministry of Agriculture and Fisheries
Pig HMW DNA was extracted from blood using the Genomic-tip 100/G kit (Qiagen
10243) following the manufacturer’s recommendations
Genomic DNA concentrations were measured using the Qubit fluorometry system with the Broad Range kit (Qiagen
32853) for detection of double-stranded DNA (Thermo Fisher
Fragment size distributions were assessed using the Femto Pulse Genomic DNA 165 kb Kit (Agilent)
Purity was evaluated using a Nanodrop system (Thermo Fisher)
All samples were purified with beads to achieve expected purity ratios i.e
the same DNA extractions were used across all three sequencing platforms (Illumina
the average gDNA molecular weight was estimated at 158 kb for quail and 174 kb for pig
DNA fragment size and quantity were carefully selected based on the specific requirements of each technology
Library preparation and sequencing were performed by the GeT-PlaGe core facility at INRAE Toulouse (https://doi.org/10.17180/NVXJ-5333)
WGBS libraries were prepared according to Bioo scientific’s protocol using the NEXTflex™ Bisulfite Library Prep Kit for Illumina Sequencing
1500 ng of DNA were fragmented by sonication
size-selected using AMPure XP beads and ligated to adaptors prior to sequencing
Bisulfite treatment was then performed for 2.5 hours using the EZ DNA Methylation-Gold™ Kit from Zymo Research
Library quality was assessed using an Advanced Analytical Fragment Analyzer (Agilent Technologies) and libraries were quantified by qPCR using the Kapa Library Quantification Kit
WGBS libraries were sequenced in paired-end mode (2 × 150 pb) on an Illumina HiSeq 3000 with the Illumina HiSeq 3000 Reagent Kits and on a NovaSeq 6000 (Illumina
USA) with the Illumina NovaSeq Reagent Kits
EM-seq libraries were prepared following NEB’s protocol using the NEBNext Enzymatic Methyl-seq Kit
200 ng of DNA were fragmented by sonication using Pixul sonicator (Active Motif)
Library quality was assessed using an Advanced Analytical Fragment Analyzer (Agilent Technologies) and libraries were quantified by qPCR with the Kapa Library Quantification Kit (Roche)
EM-seq libraries were sequenced on a NovaSeq 6000 (Illumina
USA) in paired-end mode (2 × 150 pb) with the Illumina NovaSeq Reagent Kits
ONT libraries were prepared following the manufacturer’s instructions “1D gDNA selecting for long reads (SQK-LSK109)”
DNA concentration was measured using the Qubit dsDNA HS Assay Kit (Life Technologies)
DNA purity was tested using the Nanodrop spectrophotometer (Thermo Fisher Scientific)
Size distribution and degradation were evaluated using the Fragment Analyzer (Agilent Technologies) DNF-464 HS Large Fragment Kit
Purification steps were performed using AMPure XP beads (Beckman Coulter)
5 µg of DNA were purified and sheared to 25 kb using the Megaruptor system (Diagenode)
followed by a size selection step with the Short Read Eliminator M Kit (Circulomics)
and dA-tailing of double stranded DNA fragments was performed on 2 µg of DNA
The library was loaded onto a FLO-PRO002 R9.4.1 flow cell and sequenced on a PromethION instrument (Oxford Nanopore Technologies
a nuclease flush was performed on the same flow cell
followed by reloading of the library at 20 fmol
PacBio library preparation and sequencing was performed according to the manufacturer’s instructions “Procedure & Checklist Preparing HiFi SMRTbell Libraries using SMRTbell Express Template Prep Kit 2.0”
DNA was quantified using the Qubit dsDNA HS Assay Kit (Life Technologies)
DNA purity was tested using the Nanodrop (Thermo Fisher Scientific)
and size distribution and degradation were evaluated using the Femto Pulse Genomic DNA 165 kb Kit (Agilent Technologies)
Purification steps were performed using AMPure PB beads (PacBio)
Fifteen micrograms (15 µg) of DNA were purified then sheared to 15 kb using the Megaruptor 3 system (Diagenode)
Using SMRTbell Express Template prep kit 2.0
and END repair steps were performed on 10 µg of DNA
Blunt hairpin adapters were then ligated to the library
The library was treated with an exonuclease cocktail to digest unligated DNA fragments
A size selection step using a 12 kb cutoff was performed on the BluePippin Size Selection System (Sage Science) with “0,75% DF Marker S1 High Pass 15–20 kb” protocol
Using the Binding kit 2.0 kit and sequencing kit 2.0
the primer V2 annealed and polymerase 2.0 bounded library was sequenced by diffusion loading onto 2 SMRT cells on Sequel II instrument (PacBio
USA) at 30 to 70 pM with a 2 hours pre-extension and a 30 hours movie
Methylation calling for both species was performed on ONT, EM-seq and WGBS reads. Additionally, methylation was called on PacBio reads for Coturnix japonica. The reference assemblies used were Coturnix japonica version 2.0 and Sus scrofa version 11.1
the –zymo option was applied due to the use of the Zymo kit for bisulfite conversion
The maximum insertion length was set to 800 using the –maxins parameter
All downstream analyses were performed using the stranded CpG report files
which is an output specified by the –cytosine_report option
CpG methylation calling from ONT reads was performed using megalodon v2.5.013 along with guppy v5.0.17
using res_dna_r941_prom _modbases_5mC_CpG_v001.cfg as calling model with default parameters
Methylation calling was performed on fast5_pass classified reads (quality threshold greater than Q7)
which calls methylation during basecalling
Basecalls in fastq format and read alignments can be saved on demand
The CpG methylation calling process for each sequencing run was performed with 3 GPUs (NVIDIA V100) and 40 CPUs
The time taken to complete methylation calling was approximately 12 hours for one PromethION run of Coturnix japonica
and approximately 40 hours for one PromethION run of Sus scrofa
Primrose software package in SMRT Link v11.014 was used to call CpG methylation from HiFi reads
HiFi reads were extracted from the subreads BAM file using the CCS command line tools with the –hifi-kinetics option
This generated a novel BAM used by Primrose to perform methylation calling
along with their probability of being modified
were added as additional MM and ML SAM tags to the output BAM file
This BAM file was then mapped to the reference genome using pbmm2
the modbam2bed script from ONT was used to compute and extract methylation fractions at genomic positions in bedmethyl format
The resulting files were processed to assess the quality of each methylome dataset per technology
methylation was called separately for each sequencing run (one output file per run) and then merged by technology
The methylation fraction and CpG coverage were calculated at each position by summing the values from the forward and reverse strands
Positions with a coverage higher than 100X or lower than 5X were filtered out to mitigate potential biases in the calculation of the methylation fraction
This maximum threshold accounts for the fact that it is more than twice the average coverage,helping to address challenges associated with aligning reads to repetitive regions of the genome
The minimum coverage value of 5X was set to ensure a sufficient number of reads for methylation fraction calculation
CpG structural annotation is needed for computing the average methylation fraction in CpG islands
we used the unmasked CpG island region lists available in the UCSC database for both Coturnix japonica and Sus scrofa reference genomes
Bigbeds were converted to BED format using the UCSC’s bigBedToBed tool and are available in our GitHub repository (see Code Availability section)
To compare methylation fraction trends between different sequencing technologies at the CpG island level
we focused on regions where at least 40% of CpG sites had a coverage of at least 10X
we calculated the average methylation fraction and compared it across technologies
For correlation analysis per CpG in CpG islands
we retained only the CpG positions that fell within UCSC CpG island regions
The evaluation of the correlation of CpG methylation fraction results between the four technologies using the Pearson method aims to provide more insight into the read quality and the reproducibility of the results
When computing Pearson correlation between technologies or technical replicates
only CpG sites or islands present in both datasets were retained through inner joins
The correlation values displayed in the heatmaps were rounded to two decimal places
Barplots above each correlation matrix indicate the proportion of covered reference CpG sites after coverage filtering and before the inner join (see the Code availability section for more information)
The objective was to guarantee sufficient data quality for accurate methylome analysis and to enable comparability between the methods by achieving a similar CpG site coverage across sequencing technologies
showing that enzymatic methyl-seq consistently outperforms the bisulfite-based standard in capturing candidate regions for genomic imprinting in the pig
It is believed to be caused by the DNA conformation
Despite performing a nuclease wash to empty blocked pores
quail runs still had a notably lower read count
with approximately 3-4 times fewer reads than pig runs
PacBio HiFi reads were only generated for the quail sample
The first run resulted in a limited number of reads (around 8 Gbp)
the second run produced a higher number of reads (around 22 Gbp)
pooling the data from these two runs yielded a CpG coverage of 28X
Coverage of CpG sites across technologies
The percentage of CpG sites covered at a minimum depth of coverage (a
b) and the distribution of CpG site coverage (c
and PacBio (purple) sequencing of Coturnix japonica (a
Correlation matrix of CpG per-site methylation levels across technologies. (a, c) Correlation analysis using all shared CpG positions. (b, d) Correlation analysis focusing on shared CpG sites located within CpG islands. Barplots above each matrix show the number of CpG sites covered and their percentage compared to the reference genome for each technology. Panels (a) and (b) report results for quail DNA, whereas (c) and (d) detail findings for pig DNA.
Correlation matrix of CpG islands methylation levels across technologies. Only CpG islands with at least 40% of CpG sites covered by ten or more reads were included in the correlation analysis. Barplots above each matrix display the number of CpG islands meeting this criterion and the percentage of regions covered compared to the reference genome. (a) depicts results for quail DNA, while (b) presents results for pig DNA.
Correlation matrix of CpG per-site methylation levels between technical replicates
Correlation was calculated using all shared CpG positions
Barplots above each matrix show the number of CpG positions covered by runs and the percentage of positions covered compared to the reference
we can see that both ONT and PacBio (in the case of C
japonica) had systematically higher correlation with EM-seq than with WGBS
suggesting that EM-Seq is a more accurate and reliable detection method than WGBS
potentially serving as a better reference to represent the short-read sequencing approach for long-read method benchmarking
Finally, when considering the average methylation fractions in CpG islands, all approaches showed a correlation close to 1 (0.95 to 0.99), indicating their ability to accurately assess methylation levels within CpG islands (Fig. 4)
PacBio shows a higher correlation with other sequencing technologies when comparing the mean methylation fractions of CpG islands to the methylation fractions at individual CpG sites
suggesting a potential precision shortfall in site-specific methylation detection
It’s important to note that the highest correlation is still obtained between ONT and EM-Seq
Figure 5 shows correlations between runs of the same technology
The correlation between ONT runs for both pig and quail is close to 0.90 (Fig. 5a,d), which is comparable to the observed correlation with WGBS (0.88 to 0.94) (Fig. 5b,e)
the correlation between HiSeq runs and the NovaSeq run is slightly lower (0.88 to 0.89) compared to the correlation between HiSeq runs (0.91 to 0.94)
The correlation between PacBio runs for quail was 0.74 (Fig. 5c)
One of these runs had exceptionally low CpG coverage compared to the other
which could explain the low correlation and highlights the importance of sufficient coverage when analysing vertebrate CpG methylation data
EM-seq reproducibility results were not calculated because only one run was enough to obtain 40X average coverage
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These authors contributed equally: Paul Terzian
conceived and supervised the “SeqOccIn” project
collected samples and carried out DNA extraction
performed the methylation calling for ONT and PacBio data and the post-processing of the result files
performed the methylation calling for WGBS and EM-Seq data
conceived the experimental design and supervised the technical aspects of the study
wrote the article and revised the manuscript with Ch.K
All authors read and approved the final manuscript
The authors declare no competing interests
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
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the Barkley Marathons in the hostile backwoods of Tennessee remains the world’s toughest footrace
adaptation and the struggle to survive play out
A month before the 2025 Barkley Marathons, Lazarus Lake is out on his daily eight-mile stroll along the rural roads near his Bell Buckle, Tennessee home
he fixes his gaze on a vine creeping onto the asphalt – kudzu
the invasive scourge of the American South
Laz pins it with the toe of his worn-out shoe
“That’s a common misconception – it’s war.”
Frozen Head’s relatively intact ecosystem provides a natural defense, but its steep, scarred mountains bear witness to countless other invaders – a fitting setting, as Laz sees it, for the world’s toughest footrace, where change, adaptation and the struggle to survive play out over what Outside magazine once described as “60 hours of hell”.
Read moreThe Barkley is combat – in 39 years
only 20 have finished this briar-gnarled ultramarathon
With books hidden in the woods as unmanned checkpoints
and a panic-inducing 12-hour start “window” – this run is more crucible than race
and a record-setting five finished in 2024
One factor may be Laz’s decision to intervene
he retreated the start of the race into March
Traditionally anchored to April Fool’s weekend
it was a tactical move to outflank a spring creeping ever closer
The challenge for Laz has always been keeping the Barkley on the razor’s edge of possibility –where runners suffer but still stand a chance
before the heat takes hold and the trees fill out making navigation nearly impossible
and he somehow looks younger – slightly tousled dishwater-blond hair
I remind him of his pre-race hike that year
“I don’t think I’d ever seen one that early,” mutters Kelly
“We’re not really certain what’s going on,” says a park ranger who shows up in camp
“It’s a bit of the frog in a boiling pot situation
But it’s not unusual for a snake to come out of their hole on a warm day.” He declines to be quoted – politics – and shies away from the word climate change
I’ve been hiking in Frozen Head since before it was a park,” he counters
“These snakes aren’t just sunning a bit; they’re on the move
If you actually go out every day for 60 years and pay attention
Fall is approaching spring from the other side
he sends 40 of the toughest ultrarunners in the world into the wilds by lighting a cigarette
“We might have to move it back to January,” Laz says later in the afternoon
His checked flannel jacket is off as the temperatures have now climbed into the low 70s
he’s added severe nastiness to this year’s course
then navigate their way down a treacherous 1,800-foot descent
This and other navigational trickery led one Barkley veteran to predict that three
maybe four will make three loops – “no finishers.”
View image in fullscreenIn 39 years
only 20 have finished the Barkley Marathons
Photograph: Ed Jones/AFP/Getty ImagesJohn Kelly is one of 11 who return from the first loop under the cutoff
“the course is on the verge of perfection”
and he has no idea when he returns again – it will be 15 hours later
Taps rings solemn through camp on an old bugle – honoring the many runners who’ve fallen short – while the few that remain press past rusting mining equipment wrapped in briars
“and you could find yourself at the bottom of one of those shafts.” Strip mining
But nothing has proved as indomitable as nature unleashed by man’s misguided attempts to intervene
Last year a runner was knocked sprawling by a dark
Originally brought to the Smoky Mountains for sport hunting
wild hogs bred with local hogs to create an aggressive
The state responded by introducing legislation to allow open-season hunting year-round on them
Unrestricted hunting inadvertently encouraged people to illegally transport and release hogs into new areas
hoping to create additional hunting opportunities
The ranger I spoke with had just “downed him” one the other night – more than 100
“I just want to keep them from the visitor center and the playgrounds.”
View image in fullscreenUltra-trail runner Aurélien Sanchez
who became the first ever French finisher of the Barkley Marathons in 2023
poses for a photo during a training session on the outskirts of Toulouse last year
Photograph: Ed Jones/AFP/Getty ImagesBut invasive pests
whose larvae now survive increasingly mild winters
a six-legged scourge barely 3mm long advanced from the south – the southern pine beetle
they bore through the bark and create intricate galleries in the phloem tissue
The pine responds by producing resin to “pitch out” the beetles
The next invader came in the form of innocent little tufts of what appeared to be lint – the hemlock woolly adelgid
This tiny aphid-like insect attaches itself to the base of hemlock needles and inserts specialized mouthparts to feed on the tree’s stored starches
The insects gradually drain the tree’s energy reserves
and eventual death within four to 10 years
a metallic green beetle only about half an inch long arrived in Chicago as a stowaway in wooden packing materials – the emerald ash borer
The earliest and perhaps most catastrophic scourge was the chestnut blight
It reached the park in the 1930s and killed an estimated four billion trees across the eastern United States
the fungal pathogen spread rapidly southward
the forest service sent crews wielding crosscut saws and axes through the woods to cut away a 60-mile “barrier zone.” The rhythmic chunk of steel biting wood echoed through valleys as hundred-year-old giants toppled in thunderous crashes
When Laz first hiked here in the early 70s
there were chestnut stumps “you could park a Volkswagen on.”
View image in fullscreenGary Cantrell
poses in Castanet-Tolosan near Toulouse in January
Photograph: Ed Jones/AFP/Getty ImagesBut the blight jumped the zone
and “Above the level of a man’s head,” observed naturalist Donald Culross Peattie
“the woods will never look the same again.”
each Barkley runner isn’t just battling distance
and Laz’s mind games – they’re moving through a landscape that’s fighting its own race against overwhelming odds
“The runners better be on Ball Bearing Hill if they hope to get here in time
All that’s left is a Fun Run – a special check-down finish of three loops
Laz grants a cushion of four extra hours – but no runner arriving after that extended window can continue on to loop four
it’s a consolation prize – but three are out there fighting to get it
Those in camp wait and stare into the night
Then a holler cuts through the silence and a light appears at the bottom of the hill – John Kelly
To say Laz over corrected ignores the race’s history
it took another five years before anyone finished
It’s the way of things that live on the edge
It’s been observed that though the trunks die
the roots often survive the blight to send up new shoots
Yet they persist despite near-certain failure
till one day they’ll adapt enough to thrive again
Jared Beasley is the author of the forthcoming book
about Lazarus Lake and the Barkley Marathons
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Organizers have announced that the official launch will take place on March 15
The Ghana Music Awards Europe is set to return for its fourth edition
reaffirming its role in celebrating and promoting Ghanaian music on the international stage
The official launch is scheduled for March 15
marking the beginning of an exciting journey toward the main awards ceremony
which will recognize outstanding contributions to Ghanaian music and culture
This year’s launch will feature performances from some of Ghana’s most distinguished artistes
alongside a diverse lineup that includes Samuel Sey
Their participation underscores the event’s significance as a platform for both established and emerging talents in the Ghanaian music industry
Route d’Escalquens Castanet-Tolosan in Toulouse
a venue chosen to provide an elegant and engaging atmosphere for guests and performers
As the Ghana Music Awards Europe continues to expand its reach
this edition aims to strengthen cultural ties between Ghana and the diaspora while elevating Ghanaian music to a global audience
the event will serve as a networking platform for artists
fostering collaboration and growth within the Ghanaian and European music industries
It will also highlight the evolution and influence of Ghanaian music in the global entertainment landscape
interested parties may contact the organizers via social media
the Ghana Music Awards Europe remains committed to celebrating artistic excellence and cultural heritage
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The Original Article was published on 12 February 2013
Addendum to: Nature Communications https://doi.org/10.1038/ncomms2479
In Fig. 4a and Supplementary Fig. 5 of this article, incorrect portions of the same Ponceau-stained blot are displayed as loading controls in the lower panels labelled ‘Rubisco’. Updated versions of Fig. 4a and Supplementary Fig. 5 and the original Ponceau-stained membrane and corresponding western blot images are shown in Fig. 1 below.
4a showing the correct Ponceau-stained loading controls
FLAG-tagged MYB30 or TAP-tagged MYB123 were expressed in N
benthamiana alone or with haemagglutinin (HA)-tagged MIEL1 or MIEL1m
and treated or not with the proteasome inhibitor MG132
Western blot analysis shows the expression of FLAG-tagged MYB30
TAP-tagged MYB123 and HA-tagged MIEL1 proteins
Molecular mass markers in kilodaltons are indicated on the right
5 showing the correct Ponceau-stained loading controls
benthamiana leaves transiently expressing FLAG-tagged MYB30 or the control protein GUS were mock-treated with DMSO or treated with the proteasome inhibitor MG132
Western blot analysis shows the expression of FLAG-tagged MYB30 or GUS proteins
c Original scans of Ponceau-stained membrane and corresponding anti-FLAG Western blot
4a in the original article incorrectly shows lanes 2–5 (counting from the left) rather than lanes 4–7
The loading control panel for Supplementary Fig
5 incorrectly shows lanes 4–6 instead of 10–12
In addition, the images of yeast colonies displayed in Supplementary Fig. 2 do not correspond to the strains listed in the figure labels. An updated version of Supplementary Fig. 2 showing original images of the plates used in the yeast two-hybrid assays is shown in Fig. 2 below.
All authors have approved this addendum with the exceptions of M.K.
Present address: Department of Plant Biology and Ecology
Laboratoire des Interactions Plantes-Microorganismes
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DOI: https://doi.org/10.1038/s41467-019-09341-4
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Volume 2 - 2011 | https://doi.org/10.3389/fpls.2011.00104
This article is part of the Research TopicNucleic Acid Transport in Plant-Microbe InteractionsView all 7 articles
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Traversing the Cell: Agrobacterium T-DNA’s Journey to the Host Genome
Plant immune responses depend on the ability to couple rapid recognition of the invading microbe to an efficient response
plant pathogens have acquired the ability to deliver effector molecules inside host cells in order to manipulate cellular and molecular processes and establish pathogenicity
microbial effectors may be addressed to different subcellular compartments
a significant number of effector proteins from different pathogenic microorganisms
increasing evidence highlights the crucial role played by nuclear dynamics
and nucleocytoplasmic protein trafficking during a great variety of analyzed plant–pathogen interactions
effector proteins are able to manipulate host transcription or directly subvert essential host components to promote virulence
it has been suggested that some effectors may affect histone packing and
microbial effectors may either directly activate transcription or target host transcription factors to alter their regular molecular functions
nuclear translocation of effectors may affect subcellular localization of their cognate resistance proteins in a process that is essential for resistance protein-mediated plant immunity
we review recent progress in our field on the identification of microbial effectors that are targeted to the nucleus of host plant cells
we discuss different virulence strategies deployed by microbes
which have been uncovered through examination of the mechanisms that guide nuclear localization of effector proteins
The co-evolutionary arms race between plants and pathogens has resulted in the generation of highly polymorphic repertoires of R proteins and microbial effectors
Examples of effector proteins with demonstrated nuclear localization from different phytopathogenic microorganisms
Model for nucleocytoplasmic transport of macromolecules through the nuclear pore complex
Cytoplasmic proteins with a nuclear localization signal (NLS) are translocated into the nucleus through nuclear pore complexes that are composed of nucleoporins and span the double membrane of the nuclear envelope
The NLS in cargo proteins is recognized by importin-α (Impα) that bridges the interaction of cargo proteins with importin-β (Impβ) and promote their transport into the nucleus
The directionality of transport is maintained by the Ras-related nuclear (Ran) protein through its binding to GDP (cytoplasmic side) or GTP (nuclear side)
Export receptors or exportins (Exp) recognize nuclear export signals (NES) in cargo proteins
promoting their nuclear export to the cytoplasm
we provide an overview on our current knowledge about translocation of microbial effectors into the host cell nucleus
We first discuss nuclear-targeted effectors from bacteria and viruses
whose functional characterization is rather well documented
we summarize recent data involving nuclear effectors from nematodes and filamentous pathogens
for which further functional studies are required before concluding on the specificities and commonalities of the virulence strategies that are hidden behind nuclear targeting of effector proteins
Model for integration of Agrobacterium tumefaciens T-DNA into the host cell chromatin
(A) The T-strand is transported into the host cell nucleus as an ssDNA molecule
VirE2 coats the T-strand to protect it from the attack of host nucleases
whereas VirD2 is covalently attached to the 5′ end of the T-strand
VirE2 interacts with the nuclear protein VIP1
which acts as a molecular bridge between VirE2 and nucleosomes thanks to its association with core histones
The role of VIP2 in this process remains to be determined
VirD2-intercating proteins CAK2Ms and TBP are also represented
VirF interacts with VIP1 attached to both nucleosomes and the T-complex
VirF helps uncoat the T-strand promoting proteasomal degradation of VIP1
This creates a chromatin environment favorable for T-DNA integration
Whether synthesis of the second-strand occurs before or after association of the T-strand with the chromatin remains unknown
It is possible that second-strand synthesis and integration represent coupled events
It is thus tempting to speculate that VirE2 and/or other Agrobacterium effectors may modulate histone gene expression to facilitate infection
VBF expression in and export from Agrobacterium lead to increased tumorigenesis
indicating that Agrobacterium subverts a component of the host cell proteasome
whose expression is induced during pathogen infection
it will be interesting to determine whether H3 can be modified by 6b and how potential 6b-mediated ribosylation of H3 may affect transcription
In Ralstonia solanacearum, PopB is a small basic T3SS-secreted protein that carries a functional C-terminal bipartite NLS and a predicted helix forming a coiled-coil domain, suggesting that PopB might interact with other proteins (Gueneron et al., 2000)
solanacearum T3Es of unknown function (RSp0216
and RSc3272) appear to be nuclear localized when expressed in plant cells (Anne-Claire Cazalé
The T3E from Xanthomonas campestris pathovar vesicatoria (Xcv) HpaA (for Hrp-associated) is specifically required for disease development in pepper plants. hpaA mutants are affected in pathogenicity whereas they partially retain the ability to induce an HR (Huguet et al., 1998). HpaA presents two functional NLSs that are important for full HpaA-mediated disease development in the plant (Huguet et al., 1998)
The observation that ADP-ribosylation of GRP7 by HopU1 requires two arginine residues within the RRM indicates that this modification may affect the RNA-binding ability of GRP7
the pathogen may modify the host RNA status
resulting in reduced amounts of immunity-related mRNAs available in the plant and suppression of host immunity
Whether HopAI1 is also able to target nuclear MAPKs and/or modulate histone modifications in plant cells is an exciting perspective for future research
Although in most cases the cellular activities that are manipulated by nuclear effectors remain to be determined
the action of the following nuclear T3Es has been relatively well characterized and illustrates varied pathogen strategies that lead to the establishment of a cellular environment that favors pathogen proliferation
TAL effector-DNA specificity and implications in plant disease/resistance
(A) TAL effectors contain an N-terminal domain required for T3SS-dependent secretion (T3S)
nuclear localization signals (NLS) and an acidic activation domain (AAD)
The central repeat domain confers DNA-binding specificity
One 34-amino acid repeat is shown with the variable di-residue (in red) at positions 12 and 13
The type of di-residue confers specificity for one or several DNA bases
and di-residues from each repeat define a specific DNA “TAL box.” (B) Upon binding to the TAL box in the promoter of a plant susceptibility gene
Plant resistance may result from either a molecular decoy strategy where the TAL box drives the expression of a plant “executor” (Resistance) gene (orange arrow) to counter the pathogen
or from occurrence of a mutation in the TAL box DNA sequence which prevents binding of the TAL effector
The biological implications of such differences are unclear but raise intriguing evolutionary questions since they suggest that TAL effectors containing 35-amino acid repeats independently arose by successive duplications of an initial variant repeat
recent work has identified between 1 and 4 TAL effector sequences in the genomes of 60% of a total of 55 surveyed Xcc strains (Boris Szurek and Laurent Noël
The molecular functions of these newly identified TAL effector candidates are unknown but this finding suggests that TAL-based virulence strategies are widespread among xanthomonads
These data indicate that HsvG functions as a TF in gypsophila
Examples of virulence strategies displayed by bacterial effectors in the plant cell nucleus
(A) XopD from the strain B100 of Xcc is a modular protein with an N-terminal domain of unknown function
three tandemly repeated transcriptional repressor domain of the EAR type and a C-terminal SUMO protease domain
It has been suggested that XopD DNA-binding activity through its HLH domain may provide access to chromatin and that XopD may thus modulate host transcription by altering chromatin remodeling
XopD may additionally interact with host TFs and repress their transcriptional activity directly via its EAR domains and/or by TF deSUMOylation
Recent work shows that the HLH domain of XopD targets the Arabidopsis TF AtMYB30 leading to repression of AtMYB30 trasncriptional activity and suppression of the plant HR and defense responses
solanacearum effector protein PopP2 displays acetyltransferase activity
suggesting that PopP2 may directly manipulate host transcription through chromating remodeling following acetylation of histone residues
PopP2 acetyltransferase activity may also promote the recruitment of TFs and enhance their DNA-binding affinity
resulting in modulation of host transcription
the interaction of PopP2 with host proteins
such as the cysteine protease RD19 and the resistance gene RRS1-R may additionally affect RRS1-R-meadited transcriptional reprogramming
The HLH domain of XopDXccB100 is necessary and sufficient to mediate interaction with AtMYB30 and repression of AtMYB30 transcriptional activation and plant resistance responses
the HLH domain of XopD is necessary and sufficient to suppress AtMYB30-mediated defense during Arabidopsis infection by Xcc
while the EAR motifs and Cys protease domains are not involved in this process
It is thus tempting to speculate that the EAR and the Cys protease domains in XopD are likely involved in targeting host defense-related components other than MYB30
these data suggest that different XopD host targets are involved in the outcome of the interaction between Xcc and Arabidopsis
Future work should uncover additional XopD-related molecular interactions during Xanthomonas infection
Identification of host targets of PopP2 should provide insight into the molecular mechanisms developed by R
These data highlight the importance of nucleocytoplasmic protein trafficking on the production of necrotic symptoms during BNYVV infection
Although the molecular mechanism behind p50-mediated NRIP1 nuclear relocalization remains to be determined
different hypothesis have been proposed to explain this finding
p50 might disrupt global chloroplast import by an unknown mechanism that would affect translocation of NRIP1
interaction with p50 might mask the chloroplast targeting signal in NRIP1 facilitating its nuclear import
NRIP1 may be released from chloroplasts into the cytoplasm and the nucleus following p50-induced permeabilization of the outer membrane
the close physical association between stromules and nuclei might enhance the nuclear import of chloroplastic factors
effectors that carry host-translocation signals are transported into the plant cell
effectors need to travel across two membranes
one pathogen-derived and one host cell-derived membrane surrounding the haustorium
and the nature of the mechanism directing effector delivery is still unclear
LXLFLAK-type motifs may be used in genome-wide searches to identify oomycete effector proteins
This finding confirms the importance of protein nucleocytoplasmic trafficking during defense responses against oomycete pathogens
suggesting that endogenous host proteins may facilitate nuclear delivery of these effectors
21% of the HaRxLs tested localized to the nucleolus
indicating that Hpa hijacks the plant cell transcriptional machinery perhaps by acting on RNA biogenesis
on ribosome biogenesis and thereby on protein translation to prevent de novo induction of defense responses
suggesting that virulence of this pathogen may also rely on nuclear targeting of effector proteins
Research aimed at the identification and characterization of pathogenicity effectors of plant nematodes has been intensively developed since genome sequences of these pathogenic organisms were first reported. Most nematode species are restricted to the roots in which they form specialized feeding sites to become sedentary endoparasites (Gheysen and Mitchum, 2011)
Two groups of pathogens have been particularly studied: the root knot nematodes (Meloidogyne spp.) and the cyst nematodes (Heterodera spp
Whether this differential behavior is due to artifactual mislocalization or reflects the possible existence of an effector processing mechanism within the plant cell remains to be determined
16D10 has not been visualized in the plant cell nucleus and its role in plant cell developmental processes
as well as the role of its interacting TFs
Pathogenic microorganisms use an extremely diverse panoply of effector proteins to counteract plant defense and ensure successful colonization of their hosts
Although different subcellular compartments are targeted by effector proteins following their translocation into plant cells
it is now apparent that a significant number of these effector molecules are specifically addressed to the host plant cell nucleus
Nuclear targeting of effectors appears to be a general microbial strategy since examples have been documented in each main class of pathogenic organisms from viruses to nematodes
although no nuclear effectors from bacterial Gram-positive plant pathogens have been described to date
caution should be taken when interpreting data from in silico effector analysis and functional studies of predicted effector proteins are absolutely required to validate their subcellular localization
These findings highlight the importance of the host nucleus in determining the fate of parasitic or mutualist interactions and the almost ubiquitous microbial strategy that involves direct interactions with host DNA and/or nuclear proteins
outputs resulting from these molecular interactions in the nucleus encompass various pathogenicity processes and multiple stages of host infection
such as the suppression of plant defense responses
despite significant recent advances in our field describing nuclear targeting of effector proteins
a lot remains to be done to obtain a global view on how transcriptional reprogramming of host cells or specific targeting of nuclear proteins promote pathogen infection and plant disease
Oligonucleotide microarrays or mRNA sequencing approaches should shed light on the large-scale transcriptomic changes undergone by host cells during nuclear targeting of effector proteins
integrating the contribution to virulence of nuclear effectors and that of effectors targeted to additional subcellular compartments
represents an exciting perspective for future research
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest
Our work is supported by funds from French ANR (ANR JC08_324792) and is part of the Laboratoire d’Excellence (LABEX) entitled TULIP (ANR-10-LABX-41)
and Boris Szurek for communicating unpublished results and apologize to all colleagues whose work could not be discussed because of space limitations
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Identification of the protein sequence of the type III effector XopD from the B100 strain of Xanthomonas campestris pv
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Sugar transporters for intercellular exchange and nutrition of pathogens
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Protein delivery into eukaryotic cells by type III secretion machines
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Emerging concepts in effector biology of plant-associated organisms
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vesicatoria are affected in pathogenicity but retain the ability to induce host-specific hypersensitive reaction
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RXLR effector reservoir in two Phytophthora species is dominated by a single rapidly evolving superfamily with more than 700 members
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A short amino acid sequence able to specify nuclear location
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Entry of oomycete and fungal effectors into plant and animal host cells
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A bacterial effector acts as a plant transcription factor and induces a cell size regulator
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Negative regulation of defence and stress genes by EAR-motif-containing repressors
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A secreted effector protein (SNE1) from Phytophthora infestans is a broadly acting suppressor of programmed cell death
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XopD SUMO protease affects host transcription
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Nuclear localization of NPR1 is required for activation of PR gene expression
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A secreted fungal effector of Glomus intraradices promotes symbiotic biotrophy
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Chromatin-modifying and -remodeling complexes
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Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1
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Association of the Agrobacterium T-DNA-protein complex with plant nucleosomes
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Classical nuclear localization signals: definition
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The Cauliflower mosaic virus translational transactivator interacts with the 60S ribosomal subunit protein L18 of Arabidopsis thaliana
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Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire
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Uncoupling of the functions of the Arabidopsis VIP1 protein in transient and stable plant genetic transformation by Agrobacterium
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Complementation of Tobacco etch potyvirus mutants by active RNA polymerase expressed in transgenic cells
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Functions of the Tobacco etch virus RNA polymerase (NIb): subcellular transport and protein-protein interaction with VPg/proteinase (NIa)
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Closing the circle on the discovery of genes encoding Hrp regulon members and type III secretion system effectors in the genomes of three model Pseudomonas syringae strains
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“phaseolicola” controls pathogenicity of bean plants and hypersensitivity of nonhost plants
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Nuclear trafficking during plant innate immunity
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Two host cytoplasmic effectors are required for pathogenesis of Phytophthora sojae by suppression of host defenses
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a molecular link between the Agrobacterium T-complex and the host cell chromatin
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Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus
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Chromatin configuration as a battlefield in plant-bacteria interactions
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The Xanthomonas type III effector protein AvrBs3 modulates plant gene expression and induces cell hypertrophy in the susceptible host
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Further evidence that viroplasms are the site of Cauliflower mosaic virus genome replication by reverse transcription during viral infection
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Evolution of the type III secretion system and its effectors in plant-microbe interactions
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Composition of the plant nuclear envelope: theme and variations
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Nuclear import and export of proteins in plants: a tool for the regulation of signalling
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A simple cipher governs DNA recognition by TAL effectors
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Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
The secretome of the maize pathogen Ustilago maydis
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Structural mechanism of the bromodomain of the coactivator CBP in p53 transcriptional activation
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The hypersensitive response; the centenary is upon us but how much do we know
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The repeat domain of the type III effector protein PthA shows a TPR-like structure and undergoes conformational changes upon DNA interaction
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The type III effectors HsvG and HsvB of gall-forming Pantoea agglomerans determine host specificity and function as transcriptional activators
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
In planta expression screens of Phytophthora infestans RXLR effectors reveal diverse phenotypes
including activation of the Solanum bulbocastanum disease resistance protein Rpi-blb2
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Ossareh-Nazari
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plays an important role in plant innate immunity
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Terrific protein traffic: the mystery of effector protein delivery by filamentous plant pathogens
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A plant viral “reinitiation” factor interacts with the host translational machinery
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Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex
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A secreted effector protein of Laccaria bicolor is required for symbiosis development
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Analysis of expressed sequence tags from Uromyces appendiculatus hyphae and haustoria and their comparison to sequences from other rust fungi
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Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv
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Expression of a Phytophthora sojae necrosis-inducing protein occurs during transition from biotrophy to necrotrophy
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A MYB transcription factor regulates very-long-chain fatty acid biosynthesis for activation of the hypersensitive cell death response in Arabidopsis
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A secreted SPRY domain-containing protein (SPRYSEC) from the plant-parasitic nematode Globodera rostochiensis interacts with a CC-NB-LRR protein from a susceptible tomato
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Nuclear transport of plant potyviral proteins
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Horizontal gene transfer facilitated the evolution of plant parasitic mechanisms in the oomycetes
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Nuclear dynamics during plant innate immunity
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Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Characterization of the Xanthomonas AvrXv4 effector
a SUMO protease translocated into plant cells
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Plant pathogen recognition mediated by promoter activation of the pepper Bs3 resistance gene
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A single plant resistance gene promoter engineered to recognize multiple TAL effectors from disparate pathogens
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Promoter elements of rice susceptibility genes are bound and activated by specific TAL effectors from the bacterial blight pathogen
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Mining the secretome of the root-knot nematode Meloidogyne chitwoodi for candidate parasitism genes
Pubmed Abstract | Pubmed Full Text
Identification of a nuclear localization signal and nuclear export signal of the umbraviral long-distance RNA movement protein
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Intracellular location of two groundnut rosette umbravirus proteins delivered by PVX and TMV vectors
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
The cyst nematode SPRYSEC protein RBP-1 elicits Gpa2- and RanGAP2-dependent plant cell death
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Flavin-containing monooxygenases in plants: looking beyond detox
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TAL effectors are remote controls for gene activation
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a novel AvrBs3-like effector from Xanthomonas gardneri with virulence and avirulence activity
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Ancient class of translocated oomycete effectors targets the host nucleus
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Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skp1 protein
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Rumble in the nuclear jungle: compartmentalization
and nuclear action of plant immune receptors
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How do oomycete effectors interfere with plant life
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Phytoplasma protein effector SAP11 enhances insect vector reproduction by manipulating plant development and defense hormone biosynthesis
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Diverse targets of phytoplasma effectors: from plant development to defense against insects
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Suppression of the AvrBs1-specific hypersensitive response by the YopJ effector homolog AvrBsT from Xanthomonas depends on a SNF1-related kinase
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Eukaryotic features of the Xanthomonas type III effector AvrBs3: protein domains involved in transcriptional activation and the interaction with nuclear import receptors from pepper
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Type III-dependent translocation of the Xanthomonas AvrBs3 protein into the plant cell
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RNA 3 deletion mutants of Beet necrotic yellow vein virus do not cause rhizomania disease in sugar beets
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae
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Autoacetylation of the Ralstonia solanacearum effector PopP2 targets a lysine residue essential for RRS1-R mediated immunity in Arabidopsis
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An oncoprotein from the plant pathogen Agrobacterium has histone chaperone-like activity
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New type III effectors from Xanthomonas campestris pv
vesicatoria trigger plant reactions dependent on a conserved N-myristoylation motif
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The T-DNA-linked VirD2 protein contains two distinct functional nuclear localization signals
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Agrobacterium tumefaciens T-DNA gene 6b stimulates rol-induced root formation
permits growth at high auxin concentrations and increases root size
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Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response
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Complete genome sequence and in planta subcellular localization of maize fine streak virus proteins
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Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis
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A new class of ubiquitin extension proteins secreted by the dorsal pharyngeal gland in plant parasitic cyst nematodes
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Partners-in-infection: host proteins involved in the transformation of plant cells by Agrobacterium
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an Arabidopsis protein that interacts with Agrobacterium VirE2
is involved in VirE2 nuclear import and Agrobacterium infectivity
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium
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Van den Ackerveken
Recognition of the bacterial avirulence protein AvrBs3 occurs inside the host plant cell
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
van der Biezen
Plant disease resistance proteins and the gene for gene concept
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Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defense gene expression
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
VirB/D4-dependent protein translocation from Agrobacterium into plant cells
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Nucleo-cytoplasmic shuttling of the Beet necrotic yellow vein virus RNA-3-encoded p25 protein
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Understanding and exploiting late blight resistance in the age of effectors
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Effector genomics accelerates discovery and functional profiling of potato disease resistance and Phytophthora infestans avirulence genes
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Vorhölter
Comparison of two Xanthomonas campestris pathovar campestris genomes revealed differences in their gene composition
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Molecular insights into plant cell proliferation disturbance by Agrobacterium protein 6b
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Use of geminivirus movement proteins for a cell-based export assay
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Characterization of nuclear localization signals in the type III effectors HsvG and HsvB of the gall-forming bacterium Pantoea agglomerans
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Nucleocytoplasmic transport: cargo trafficking across the border
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
A translocation signal for delivery of oomycete effector proteins into host plant cells
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Nucleocytoplasmic trafficking in plant innate immunity
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Os8N3 is a host disease-susceptibility gene for bacterial blight of rice
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Xanthomonas avirulence/pathogenicity gene family encodes functional plant nuclear targeting signals
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
The bacterial pathogen Xanthomonas oryzae overcomes rice defenses by regulating host copper redistribution
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Agrobacterium induces expression of a host F-box protein required for tumorigenicity
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
A Pseudomonas syringae effector inactivates MAPKs to suppress PAMP-Induced immunity in plants
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
A putative nucleoporin 96 Is required for both basal defense and constitutive resistance responses mediated by suppressor of npr1-1
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Shigella flexneri type III secretion system effectors OspB and OspF target the nucleus to downregulate the host inflammatory response via interactions with retinoblastoma protein
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
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The transition from a milk-based diet to exclusive solid feeding deeply modifies microbiota-host crosstalk
early ingestion of plant polysaccharides would be one of the main nutritional components to drive host-microbiota-interaction
To capture the effects of polysaccharides early-life nutrition (starch vs rapidly fermentable fiber) on the holobiont development
we investigated on the one hand the gut bacteriome and metabolome and on the other hand the transcriptome of two host gut tissues
Rabbit model was used to study post-natal co-development of the gut microbiota and its host around weaning transition
The assessment of the microbial composition of the gut appendix together with the caecum was provided for the first time
Gene expression signatures were analyzed along the gut (ileum and caecum) through high-throughput qPCR
The data collected were completed by the analysis of animal growth changes and time-series assessment of blood biomarkers
Those accessible and reusable data could help highlight the gut development dynamics as well as biological adaptation processes at the onset of solid feeding
If this diet transition is recognized as a major step in the young’s development
the mechanistic understanding of the changes occurring in the gut and the long-term consequences of pre-weaning experiences remain unknown
with a dynamic follow-up appears necessary to capture the process occurring at the suckling-weaning transition
rabbits present a specific behaviour pattern allowing mother-offspring separation most of the time
Easy control of early-life ingestion (milk and food) can therefore be performed to investigate post-natal food ingestion apart from nursing influence
a better understanding of the multidimensional aspects of gut health in rabbits appears essential to improve rabbit breeding practices
Experimental design (a) and data collection (b)
This study aimed to provide insights into host and gut microbiota responses to changes in the timing of solid food introduction (groups STA-/STA+) when two different diets were provided throughout the life (diets STA and RFF)
STA-: access to starch-enriched food in a pellet form from 15 to 70 days-old
STA+: access to starch-enriched food from 3 to 18 days-old in a gel form and from 15 to 70 days-old in a pellet form
RFF+: access to food rich in rapidly fermentable fibers from 3 to 18 days-old in a gel form and from 15 to 70 days-old in a pellet form
with this work intended to provide further methods descriptions and validation to facilitate re-use
New data are presented compared to our previous publication
technical replicates for 16S rDNA sequencing
rabbit nest quality evaluation and ileal gene expression
we demonstrated that the ingestion of small quantity of solid food in early life while maintaining milking could effectively accelerate gut microbiota maturation
Increasing the ingestion of rapidly fermentable fibers contributed to a specialization of the microbiota towards fibers-degradation activities
Effects of those strategies on the gut transcriptome and the host phenotype were limited in a context of good sanitation facilities
data analysis was restricted to single-omics approach
Time-monitoring of gut bacterial communities with associated host responses can allow researchers to extract a better picture of early-life developmental process
This comprehensive and reusable collection of data can be further explored by integrating the different types of datasets provided (multi-omics approaches)
This could help revel some interplay mechanisms
some data were generated for the first time with this experiment
thus providing new information to scientific community (e.g
investigation of caecal and appendix microbiomes in the same individuals or evaluation of plasmatic fatty acids and protein contents in rabbits at different stages)
Controls for technical and biological reproductibility of the data were generated
thus leading to valuable data resources for microbiologists and physiologists
We provided herein the description of the methods used together with raw and processed data to ensure easy sharing and re-processing thanks to analysis script provided on the Github platform
Detailed procedures or additional information such as complete lab steps for performing NMR measurements and RNA extraction are provided here to ensure that our datasets are understandable and reusable
the cages used for the experiment are described in more detail
as the housing system was specifically designed for this study
Additional data collection on nest quality is also presented
3D model of the experimental housing system used before weaning
View from the top of a nursing cage where the doe and its kits are raised together for 35 days
Blue arrows represent possible movements of the kits while red arrow represents possible path for the doe
the nest was only opened once a day for suckling (dottled arrows)
The sampling dates were chosen based on the feed transition process11:
18 days of age (solid food consumption remains minor)
25 days (dietary switch with marked increase of solid food ingestion proportion compared to milk)
30 days (solid-based diet with low amounts of milk consumed)
38 days (beginning of exclusive solid diet related with high susceptibility to digestive diseases)
58 days (settled exclusive solid feeding with more stable sanitary status)
pups that exhibited interest for gel food were preferably selected while at days 25
the killing procedure was performed 1 to 2 hours after suckling
while after weaning the rabbits were sampled 2 to 3 hours after feed distribution to obtain similar postprandial state
After the determination of sex and body weight of the animals chosen
they were killed by electronarcosis and exsanguination
Blood was collected at exsanguination in EDTA and dry tubes for plasma and serum preparation respectively
EDTA tubes were immediately put on ice until centrifugation (800 g for 10 min at 4 °C)
dry tubes were let undisturbed at room temperature for 20 minutes
The clot was then removed by centrifugation (1 800 g for 10 min in a regular centrifuge)
Resulting supernatants were collected and stored at −20 °C
After isolation of the caecum and the appendix vermiformis
USA) and up to 500 mg of luminal contents were collected in sterile tubes stored at −80 °C until the extraction step
protective gloves were replaced and the material was sterilized to prevent crossed contamination
0.5 cm of tissues from proximal caecum and distal ileon were quickly extracted at 3 cm up and down the Sacculus Rototondus
they were immediately snap-frozen in liquid nitrogen and stored at −80 °C prior to further processing
1 g of caecal content was collected and diluted in H2SO4 (at 2% w/v) to quantify ammoniac (NH3) concentrations at days 25
38 and 58 (insufficient quantities at day 18)
Caecal pH was measured after previous samplings by introducing a glass electrode at the ileocecal junction (VWR Collection SP225
around 2 g of caecal contents were collected to determine dry matter level after heating at 103 °C for 24 h
Caecal contents (100 mg) were homogenized in 500 µL phosphate buffer (sodium phosphate 0.2 M
and 20% water) in 2 mL FastPrep tubes (Lysing D matrix) by using a FastPrep Instrument (MP biomedicals
the supernatant was transferred to a new tube
The pellet was resuspended in 500 µL phosphate buffer and the homogenization and centrifugation steps were repeated
Supernatants from both extraction steps were pooled and centrifuged twice to remove particles (18 000 g
The resulting supernatants (600 µL) were transferred to 5 mm NMR tubes
NMR spectra were obtained with an Avance III HD NMR spectrometer operating at 600.13 MHz for 1H resonance frequency using a 5 mm inverse detection CryoProbe (Bruker Biospin
Germany) in the MetaboHUB-MetaToul-AXIOM metabolomics platform (Toulouse
The 1H NMR spectra were acquired at 300 K using the Carr-Purcell-Meiboom-Gill (CPMG) spin-echo pulse sequence with presaturation
A total of 128 scans (16 dummy scans) were collected in 32 K data points using a spectral width of 20 ppm and an acquisition time of 1.36 s
To improve the reproducibility of our work
we provide below new information on our RT-qPCR protocol
Caecal (n = 149) and ileal (n = 149) tissues were homogenized in 800 μL TRI reagent (ZymoResearch) with one sterile stainless steel 5 mm diameter bead (Qiagen
Germany) by using a TissueLyzer (Qiagen) with two 3 min cycles at 30 Hz
300 μL of supernatant was used for RNA extraction by using Direct-zol kit (ZymoResearch) following the manufacturer instruction
including a DNAse I treatment with DNA digestion buffer
RNA concentration and quality (260:280 and 260:230 ratios) were analyzed with NanoDrop 8000 (Thermo Fisher Scientific)
1 μL Oligo(dT) (100 µM) and 1 μL dNTP Mix (10 mM each) (Promega
After heating the mixture to 65 °C for 5 min and quick chill on ice
the contents of the tube was mixed with 4 μL 5X First-Strand Buffer (Invitrogen
1 μLSuperscript II Reverse Transcriptase (ThermoFisher Scientific) and 1 μL RNasin®Ribonuclease Inhibitor (Promega)
the reaction was inactivated by heating at 70 °C for 15 min
cDNA synthesis was checked with the house keeping gene GAPDH by using real-time quantitative PCR performed in QuantStudio 6 (Thermo Fisher Scientific
USA) with a reaction solution containing 2.5 μl of SybrGreen fluorescent DNA-binding dye (ThermoFisher Scientific)
0.9 μM of sterile water and 1 µL of DNA diluted at 1:10
We performed 40 PCR amplification cycles with an annealing temperature of 60 °C
high throughput real-time qPCR was performed for each tissue using the Biomark microfluidics system using a 96.96 Dynamic Array™ IFC for gene expression (Fluidigm
1.3 μL of cDNA (5 ng.μl−1) was added to the array and processed on the fluidics system according to the guidelines of the GENOTOUL platform
an initial high temperature activation step of 10 minutes was performed into the PCR reaction with 35 amplification cycles at 60 °C later on
The sequences of the primers used are presented in our companion paper
good efficiency (>90% and < 110%) and linearity were kept for statistical analysis
GAPDH and TOP1 gene were selected as housekeeping genes for caecal and ileal tissue respectively
based on their stability over time and between groups
in order to calculate gene expression using the 2−ΔΔCt method
such as the construction of the calibration curve
are given below for better reproducibility
In order to extract the immunoglobulins A (IgA) from the caecal content
200–500 mg of digesta was diluted at 50 mg/mL in cold TBS
and centrifugation at 3000 g for 10 min at 4 °C
the supernatant was collected for measurement
Total plasma IgG or caecal content IgA levels were determined in duplicates by sandwich ELISA in 96-well plates coated with specific polyclonal goat anti-rabbit IgG or IgA antibody (Bethyl Laboratories
USA) with further plate reading at 450 nm after fixation of the reaction between HRP conjugated antibodies (Bethyl Laboratories) and TMB
IgG were quantified using a reference IgG serum (Bethyl Laboratories)
12 samples were pooled to build a reference sample for the standard curve construction
seven calibrator points and water blank were added in duplicate to the microwell plates along with the samples
While linear standard curves were sufficient to fit the seven calibrators used for IgG quantification in plasma
a 4-parametric logistic model (4-PL) was used to improve the fit of caecal IgA calibration curve (average R2 with the 7 calibrators points = 0.98)
26 µL of plasma per sample were collected for the dosage of free fatty acids
the dosages were performed with Pentra 400 device (HORIBA Medical
France) at the Anexplo Phenotypage GENOTOUL platform (Toulouse
One sample (ID: 334) was diluted at 1:30 to fit the absorbance linearity range
A summary of all the data collected during this experiment and their repository access are given in Table 1 and Fig. 1b
Assessment of microbiota variability due to the DNA extraction step
Bray Curtis dissimilarities were computed using 16S rDNA from caecal contents extracted several time with the same procedure and similar follow-up processing (sequencing and bioinformatics analysis)
The numbers refer to the individuals sampled (rabbits of different litters aged from 18 to 58 days) and the letters a
Rarefaction curves, constructed for each individual sample, generally approached saturation which indicated sufficient read depth (Fig. 4). Furthermore, rarefaction curves showed different asymptotes according to rabbit physiological development, which was expected given the limited amount of plant polysaccharides in the diets of young suckling rabbits.
Rarefaction curves for caecal and appendix sites of each individuals (n = 30 individuals per sampling time with two gut sections investigated
Biochemical measurements of blood content were performed on two batches with a repeated control of known concentration (human serum) and a repeated pool of samples (two repetitions per batch)
The control measurements fell within appropriate confidence limits and the CV values for the pool repetitions were between 2 and 4% thus ensuring comparable results between batches for the three analytes measured
Mutualistic fermentative digestion in the gastrointestinal tract: diversity and evolution
Role of the gut microbiota in immunity and inflammatory disease
Succession of microbial consortia in the developing infant gut microbiome
introduction of solid feed and weaning are more important determinants of gut bacterial succession in piglets than breed and nursing mother as revealed by a reciprocal cross-fostering model
From pre- to postweaning: Transformation of the young calf’s gastrointestinal tract
Regulation of intestinal epithelial permeability by tight junctions
Feeding strategy for young rabbit around weaning: a review of digestive capacity and nutritional needs
The Locus of Enterocyte Effacement-Encoded Effector Proteins All Promote Enterohemorrhagic Escherichia coli Pathogenicity in Infant Rabbits
Yum, L. K. & Agaisse, H. Mechanisms of bacillary dysentery: lessons learnt from infant rabbits. Gut Microbes 1–6 https://doi.org/10.1080/19490976.2019.1667726 (2019)
Diversity and Co-occurrence Pattern Analysis of Cecal Microbiota Establishment at the Onset of Solid Feeding in Young Rabbits
Paës, C. et al. Early Introduction of Plant Polysaccharides Drives the Establishment of Rabbit Gut Bacterial Ecosystems and the Acquisition of Microbial Functions. mSystems https://doi.org/10.1128/msystems.00243-22 (2022)
Mesure quantitative de la production laitière chez la lapine
Le système d’information Sicpa Experimentations
Les systèmes d’Informations de phénotypage des animaux à l’Inra
Workflow4Metabolomics: a collaborative research infrastructure for computational metabolomics
Paës, C. et al. Source code for present data analysis. Github. https://github.com/charlottepaes/early_life_nutrition_rabbit (2020)
NCBI Sequence Read. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA615661 ID 615661 - BioProject
Paës, C. & Combes, S. Impact of feeding strategies on young rabbit and its gut microbiota. Data INRAE. https://doi.org/10.15454/QSTXWF (2021)
Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing
Download references
The authors would also like to thank the metabolomics platform Metatoul-AXIOM and the GENOTOUL phenotypic platform in Toulouse
summarized the data results and drafted the manuscript
carried out the in vivo experiments and measured the phenotypes with the data recording system built by F.L
contributed to the sequencing of 16S rDNA amplicons
helped to perform the Galaxy-supported pipeline FROGS
The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest
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DOI: https://doi.org/10.1038/s41597-024-03471-1
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Inspired by the production of reference data sets in the Genome in a Bottle project
we sequenced one Charolais heifer with different technologies: Illumina paired-end
In order to generate haplotypic assemblies
we also sequenced both parents with short reads
we built two haplotyped trio high quality reference genomes and a consensus assembly
The assemblies obtained using PacBio HiFi reaches a size of 3.2 Gb
which is significantly larger than the 2.7 Gb ARS-UCD1.2 reference
The BUSCO score of the consensus assembly reaches a completeness of 95.8%
We also identified 35,866 structural variants larger than 50 base pairs
This assembly is a contribution to the bovine pangenome for the “Charolais” breed
These datasets will prove to be useful resources enabling the community to gain additional insight on sequencing technologies for applications such as SNP
is the leading suckling cattle breed in Europe
the population has an international extension
It is particularly developed on grazing and extensive production systems with excellent maternal qualities combined with a high growth potential and an excellent beef conformation
In spite of the economic and social importance of this breed
the specificities of its genome remain poorly known and justify an in-depth characterization effort
Genome sequencing has evolved at a very fast pace in decades
Whereas only short reads were available less than ten years ago
which make it possible to build high quality de novo genome assemblies
these long reads are produced by two sequencing technologies
The Oxford Nanopore Technology (ONT) sequencers produce very long reads
The Pacific Biosciences (PacBio) sequencers offer two sequencing modes: Continuous Long Read (CLR)
which provides long reads with high error rate; and Circular Consensus Sequences (CCS)
which provides reads somewhat shorter and almost devoid of errors
While both these technologies do improve genome assembly quality
we still observe that some regions are missing or cannot be resolved
due to repetitiveness distance between them
We now have access to more advanced technologies
Choosing the best mix of technologies for a desired assembly quality requires a deep knowledge of the field
incessant improvements in sequencing technologies usually make previous ones obsolete
and drive users to switch from one mix of technologies to another
The type of repeated sequences present in the genome strongly influences the ability to establish a complete genome assembly
There was therefore a great interest in having another type of dataset next to the already available benchmarks such as the human dataset proposed by GIAB
The SeqOccIn project aims to explore the use of new sequencing technologies in agronomic research
We created reference datasets for genome assembly
and variability discovery for species of agronomic interest
we describe the datasets corresponding to the sequencing of a Charolais heifer
The parents and the heifer were sequenced with Oxford Nanopore Technologies on GridION and PromethION
The heifer was additionally sequenced with Illumina 2 × 250 bp on NovaSeq 6000 and PacBio Sequel II (CLR and CCS (i.e HiFi reads) mode)
parent reads (2 × 150bp) from 10X Genomics Chromium datas were used
The 29 autosomes and the X chromosome are covered by 1.7 scaffolds on average
we used Illumina reads from the parents in order to produce phased assemblies with similar metrics: an N50 of 70 Mb
and 3.15 Gb total size with less than 3% missphased k-mers
With the current state of sequencing technologies
the approach we currently recommend is a combination of short fragment sequencing on both parents with PacBio Hifi sequencing of the progeny to be assembled
allowing the separation of reads from each parental chromosome set
Although made from reads of a limited size of 12–15 kb
the very low sequencing error rate allows the inclusion of most of the repeated areas in the assembly thanks to the few SNPs making the repeated sequences present in the genome almost unique
Parent short reads data allowing identification of the correct parental origin of independent contigs
We used ARS-UCD1.2 assembly as the reference to evaluate the quality of our assemblies
The Wizard genomic DNA extraction kit (Promega) was used to extract frozen DNA from the three individuals
Genomic DNA concentrations were measured using the Qubit fluorimetry system (Life Technologies) with the High Sensitivity(HS) kit for detection of double-stranded DNA (Thermo Fisher
Fragment size distributions were assessed using the Femto pulse Genomic DNA 165 kb Kit (Agilent
Purity was measured using a Nanodrop system (Thermofisher)
All samples were purified with AMPureXP beads (Beckman Coulter
Degraded samples were re-extracted or sized using BluePippin Size Selection system (Sage Science) or circulomics kit
All sequencing processes were performed at the GeT-PlaGe core facility at INRAE Toulouse, https://doi.org/10.17180/NVXJ-5333
DNA-seq libraries were prepared according to the Illumina protocol TruSeq DNA PCR-Free High Throughput Library Prep Kit (96 samples
DNA was fragmented by sonication on Covaris M220
size selection was performed using Sample Purification Beads of the library kit (ratio 1/1 beads water) and adaptators IDT for Illumina – TruSeq DNA UD Indexes (96 Indexes
Library quality was assessed using a Fragment Analyzer (Agilent)with High Sensitivity NGS Kit (DNF-474-0500)
Libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche
DNA was sequenced on two SP NovaSeq 6000 lanes using a paired-end read length of 2 × 250 bp with the Illumina SP Reagent kit (500 cycles)
Both lanes produced respectively 118 Gb and 219 Gb
and cell pellet was resuspended and fixed in 1% formaldehyde
5 million cells were processed for the Hi-C library
After overnight digestion with HindIII (NEB)
DNA ends were labeled with Biotin-14-DCTP (Invitrogen) using the klenow (NEB) and religated
1.4 microgramme of DNA was sheared to an average size of 550 bp (Covaris)
A Qubit Fluorometer (HS kit) and a Fragment analyzer (NGS kit) were used to control DNA concentration and size
Biotinylated DNA fragments were pulled down using M280 Streptavidin Dynabeads (Invitrogen) and ligated to PE adaptors (Illumina)
The Hi-C library was amplified using paired-end primers (Illumina) for 10 cycles
Sequencing was performed on on one S4 lane NovaSeq 6000 lanes using a paired-end read length of 2 × 150 bp
Linked reads libraries were prepared for the heifer and her parents according to 10XTM Genomics protocols using the Genome Library Kit & Gel Bead Kit v2
Sample quantity and quality controls were validated on Qubit (HS kit)
Libraries were prepared from 3 µg of High Molecular Weight gDNA (cut off at 50 kb using BluePippin system
with “0.75% DF Marker U1 high pass 30–40 kb VS3” protocol
a library of Genome Gel Beads is combined with HMW template gDNA in Master Mix and partitioning oil to create Gel Bead-In-EMulsions (GEMs) in the Chromium
Each Gel Bead is functionalized with millions of copies of a 10XTM Barcoded primer (i7 Multiplex Kit PN-120262)
Upon dissolution of the Genome Gel Bead in the GEM
primers containing (i) an Illumina R1 sequence (Read 1 sequencing primer)
and (iii) a 6 bp random primer sequence are released
Read 1 sequence and the 10XTM Barcode are added to the molecules during the GEM incubation
and Sample Index are added during library construction
10 PCR cycles were applied to amplify libraries
Library quality was assessed using a Fragment Analyser (NGS kit) and libraries were quantified by qPCR using the Kapa Library Libraries were sequenced on a Illumina MiSeq with MiSeq Reagent Nano Kit v2 (300-cycles
MS-103-1001) to check equimolarity and quality then on a Illumina Novaseq 6000 S4 lane using a paired-end read length of 2 × 150 bp with the S4 Illumina Novaseq 6000 sequencing kits (300 cycles)
Reads produced on the heifer and its parents were simultaneously sequenced of a S4 line
producing 801 Gb so on average 89 X par individual
DNA purity was tested using a Nanodrop and size distribution and degradation assessed using the Femto pulse DNA 165 kb Kit
Purification steps were performed using AMPure PB beads (PacBio 100-265-900)
Library preparation was performed according to the manufacturer’s instructions “Procedure & Checklist Preparing HiFi SMRTbell Libraries using SMRTbell Express Template Prep Kit 2.0”(100-938-900)
For CLR library: 30 µg of DNA was purified then sheared at 60 kb using the Megaruptor3 system (Diagenode)
a DNA and END damage repair step were performed on 10 µg of sample
Then blunt hairpin adapters were ligated to the library
A size selection step using a 30 kb cutoff was performed on the BluePippin Size Selection system with “0.75% DF Marker U1 high pass 30–40 kb VS3” protocol
Using Binding kit 2.0 kit (101-789-500) and sequencing kit 2.0 (101-820-200) with 1 h of annealing and 4 h of binding
the primer V4 annealed and polymerase 2.0 bounded library was sequenced by diffusion loading onto 2 SMRTcells 8 M (101-389-001) on Sequel II instrument at 50 to 70 pM with a 15 hours movie
For HiFi library: 30 µg of DNA was purified then sheared at 15 kb using the Megaruptor3 system (Diagenode
A size selection step using a 12 kb cutoff was performed on the BluePippin Size Selection system with “0.75% DF Marker S1 3–10 kb Improved Recovery” protocol (BLF7510)
the second was recovered in manual mode to obtain a 20 kb library
Using Binding kit 2.0 kit and sequencing kit 2.0 with 1 h for annealing and 1 h for binding
the primer V2 (101-847-900) annealed and polymerase 2.0 bounded library was sequenced by diffusion loading onto 6 SMRTcells on Sequel II instrument at 50 to 70 pM with a 2 hours pre-extension and a 30 hours movie
DNA purity was tested using the nanodrop and size distribution and degradation assessed using the Fragment analyzer (AATI) High Sensitivity DNA Fragment Analysis Kit
Library preparation was performed according to the manufacturer’s instructions “1D gDNA selecting for long reads (SQK-LSK109)”
5 µg of DNA was purified then sheared at 20 kb using the Megaruptor system (Diagenode)
A 10 kb size selection step using Short Read Eliminator XS Kit (circulomics) or using the BluePippin Size Selection system was performed
A one-step DNA damage repair + END-repair + dA tail (NEB) of double stranded DNA fragments was performed on 1 µg of sample
The library was loaded onto three FLO-MIN106D and two FLO-PRO002 (R9.4.1) flowcells and sequenced on GridION and PromethION instruments at 20 pmol within 72 h
Nuclease flush steps were done when necessary and possible
when only 30% of the pores were still in sequencing
Details of the 5 pipelines used to produce our assemblies
a-Long reads assemblies from Oxford Nanopore Technologies and Pacific Biosciences followed by polishing step for erroneous assemblies and scaffolding step
b-10X Chromium assembly and scaffolding with Supernova
c-Phased assembly with HiFi and parental illumina reads
d-Phased assembly with HiFi and and Hi-C data
Read sets technical validation was performed first by producing read metrics
second by assessing read assembly quality and last by checking the correspondence of the variants found using each technology
Illumina data were basecalled with bcl2fastq2.10
and PacBio data with the current version of SMRTLink at the time of sequencing
the basecaller version is indicated in the metadata
Plot of the length and quality of our long reads datasets
The main graph of each technologies shows the histogram of size distribution from the randomly chosen 100 000 reads
in the corner of this graph is a boxplot of the quality of theses reads
Metrics are based on fastq file output and are technology dependent
CLR quality is not computed during fastq production and therefore not exploited
Mapping the reads to the ARS-UCD1.2 reference assembly resulted in 119X coverage and more than 99% of genome covered by at least 50 reads for Illumina
54X and more than 99% of genome covered by at least 10 reads for ONT
42X and more than 99% of genome covered by at least 10 reads for HiFi
and 34X and more than 99% of genome covered by at least 10 reads for CLR
In addition, Juicer12 was used to align Hi-C reads on the ONT wtdbg2 polished assembly
From around 250 million read pairs sequenced
more than 94% are considered as alignable (Normal paired + Chimeric paired)
More than 72% are unique and for the final Hi-C map elaboration
we obtained 151,865,989 pairs which represent 15X useful coverage
BUSCO plot of different Heifer assemblies. Summary plot produced with BUSCO V5.1 and mammalia odb10 dataset.
Comparison and evolution of the heifer assemblies size metrics after different steps in different assemblies pipelines
The alignment of these assemblies against the reference gave a coverage greater than 94.2% and read alignment against the assemblies gave alignment rates greater than 99.75%
These values attest a good read production
allowing to produce high quality assemblies
The best assembly being the one produced hifiasm using HiFi reads
the assembly BUSCO score increased from 70.2% to 95.2% which represents an increase of 25%
less than 5% of expected genes are still missing
which represents 317 of the 9226 genes evaluated by BUSCO
With a BUSCO scores above 95% and few errors in the reads
HiFi and 10X assemblies do not require polishing
These alignments showed concordance between scaffolds and reference chromosomes over the entire genome
representing possible intra-contig reorganizations
156 Mb for the longest chromosome and a 101 Mb of N50
The HiFi hifiasm assembly is around 17% larger than the reference assembly
mainly due to additional repeated sequences assembled
haplotype2 was 100 Mb larger than haplotype1
mainly due to misphased contigs creating duplication in haplotype2
Haplotype2 also had 9 Mb larger N50 but BUSCO scores were similar between both haplotypes (95.7% and 95.8%)
both haplotypes metrics were close (3.16 Gb vs 3.11 Gb and 71 Mb vs 69 Mb N50)
but BUSCO scores were slightly different (95.8% for haplotype1 and 95.3% for haplotype2)
Having no phased reference to evaluate these assemblies
we tested an in-house protocol (see code availability section) to estimate phasing quality as the proportion of misplaced haplotypes in each assembly
k-mer dictionaries was created for each assembly (HiFi hifiasm parent hap1 and hap2) and each origin (paternal reads
Ambiguous and homozygous k-mers were discarded: all the k-mers with low coverage in reads and those present in both paternal and maternal reads or in both haplotype1 and haplotype2 assemblies
assembly and read k-mer dictionaries were then pairwise compared
A phasing value of each assembly was then computed corresponding to \(\frac{{n}_{{\rm{maj}}}}{{n}_{{\rm{maj}}}+{n}_{{\rm{\min }}}}\times 100\)
where nmaj and nmin is the number of paternal k-mers in the supposed paternal haplotype (majority) and the number of maternal k-mer (minority)
Haplotype separations of 97.3% and 96.7% for HiFi hifiasm parent assemblies and 62.6%
60.5% for HiFi hifiasm Hi-C assemblies were obtained
The lower HiFi hifiasm Hi-C assembly phasing quality comes from the fact that Hi-C data is not optimal to separate contigs with distinct parental origins
Therefore the same calculation was performed on the contig level
which produced intra-contig separations are 84.6% and 85.6% showing that the Hi-C separation is less efficient than parental separation
but works fine on a contig level even if parental data is not available
contig haplotyping worked with both approaches
HiFi hifiasm parent method being more efficient than HiFi hifiasm Hi-C
HiFi hifiasm Hi-C required additional processing
such as manual reorganization of the contigs in the haplotypes
in order to obtain good quality assemblies
Upset plot of the identified deletions and insertions. Most of the variants are identified by all the long read technologies as well as for the assembly comparison based variant detection. This Upset plot underlines again the added value of long reads for variant detection.
a-Hifi assembly from heifer Hi-C contact matrix visualization with Juicebox
blue squares the manually produced scaffolds and each red dot an Hi-C contact
b-Dgenies Dot-Plot of HiFi Final assembly against bos taurus reference ARS-UCD1.2
Scaffolded assembly (Y-axis) were aligned to ARS-UCD1.2 chromosomes (X-axis) using minimap2 c-RepeatMasker/RepeatModeler representation of HiFi assembly and ONT assembly
The additionnal information in HiFi assembly is mainly Complex duplications
The scripts enabling to reproduce the assemblies presented in this manuscript are available on a dedicated Web page: [https://github.com/GeTPlaGe/SeqOccIn/tree/main/Data%20paper/Bos%20taurus%20data%20paper]
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Juicer provides a one-click system for analyzing loop-resolution hi-c experiments
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Download references
The SIGENAE team carried out part of the analyses
conceived and supervised the “SeqOccIn” project; C.D.
conceived the experimental design and supervised the technical aspects of the project; Ce.G
collected samples and realized extraction; C.I.
detected and analysed structural variation; Cl.K submitted the data; C.E.
contributed equally to this work; all authors reviewed the manuscript
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DOI: https://doi.org/10.1038/s41597-023-02249-1
Volume 9 - 2018 | https://doi.org/10.3389/fpls.2018.01780
Resistance to downy mildew (Plasmopara halstedii) in sunflower (Helianthus annuus L.) is conferred by major resistance genes
Twenty-two Pl genes have been identified and genetically mapped so far
wide-scale presence of only a few of them in sunflower crops led to the appearance of new
more virulent pathotypes (races) so it is important for sunflower varieties to carry as wide a range of resistance genes as possible
We analyzed phenotypically 12 novel resistant sources discovered in breeding pools derived from two wild Helianthus species and in eight wild H
All were effective against at least 16 downy mildew pathotypes
We mapped their resistance genes on the sunflower reference genome of 3,600 Mb
in intervals that varied from 75 Kb to 32 Mb using an AXIOM® genotyping array of 49,449 SNP
Ten probably new genes were identified according to resistance spectrum
hypersensitive response to the transient expression of a P
or the ecotype/species from which they originated
The resistance source HAS6 was found to carry the first downy mildew resistance gene mapped on chromosome 11
whereas the other resistances were positioned on chromosomes 1
and 13 carrying already published Pl genes that we also mapped physically on the same reference genome
The new genes were designated Pl23–Pl32 according to the current nomenclature
since sunflower downy mildew resistance genes have not yet been sequenced
This is the first large scale physical mapping of both 10 new and 10 already reported downy mildew resistance genes in sunflower
Studies have been made of quantitative, perhaps pathotype-non-specific, resistance (Tourvieille de Labrouhe et al., 2008; Vear et al., 2008b; Vincourt et al., 2012) but this is not easy to use in breeding
so control of downy mildew in sunflower still depends largely on major resistance genes
It is thus most important to find a wide range of genes giving resistance to the most prevalent pathotypes in areas where sunflower is an important crop
It is therefore not easy to localize precisely the different genes relative to each other
especially if different polymorphic markers are used to map them
Physical mapping of both known and novel resistance Pl genes on the reference genome sequence is therefore a major progress and should be an asset for breeders
We used this property to distinguish some closely linked resistances
This paper reports genetic mapping of the broad spectrum resistance genes in 12 sunflower lines developed from crosses of cultivated and wild Helianthus spp.
and their physical mapping on the sunflower reference genome using an AXIOM® SNP genotyping array
Ten putatively new downy mildew resistance genes were located on the sunflower genome sequence together with 10 already published Pl genes mapped on the same chromosomes
The question of what constitutes a new resistance gene is discussed
halstedii and a Spanish pathotype 330 not present in France
Pedigrees of known and new inbred sunflower lines with downy mildew resistance
Reaction of sunflower resistance sources to 15 Plasmopara halstedii pathotypes found in France and P
this line was not used to produce test crosses
The F1 hybrids were crossed with downy mildew susceptible lines to obtain test cross progenies which were tested for their downy mildew resistance to determine segregations indicating allelism or independent inheritance
In addition, to prepare for molecular mapping, the new lines were crossed with lines susceptible to at least some downy mildew pathotypes. F1 and F2 generations were selfed in order to develop F3 progenies which were phenotyped to determine the genotype of the F2 plants from which they were derived (Table 3)
Downy mildew reactions for F3 progenies from crosses of new resistance sources with lines susceptible to the pathotypes tested
Classic seed immersion downy mildew tests (Mouzeyar et al., 1993) were used to phenotype both test cross and F3 progenies. These tests were made in confined growth chambers in agreement with European quarantine regulations. Pathotypes (710, 304, 334, 714) were among those known to be present in France, and chosen to permit determination of the presence or absence of the resistance genes studied. As in previous studies (Vear et al., 1997)
plants with no sporulation (R) or with sporulation only on cotyledons (RII) were counted as resistant; those with sporulation on cotyledons and leaves
whether profuse (S) or rare (SII) were considered as susceptible
For each test cross progeny, the number of plants tested varied according to the number of seed available. All the results were summed, such that the total number of plants tested with one pathotype for one progeny varied between 91 and 763, except for one progeny with 51 plants, which showed no segregation for resistance (all numbers of plants tested are given in Supplementary Table 2)
Chi square tests were made for segregation patterns: either no segregation (same gene or closely linked genes)
3R:1S (two independent genes) or 7R:1S (three independent genes)
For each F3 progeny, 20–25 seedlings were observed and, 72 to 177 F3 progenies for each resistance origin were tested using one pathotype and between 41 and 95 when tests were made with a second pathotype (Table 3)
Chi-square tests were made for segregation patterns for one gene (1RR : 2SEG : 1SS) or for two genes (7RR : 8SEG :1SS)
Progenies chosen for molecular analyses were preferentially those homozygous for resistance or susceptibility
following the Best Practices Genotyping Workflow
Samples with a dish quality control (DQC) value ≥ 0.9 and QC call rate ≥ 0.90 thresholds were considered to have passed the quality control assessment
SNP quality control parameters and thresholds were defined as follows: polyploid species-type; cr-cutoff ≥ 90; fld-cutoff ≥ 3.6; het-so-cutoff ≥ -0.3; het-so-otv-cutoff ≥ -0.3; hom-ro-1-cutoff ≥ 0.6; hom-ro-2-cutoff ≥ 0.3; hom-ro-3-cutoff ≥ -0.9
only PolyHighResolution SNPs considered to be reliable and informative were retained
A final SNP filter focused on genotype data of parental lines was applied: for either or both parents
or showed divergences of allele calling between technical replicates were discarded from the analysis
Supplementary Table 2 presents the results of downy mildew resistance tests on all the test crosses
ordered according to the chromosome on which the downy mildew resistance has been mapped (as described below)
The X2 were calculated for expected segregations in the case of two or three independent genes (3R:1S or 7R:1S)
Ninety four of the 122 progeny segregations agreed with those expected (non-significant X2)
SII or RII plants could have caused some misinterpretation
Pathotype 710 was used for the majority of tests and gave the highest percentage of rejection of the null hypothesis (significant X2): 33% compared with 27% for pathotype 304 and 12% for pathotype 334
COQ (HA458) and HAS40 gave the largest numbers of significant X2
there were more segregations with unexpectedly low numbers of susceptible plants than those with excess susceptible plants
All X2 agreed with the hypotheses proposed for the line HIS32
INTER35 and HAS62 showed linkage to PlARG on chromosome 1
HIS36 and HAS85 linkage to Pl17 on chromosome 4 and those of HAS42 and HAS54 linkage to Pl5 on chromosome 13
whereas resistances of HAS103 and HAS6 appeared independent of all the known Pl genes tested
It was only for HAS40 that no conclusion could be drawn; it showed few susceptible plants in crosses with PlARG but also in crosses involving Pl17
Table 3 presents a summary of the reactions of F3 progenies
or pathotype 334 if the “susceptible” parent was VKQ
carrying Pl6 conferring resistance to pathotype 710
and HAS54 were found to show segregations indicating the presence of two genes for resistance to pathotype 334 although Pl6 is not effective against this pathotype
The three lines are from quite different origins
but they appear to contain a Pl2-type gene in addition to that giving resistance to a large number of pathotypes
the progenies showed segregations agreeing with the hypothesis for a single dominant gene
These segregations were used to define the resistance genotype of each F2 plant used for mapping
Transient expression assays with the PhRXLR-C01 effector of P. halstedii showed that only three of the 12 resistant lines responded by an HR: HAS103, HAS6 and HAS85, as well as the positive control line PMI3 (Pecrix et al., 2018) (Figure 1)
All the other resistant lines showed no HR to the infiltration of the PhRXLR-C01 effector
suggesting that the resistances they carry do not recognize this specific effector
XRQ showed a discoloration phenotype with both YFP negative control and PhRXLR-C01 constructs
suggesting a stress response to the Agroinfiltration process in this ecotype but no specific recognition of the P
Sunflower leaf size varied from 5 to 6 cm long and 2 to 3 cm wide
Mapping was not precise for the gene in the line HAS103 since we found 219 SNP markers of chromosome 2 (on a total of 345) co-segregating with resistance
and a physical interval for the resistance of 114 Mb
The resistance of HAS40 was quite precisely mapped on the genome sequence in a region of 75 Kb
but surprisingly corresponding to a large genetic region of 16.8 cM
This genomic region does not appear to include any RGC in XRQ
its resistance would be distinguished from that of Pl18
if lines containing this gene did not respond to the effector
One downy mildew resistance maps to chromosome 2 of sunflower
Genetic (yellow) and physical (gray) map of the Pl26_HAS103 resistance gene on chromosome 2
The cross used to map the resistance is indicated on top of the map
Only the non-redundant SNP markers are indicated with their genetic (left map) and corresponding physical positions on the XRQ genome (right map)
The polymorphic SNP markers of the sunflower AXIOM® array denominated AX-xxxxxxxxx are indicated with their physical positions on the sunflower XRQ genomic sequence
halstedii PhRXLR-C01 effector and resistance gene names
The downy mildew resistance of HAS6 was the first to be mapped on chromosome 11, in a short region spanning 1.3 Mb (Figure 5). This resistance also shows HR with PhRXLR-C01 (Figure 1 and Table 4)
The downy mildew resistance of HAS6 is the first to be localized on chromosome 11 of sunflower
Genetic (yellow) and physical (gray) map of the Pl30_HAS6 resistance gene on chromosome 11
The resistances of HAS42 and HAS54 both mapped in a small area on chromosome 13 where Pl5, Pl8, Pl21, and Pl22 had already been mapped, closest to Pl8 (Figure 6). However, they differ from all these genes, either by their reaction to certain downy mildew pathotypes (Pl5, Pl21, and Pl22) or by their resistance phenotypes (Pl8 always shows RII type reactions whereas they show R) (Gascuel et al., 2015)
so it is perhaps not surprising that many Pl genes map in this area
the physical mapping of 11 of the 12 resistance genes was consistent with the conclusions of the allelism tests performed initially and the only resistance (HAS40) that could not be localized by phenotypic studies was mapped successfully with the array on chromosome 1
The results presented here describe mapping, on the sunflower reference genome (Badouin et al., 2017)
using a high-throughput mapping SNP AXIOM® array
of 12 sources of resistance effective against at least 16 downy mildew pathotypes
The results suggest that these sources may be considered as providing new resistances carried by 10 novel resistance genes
Before the availability of molecular analyses
determination of linkage or independence of downy mildew resistance genes depended on resistance tests on seedlings with adequate pathotypes
The present results show that a large number of crosses and progenies must be tested for analyses of segregations to permit decisions as to whether Pl genes are in one or different clusters
Seventy seven percent of tests reported here did not differ from the Mendelian segregations 3R: 1S
some were probably due to the wide use of the highly aggressive pathotype 710
has occasionally been found to give symptoms in plants containing resistance genes but with background genotypes that are unfavorable for expression of resistance
COQ (HA458) and HAS40 showed the highest proportion of “abnormal” segregations (6/13 and 8/17 respectively)
there was an excess of susceptible plants in five tests out of the six
and it may be suggested that this is due to background susceptibility to pathotype 710
five of the eight “abnormal” segregations showed an excess of resistant plants
In three cases they were in crosses with lines (PAA1
and HAS62) that were confirmed by the SNP genotyping to have resistance genes on the same region of chromosome 1
These segregations thus probably corresponded to loose linkage between the genes
which could not be estimated precisely with the numbers of plants phenotyped for their downy mildew resistance
It was concluded that phenotyping by resistance tests provided first indications of the genetics of the downy mildew resistances studied but that molecular studies were necessary to confirm distinction and precise location
The AXIOM® array used for genotyping has theoretically 49,449 SNP
we found an average of 5953 high-quality polymorphic SNPs
between a minimum of 4138 for HAS40 and a maximum of 8548 for HIS33 (i.e.
17% of the total SNPs present on the array)
Although some physical information was lost according to the cross
there were always sufficient markers to map correctly the resistances in regions of 10s of Mb
This was quite surprising since HAS40 had a small number of polymorphic markers on the chromosome concerned (221)
the F3 population used for mapping was large (70 plants) making it possible to take account of a sufficient number of recombination events
the segregation ratios observed for resistance were close to theoretical values
suggesting that mapping population size and phenotyping quality are important criteria for precise fine mapping
the cross involving HAS103 had 50% more polymorphic markers on the chromosome concerned
but the mapping population was smaller (45) and segregation for resistance not optimal
Together with a lack of recombination events on chromosome 2 in the HAS103 x XRQ population
comparisons between genetic and physical maps in sunflower
for 5 of the total 17 chromosomes and highlights several interesting features
we observed the presence of differences between the two maps
On the physical map of the reference genome
there appear to be some large genetic regions (10s of cM) devoid of SNPs
whereas the SNPs selected for the study provide relatively regular chromosome coverage
the recombination frequency may be high in these regions compared with the reference genome
there may have been deletions in the reference genome compared with the genotypes used for mapping
we observed inversions between genetic and physical maps
again suggesting differences between the parental genotypes used to map the resistances and the reference genome
It would be interesting to have the sequence of more than one genome to allow better comparisons with genotypes of various origins and to understand sunflower genome evolution
It is therefore highly probable to find closely linked but different resistance genes in such large regions
and one of the reasons why we propose two new genes
it should be kept in mind that gene order in some genotypes could be slightly different from that in the reference genotype
Even with the availability of a sunflower genome sequence
all the questions regarding the genetic and molecular architecture of resistance to downy mildew will not be solved in the near future
so it appears of interest to fix a rule to designate genes in sunflower involved in the interaction with P
until their sequences and their possibility of reacting differently to different pathogen genotypes
it is difficult to know whether all merit new names
Mapped regions vary from 0.07 Mb (about two genes) for the gene in HAS40 to 32 Mb (640 genes) for the other resistances on chromosome 1
we cannot conclude between close genes or one gene so we have tried to fix some rules for naming the genes identified in the present study
They could make a total of 10 additional genes available in breeding for resistance to sunflower downy mildew
With more than 30 downy mildew resistance genes identified and positioned on the sunflower genome, simple numbering according to order of identification is not helpful to avoid errors. It might be useful to include at least chromosome number in gene names. Of course, international agreement would be necessary, as for the triplet denomination of downy mildew pathotypes (Gulya et al., 1998)
We performed the colinearity studies of the genetic maps using the genome sequence of the cultivated sunflower line XRQ (Badouin et al., 2017)
although this resource was a major breakthrough for genetic studies in sunflower such as for the mapping of the resistance genes to downy mildew of our study
this reference sequence does not represent the diversity in cultivated sunflower and even less the diversity in wild Helianthus
It is now relatively easy to re-sequence genomes and identify SNP polymorphisms by comparing the re-sequencing data with a reference sequence
or copy number variation) that can range from a few nucleotides to 100s of kb
By aligning genetic maps on the reference genome sequence
we identified such polymorphisms which suggest that some resistances
In order to facilitate the sequencing of the downy mildew resistance genes or other loci of interest
it would be very useful to produce new tools or new genomic resources in sunflower
it would be interesting to develop a genotyping tool based on SNPs identified on this pan-genome in order to identify large structural variations
making the optical maps of the genomes from 10s of accessions would be a useful resource too
assembling high quality genome sequences of sunflowers and wild Helianthus would considerably speed up research in sunflower
which could become a model plant species in many scientific fields (genetics
responses to biotic and abiotic stresses...)
The present results suggest that Helianthus species
annuus are quite rich in broad spectrum downy mildew resistance genes
Use of combinations of those described here should help to provide some reasonably durable resistance
protecting the sunflower crop against changes in downy mildew pathotypes
Sunflower plant material used in this study was grown from seeds made available by INRA as part of INRA sunflower genetic resources
COQ and U8Q were obtained by selfing from USDA sources
FV produced the plant material and carried out phenotyping
CP-S and YP produced the plant material for genotyping
SM designed the 50K-SNP AXIOM® array
All authors were involved in revising the manuscript critically and approved it
This work was supported by the French Laboratory of Excellence project ‘TULIP’ (Toward a unified theory of biotic interactions: role of environmental perturbations) (ANR-10-LABX-41 and ANR-11-IDEX-0002-02) and by PROMOSOL (“Association pour la promotion de la sélection des plantes oléagineuses”)
The authors thank CRB sunflower for providing seeds (LIPM
Sylvie Roche and Frédéric Serre (INRA
France) for technical help in plant experiments and Clémence Paris (Syngenta seeds
France) for her help in genotyping analyses
The authors also thank the Gentyane Platform (Clermont-Ferrand
They acknowledge Denis Tourvieille de Labrouhe (INRA Clermont-Ferrand
France) and Emmanuelle Mestries (Terres Inovia
France) for being members of the “Milvarsunres” project steering committee
and Patrick Vincourt for critical reading of the manuscript
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01780/full#supplementary-material
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Copyright © 2018 Pecrix, Penouilh-Suzette, Muños, Vear and Godiard. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY)
provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited
*Correspondence: Felicity Vear, ZmVsaWNpdHkudmVhckB3YW5hZG9vLmZy Laurence Godiard, bGF1cmVuY2UuZ29kaWFyZEBpbnJhLmZy
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Genomic imprinting represents an original model of epigenetic regulation resulting in a parent-of-origin expression
Despite the critical role of imprinted genes in mammalian growth
there is no molecular tool specifically targeting them for a systematic evaluation
We show here that enzymatic methyl-seq consistently outperforms the bisulfite-based standard in capturing 165 candidate regions for genomic imprinting in the pig
This highlights the potential for a turnkey
fully customizable and reliable capture tool of genomic regions regulated by cytosine methylation in any population of interest
it opens up the possibility of detecting multilocus imprinting variations across the genome
Although knowledge about GI has significantly advanced so far
some technological bottlenecks remain to tackle challenging scientific insights
we optimized and compared capture-based methylation sequencing technologies aiming for an exhaustive detection of evolutionary conserved imprinted loci across the genome
We propose here in the pig the first global evaluation of methylation patterns related to genomic imprinting in livestock
(b) Distribution and size of final designed panels by the two manufacturers
duplication rate (d) and GC percentage (e)
that is represented as the mean + /- standard deviation of the fraction of targets covered at a specific depth (f)
that is represented as the mean + /- standard deviation of depth coverage for the 165 targeted regions (g)
that is represented as percentage (h) and density (i) of off-target reads
which mapped outside of the 165 targeted regions
Correlation of the mean coverage with either the size (j) or the GC percentage (k) of the 165 targeted regions
the AG classical protocol is in green and the two TB protocols (TB1 and TB2) are in light and dark purple
Feature annotation of region per technology
h) Annotation of the pig genome using Sus_scrofa.Sscrofa11.1.104.gtf showing that KCNQ1OT1 was missing
l) Variants identification and informativity with parental origin in the offspring of reciprocal crosses
m) Methylation evaluation in blood and sperm tissues and detection of PofO methylation
hemiR100: occurrence of ≥ 5 hemi-methylated CpGs within 100 bp; hemiR5: occurrence of ≥ 5 consecutive hemi-methylated CpGs
The study included 10 pigs, 8 pigs were bred at the INRAE experimental farm (https://doi.org/https://doi.org/10.15454/1.5572415481185847E12) and 2 pigs come from breeding organizations in accordance with the French and European legislation on animal welfare
Animals were produced in a reciprocal cross design between Large White and Meishan pig breeds
Ten biological samples were used in the experiment
Nine of them are blood samples collected on EDTA and were stored frozen nine months at − 20 °C
One biological sample is a sperm sample from dose for artificial insemination and was stored two years at − 20 °C
Biological samples were collected at adult developmental stage for all the parents (n = 5) of the reciprocal cross design while biological samples were collected at 1d after birth for all offspring (n = 5) of the reciprocal cross design
Genomic DNA was extracted from blood using the Genomic-tip 100 DNA kit (Qiagen
10,243) or using MagAttract HMW DNA kit (Qiagen
67,563) following manufacturer’s instructions
Genomic DNA was extracted from sperm using standard phenol/chloroform method
DNA purity was determined using the Nanodrop 8000 spectrophotometer (Thermo Fisher Scientific)
DNA concentration was determined using the DS DNA Broad Range Assay kit (Invitrogen
Q32850) and was measured with the Qubit3 fluorometer (Invitrogen
All the procedures and guidelines for animal care were approved by the local ethical committee in animal experimentation (Poitou–Charentes) and the French Ministry of Higher Education and Scientific Research (authorizations n°2,018,021,912,005,794 and n°11,789–2,017,101,117,033,530)
All animal and sample information is available at the European Nucleotide Archive (ENA) as accession number PRJEB58558
Sequences not annotated in the pig genome were subjected to BLAST searches against the Sscrofa11.1 reference
A total of 165 regions ranging from 458 bp to 2.3 Mb
the X chromosome and 4 scaffolds of the pig reference
were submitted to the two commercial platforms
Each platform used its own confidential algorithm for panel design
The sizes of custom panels from TB and AG were 20.5 Mb and 19.7 Mb
with all the 165 candidate regions for GI represented
The TB2 experiment was performed by Twist Bioscience company (Twist Bioscience
Eight library preparations were carried out using the SureSelect Methyl-Seq Target Enrichment kit (Agilent
G9651) following the manufacturer’s protocol (User guide: SureSelect
Genomic DNA (1 µg) was first fragmented using a Covaris M220 focused ultrasonicator in micro-TUBE 50 AFA Fiber screw cap (Covaris
520,166) for a target insert size of 200 bp under the following conditions: peak power 75W
An additional 0.8X AMPure beads purification step was done to eliminate adaptor dimers
Sixteen library preparations were carried out using an in-house combination of two protocols: NEB-Next Enzymatic Methyl-seq Library Preparation and Twist Bioscience Targeted Methylation Sequencing, using a methyl custom panel. The whole detailed and optimized protocol has been deposited to Protocol Exchange open repository (https://doi.org/https://doi.org/10.21203/rs.3.pex-2159/v1)
eight library preparations were carried out with a first similar development protocol (TB1) in which some adjustments have not yet been made
Differences between protocolTB1 and protocolTB2 are referenced in the procedure deposited in Protocol Exchange
All library quantifications were performed on a Qubit 3.0 fluorometer with High Sensitivity DNA Quantitation Assay kit according manufacturer’s recommendations (Agilent
All library validations were performed on a 2100 Bioanalyzer with High Sensitivity DNA kit according to manufacturer's recommendations (Agilent Technologies
All libraries were quantified by qPCR on QuantStudio 6 device (Applied Biosystems
using the Kapa Library Quantification Kit (Roche
Agilent libraries and experiment TB1 libraries were each sequenced on one lane of an Illumina SP NovaSeq 6000 flow cell
using the SP Reagent kit v1.5 300 cycles (Illumina
according to the manufacturer's recommendations
The loading concentration was 2 nM 25% phiX
Experiment TB2 libraries were sequenced on Illumina P2 NextSeq 2000 flow cell
using the SP Reagent kit v3 300 cycles (Illumina
The loading concentration was 1000 pM 5% phiX
All sequences are available at ENA under study accession PRJEB58558
identifying when possible the parental origin of methylation in the progeny of the reciprocal cross
A complete list of software versions used in this study is provided in the next section
Cutadapt (v2.9)31
Nextflow (v20.01.0)32
FastQC (v0.11.9, https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)
Preseq (v2.0.3)35
R base (v4.1.1) with dplyr (v1.0.9), ggplot2 (v3.3.6), RIdeogram (v0.2.2), scales (v1.2.1) and tidyr (v1.2) packages (https://cran.r-project.org/)
Samtools (v1.9)36
Trim Galore! (v0.6.4_dev, https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/)
The dataset generated during the current study is available in the ENA data repository (https://www.ebi.ac.uk/ena/browser/home
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New England Biolabs and Twist Bioscience for support throughout the optimization
This protocol was funded by the ANR PIPETTE (ANR-18-CE20-0018) and FEDER-FSE SeqOccIn projects
Jean-Noël Hubert is partly funded by the ANR PIPETTE and the Animal Genetics division of INRAE
These authors contributed equally: Jean-Noël Hubert and Nathalie Iannuccelli
Céline Vandecasteele & Cécile Donnadieu
analyzed methylation data and wrote the manuscript
performed the experiments and wrote the protocol submitted to Protocol Exchange
conducted analyses and visualization for the IGF2-H19/KCNQ1-CDKN1C region
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
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DOI: https://doi.org/10.1038/s41598-024-52114-3
Volume 4 - 2013 | https://doi.org/10.3389/fpls.2013.00098
This article is part of the Research TopicNuclear components and dynamics during plant innate immunityView all 12 articles
Transcriptional regulation in host cells plays a crucial role in the establishment of plant defense and associated cell death in response to pathogen attack
we review our current knowledge of the transcriptional control of plant defenses with a focus on the MYB family of transcription factors (TFs)
the Arabidopsis MYB protein AtMYB30 is a key regulator of plant defenses and one of the best characterized MYB regulators directing defense-related transcriptional responses
The crucial role played by AtMYB30 in the regulation of plant disease resistance is underlined by the finding that AtMYB30 is targeted by the Xanthomonas type III effector XopD resulting in suppression of AtMYB30-mediated plant defenses
the function of AtMYB30 is also tightly controlled by plant cells through protein-protein interactions and post-translational modifications (PTMs)
AtMYB30 studies highlight the importance of cellular dynamics for defense-associated gene regulation in plants
we discuss how AtMYB30 and other MYB TFs mediate the interplay between disease resistance and other stress responses
Among the closest paralogs of AtMYB96 is AtMYB30
which was the first R2R3 MYB gene to be associated with the regulation of defense response in Arabidopsis and one of the best defense-related MYBs characterized to date
Although the mechanisms by which MYB TFs control defense responses are still enigmatic
recent advances in our understanding of AtMYB30 function summarized in this review shed new light on the regulation of plant immunity by this family of TFs
These data identify AtMYB30 as a positive regulator of the signaling pathway controlling the establishment of cell death responses to pathogen attack
the study of AtMYB30 regulatory mechanisms has increased our knowledge about the mode of action of this TF
These studies have uncovered a tight control of the activity of AtMYB30 through protein-protein interactions and post-translational modifications (PTMs)
we summarize our current knowledge of the AtMYB30 interaction and regulatory network involved in the control of plant defense responses
Additional roles of AtMYB30 during the integration of other environmental cues are also discussed
modifications of this kind are critical for the regulation of AtMYB30 activity
It is therefore tempting to speculate that the differential activation of the N-termini of MYB TFs of the sub-group S1 may integrate signals arising from multiple stresses to regulate a partially common set of genes
Whether and how the interplay between AtMYB30 and AtMYB96 fine-tunes the activation of VLCFA-mediated responses remains to be investigated
Whether other MYBs of sub-group S1 are able to activate the VLCFA pathway is also unknown
Shared and specific functions of related MYB TFs may explain how expansion and diversification in this family contributed to the emergence of an integrated stress-response machinery in plants
Simplified model for the simultaneous regulation of AtMYB30-mediated HR cell death through interaction with AtsPLA2−α and MIEL1
The action of with AtsPLA2−α and MIEL1 on AtMYB30-mediated HR development is presented in cells challenged with bacterial inoculation (A) and peripheral cells (B)
Activity of the bacterial XopD effector is shown in red
Figure 2. AtMYB30 sequence analysis: relationship with other MYBs, protein motifs and predicted structure. (A) Relationship between MYB TFs of the subgroup S1 (from Dubos et al., 2010)
(B) Predicted structure of AtMYB30 DNA binding domain bound to DNA (gray)
The model was predicted using the I-TASSER server and rendered with UCSF Chimera
The conservation between members of subgroup S1 was inferred from a MUSCLE alignment and colored using JALVIEW
Alpha helices and DNA binding sites were predicted using the I-TASSER server
MYB domains were identified using INTERPROSCAN
sumoylation and ubiquitation sites were predicted using PhosphAt
AtMYB30 being a positive regulator of plant defense and associated cell death responses
several mechanisms of negative regulation of its activity have been described
whether and how SUMOylation of AtMYB30 affects AtMYB30-mediated defense responses remains to be determined
the promoters of AtMYB30 and BES1 common target genes harbor boxes bound by each TF
this data shows that AtMYB30 functions to amplify BR signaling through cooperation with BES1 to promote BR target gene expression
Cellular responses to environmental or physiological cues rely on transduction pathways that must discriminate between different signals and ensure a combinatorial regulation
combinations of different PTMs and protein-protein interactions provide different layers of information that may allow the integration of several transduction pathways and warrant highly specific cellular outputs
Accumulating evidence shows that the Arabidopsis MYB regulator AtMYB30 is a multi-regulated protein that is involved in the integration of various environmental stimuli
likely through the activation of shared and specific sets of target genes
How simultaneous and diverse stress signals are integrated into a unified cellular response is a major unknown in cell signaling
The acceleration of large data set acquisition and the development of systems biology approaches promise to offer new insights into the functioning of such complex regulatory networks
The wealth of knowledge gained in recent years on Arabidopsis R2R3 MYB TFs provides an excellent framework toward this end
Our work is performed at the LIPM that is part of the Laboratoire d'Excellence (LABEX) entitled TULIP (ANR-10-LABX-41)
A post-translational modification code for transcription factors: sorting through a sea of signals
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Programmed cell death in the plant immune system
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A novel myb oncogene homolog in Arabidopsis thaliana related to the hypersensitive cell death
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Recently duplicated maize R2R3 Myb genes provide evidence for distinct mechanisms of evolutionary divergence after duplication
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Evolutionary and comparative analysis of MYB and bHLH plant transcription factors
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Altered patterns of gene expression in Arabidopsis elicited by cauliflower mosaic virus (CaMV) infection and by a CaMV gene VI transgene
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Hammond-Martel
Roles of ubiquitin signaling in transcription regulation
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The transcription factors WRKY11 and WRKY17 act as negative regulators of basal resistance in Arabidopsis thaliana
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Translocation of phospholipase A2alpha to apoplasts is modulated by developmental stages and bacterial infection in Arabidopsis
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Xanthomonas type III effector XopD desumoylates tomato transcription factor SlERF4 to suppress ethylene responses and promote pathogen growth
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Comparative analysis of the XopD type III secretion (T3S) effector family in plant pathogenic bacteria
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Identification of new early markers of the hypersensitive response in Arabidopsis thaliana
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Citation: Raffaele S and Rivas S (2013) Regulate and be regulated: integration of defense and other signals by the AtMYB30 transcription factor
Received: 04 March 2013; Paper pending published: 21 March 2013; Accepted: 28 March 2013; Published online: 11 April 2013
Copyright © 2013 Raffaele and Rivas. This is an open-access article distributed under the terms of the Creative Commons Attribution License
distribution and reproduction in other forums
provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc
*Correspondence: Susana Rivas, Laboratoire des Interactions Plantes-Microorganismes, UMR CNRS/INRA 2594/441, 24 Chemin de Borde Rouge-Auzeville, CS 52627, Castanet-Tolosan cedex 31326, France. e-mail:c3VzYW5hLnJpdmFzQHRvdWxvdXNlLmlucmEuZnI=
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Volume 5 - 2023 | https://doi.org/10.3389/fagro.2023.1223639
This article is part of the Research TopicCover Crops and Green Manures: Providing Services to AgroecosystemsView all 10 articles
Cover crops are often mentioned as a way to decrease nutrient losses during the fallow period
Species mixtures of crucifer-legume have been shown to effectively take up nitrogen (N) and sulphur (S) to decrease losses due to leaching
but their ability to simultaneously take up other key macro- and micronutrients remains unknown
Our study assessed the performances of a wide variety of bispecific crucifer-legume mixtures to provide synergetic uptake of multiple nutrients to target nutrient-recycling ecosystem services
Cultivars from eight crucifer and seven legume species were tested as sole and bispecific cover crops at an experimental site near Toulouse
Crucifer-legume mixtures showed synergetic uptake of nutrients per ha compared to sole cover crops for some nutrients through niche complementarity and facilitation processes and net competition for other nutrients
Species mixtures induced both i) higher nutrient concentrations for Mn and Fe in crucifers and Ca and B in legumes) and ii) lower concentrations for P
Ca and B in crucifers and Cu and Fe in legumes
indicating that the interactions differed among the species mixtures
the nutrient uptake measured in shoots were always higher in species mixtures than in sole crops (LER > 1) demonstrating the compatibility and complementarity of crucifer and legume mixtures in providing multiple-nutrient catch-crop and recycling related ecosystem services
Despite overall positive interactions and synergetic complementarities
the results also highlight that some net negative competition occurred for some nutrients
further investigation is still necessary to completely understand the processes behind multiple-nutrient acquisition in species mixtures
An effective option to encourage synergetic uptake of multiple nutrients by cover crops combines a variety of families that have different characteristics and performances
species mixtures of cover crops could be a solution to increase nutrient uptake over that of sole cover crops due to niche complementarity and facilitation processes
Ca and Mn concentrations in cucumber shoots and roots were significantly higher when grown in a species mixture with green garlic
but Mg concentrations were lower than those in sole crops
Such trade-offs in simultaneous nutrient acquisition require assessing cover crop mixtures to determine their potential to provide nutrient ecosystem services for leaching mitigation and recycling to the next cash crop
cooperation (facilitation) and compensation in species mixtures grown in agroecosystems for providing ecosystem services
We tested two hypotheses for using cover crop sole crops and species mixtures in short (summer to mid-autumn) fallow periods:
1) The relation (complementarity or competition) between crucifers and legumes depends on the nutrient considered and the species mixed
2) Bispecific mixtures mutualise their acquisition of multiple nutrients
mainly due to the niche complementarity of nutrient uptake capacity and root systems when exploring the soil
To avoid confounding effects of plant-plant interactions between adjacent treatments
only the six rows in the middle of each plot were harvested and used for soil measurements
Species grown as sole cover crops were sown at densities recommended for cover crops to obtain plant cover with similar densities and rapid growth and soil coverage
Sowing densities for sole cover crops were 800 seeds m-2 for all clover species; 150 plants m-2 for Ethiopian mustard; 100 plants m-2 for white mustard and all vetch species; 80 plants m-2 for rape
radish and turnip rape; 70 plants m-2 for soya bean and 40 plants m-2 for faba bean
Sowing densities for bispecific mixtures were half of the corresponding sole cover crop density of each species (50% crucifers and 50% legumes) to create a substitutive design of species mixtures
Seeds of both species were mixed in the row and sown with an experimental seeding machine
to obtain a homogenous mixed-plant cover crop in the row to maximise plant-plant interactions
We thus assumed that nutrients in the soil were not a limiting factor in our experiments
which have a shorter growing period and thus produce less biomass
An anti-grass herbicide (Axéo®) was sprayed at 1.2 litre per hectare to control a strong emergence of weeds (mainly annual grasses) in September to decrease the influence of extraneous factors
Cover crops were sampled 2.5 months after sowing
which is consistent with the usual practice of incorporating cover crops into the soil before sowing the next winter crop
as autumn is warm and rainy in this region
which promotes rapid growth of cover crops
was collected from 1 m2 per replicate (more precisely
two 0.5 m2 quadrats were randomly put in each 12 m plot length)
The roots of legume species were not considered in the results due to technical difficulties in sampling fine roots in the silty clay loam soil of the study site
Most of the root biomass of legumes is composed of fine roots
while the roots of crucifers are generally tuberous
which makes them easier to collect and measure more accurately
Shoots and roots were separated using a sharp knife and secateurs
and the root systems were carefully washed with cold water to remove the remaining soil
and Zn via elemental analyses based on the Dumas method (Elementar MicroVario Cube
and by inductively coupled plasma - atomic emission spectroscopy (ICP-AES) for the other nutrients (at the SADEF-accredited laboratory
The land equivalent ratio (LER), defined as the sole crop area required to reach the same biomass as a multi-species mixture or intercrop (Willey, 1979), equals the sum of the partial LER (pLER) of each mixed species. We used the LER index to assess nutrient uptake as Couëdel et al., 2018a; Couëdel et al., 2018b did for N and S acquisition
and applied here for all nutrients analysed:
where pLER_C and pLER_L are the pLER of mixed crucifers and legumes
NutrientupIC is the nutrient uptake of the given nutrient for a given species (crucifer or legume) in the mixture
and NutrientupSC is the nutrient uptake of the same species as a sole cover crop
and differences among treatments were considered significant at P< 0.05
Cultivar data were pooled as no significant differences in nutrient concentrations or acquisition were observed between cultivars
Results were then analysed by crop family and species to increase the robustness of the results and to highlight key points
Figure 1 Macronutrient (%) and micronutrient (ppm) concentrations in shoots and roots of crucifers and shoots of legumes
Values correspond to the mean of sole (SC) and mixed (Mix) crucifers or legumes
Asterisks indicate a significant difference in macro- and micronutrient concentrations between a given family in a sole crop and in mixtures (P< 0.05)
Table 1 Macronutrient (%) and micronutrient (ppm) concentrations in shoots of crucifer and legume sole crops (SC) and species mixtures (Mix)
Table 2 Macronutrients (kg ha-1) and micronutrients (g ha-1) uptake in shoots of crucifer and legume sole crop (SC) and species mixtures (Mix)
suggesting that the legumes may have outcompeted the crucifers for Ca and B
Figure 2 Macronutrients (kg ha−1) and micronutrients (g ha−1) acquired in shoots of crucifers and legumes in sole crops and species mixtures
Values correspond to the mean of crucifer (Cr) and legume (Le) sole crops or the mean of both species included in a mixture (Mix)
Different letters indicate treatments with significant differences at P< 0.05
Only shoots of crucifers and legumes were included in the analysis
The pLER_L did not differ significantly from 0.5 for all other nutrients indicating that mixture had no effect on legume acquisition per plant for these nutrients
Figure 3 Partial land equivalent ratio (pLER) of crucifers (red) and legumes (blue)
The land equivalent ratio (LER) equals the sum of the two partial LERs
Each value represents the mean of species mixtures
Asterisks indicate that pLER and LER were significantly greater than 0.5 and 1.0
Among legumes, some species had a pLER_L< 0.5 such as hairy vetch (for nearly all nutrients), purple vetch (for S and Cu), faba bean (for S, P, K and Cu) and common vetch (for Mn and Fe), while other legume × nutrient combinations had a pLER_L > 0.5 (Figure 4)
None of species mixtures had both pLER_C and pLER_L< 0.5, indicating that overall competition in mixtures was never stronger than complementarity and compensation. In comparison, approximately half of the species mixtures had a pLER_L and pLER_C > 0.5, indicating that complementarity and facilitation were stronger than competition (Figure 4)
Cross-analysis of nutrient concentrations in plants could help detect antagonisms or facilitation between species for a given nutrient that would decrease acquisition of other nutrients and thus the performance of the entire mixture (i.e. Liebig’s Law). Rietra et al. (2017) found synergy between P and K acquisition and antagonism between K and Mg acquisition
Our study confirms these results in crucifers
as lower K concentrations in mixtures than those in sole crops were associated with lower P concentrations but higher Mg concentrations
cross-analysis of nutrient acquisition requires further studies at more sites and for additional years to definitively validate the net effect of nutrient-acquisition processes by cover crops as synergy or antagonism
B and Zn uptake were also slightly higher in mixtures
than in both crucifer and legume sole cover crops
suggesting potential complementarity of mixtures in providing catch-crop effects for these nutrients as well
By assessing the multi-nutrient uptake capabilities of species mixtures composed of crucifers and legumes
our study contributes valuable insights for assessing their performance across a broader spectrum of ecosystem services
crucifer-legume cover crop mixtures provide synergetic multiple-nutrient catch-crop services by acquiring as much or more of each nutrient than the best sole cover crop
crucifers acquired certain nutrients in much larger quantities
Crop nutrition status revealed strong niche complementarity and/or facilitation in crucifer-legume mixtures
in which crucifers had higher Mn and Fe concentrations
and legumes had higher Ca and B concentrations
Net competition and/or dilution effects of nutrient concentration were also identified in the cover crop mixtures tested
as legumes tended to have a negative impact on crucifer concentrations of Ca
while crucifers had a negative impact on legume concentrations of Cu and Fe
Our work validated the potential of species complementarity for multi-nutrient uptake using a wide range of crucifer-legume species and cultivars
However further studies should include additional sites with a variety of soil and climate conditions to further validate these first results and help design more efficient cover crop mixtures to achieve high levels of multiple nutrient-related ecosystem services under different growing conditions
The raw data supporting the conclusions of this article will be made available by the authors
AC prepared the data and performed all preliminary and final analyses
All authors were involved in the interpretation of the results and contributed to writing the original version of the manuscript and improving the subsequent ones
All authors contributed to the article and approved the submitted version
This study was part of the CRUCIAL project
which was financially supported by the French Ministry of Agriculture (CASDAR project no
C-2013-05) and the Occitanie Region (CLE project no
This project has received funding from the European Union’s Horizon Europe research and innovation programme under grant agreement No 101081973 - IntercropValuES programme
The authors thank Annick Basset from Jouffray Drillaud and Cedric Monprofit from RAGT Semences for their expert advice in selecting cover crops for the project
We also thank François Perdrieux and Gaël Rametti for their effective technical help at the Domaine de Lamothe site of INP-EI Purpan
and Eric Lecloux and Didier Raffaillac for their support in sampling plants and laboratory analysis
Gaëlle van Frank and Antoine Parisot for their helpful discussions
We thank Michelle and Michael Corson for reviewing the English language
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations
Any product that may be evaluated in this article
or claim that may be made by its manufacturer
is not guaranteed or endorsed by the publisher
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fagro.2023.1223639/full#supplementary-material
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Received: 16 May 2023; Accepted: 09 August 2023;Published: 29 August 2023
Copyright © 2023 Couëdel, Alletto and Justes. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY)
*Correspondence: Antoine Couëdel, YW50b2luZS5jb3VlZGVsQGNpcmFkLmZy
†ORCID: Antoine Couëdel, orcid.org/0000-0002-5786-0939Lionel Alletto, orcid.org/0000-0003-0933-9476Eric Justes, orcid.org/0000-0001-7390-7058
Metrics details
Circular representation of the pseudomolecules
Density of two families of long retrotransposon terminal repeats (5.25–9.5 kb in blue and 9.5–12.25 kb in red) and genes (purple)
SNP density in 80 lines of the domesticated sunflower
Locations of genes mapping to oil metabolic pathways
whereby the colour (from light to dark) indicates the trait: palmitate
Regions associated with flowering time in domesticated sunflowers
Expression of organ-specific genes (from outside to inside tracks: pollen
These findings highlight the creative potential of transposons and provide tools for understanding gene function in this model system
Evolutionary scenario of the Asterids (sunflower
lettuce and coffee) from the AEKs of 21 (post-WGT-γ) and 7 (pre-WGT-γ) protochromosomes
The modern genomes are illustrated at the bottom with the different colours reflecting the origin from the seven ancestral chromosomes from the n = 7 AEK (top)
Polyploidization events are shown with coloured dots (duplications) and stars (triplications)
along with the shuffling events (fusions and fissions)
The time scale is shown on the left (million years)
sunflower paralogues (black); right y axis
artichoke paralogues (blue) and sunflower–coffee orthologues (purple)
WGD-2 and WGT-γ) and speciation (sunflower–coffee) events are referenced on the x axis
WGD-2 (1–2 chromosomal relationships in red circles)
WGT-1 (1–3 relationships in blue circles) and WGT-γ (1–3 relationships in brown circles) events
There were several sunflower in-paralogues for 180 Arabidopsis genes
illustrating the complexity of regulatory networks in sunflower
This analysis therefore provides insights into the architecture of flowering time in domesticated sunflowers and provides a major resource for breeding programs
demonstrating the sensitivity of the approach
The sunflower now has the potential to become a model crop for climate change adaptation
which can be achieved by exploiting genome-enabled systems biology and multi-disciplinary analyses of interactions between abiotic stressors
A full description of the Methods can be found in the Supplementary Information
No statistical methods were used to predetermine sample size
The genome-wide association experiments were fully randomized and the investigators were not blinded to allocation during experiments and outcome assessment
In total 32.8 million subreads were generated with an N50 of 13.7 kb and a mean length of 10.3 kb
The targeted genome coverage of 102× was obtained with 367 Gb of raw sequence (340 Gb of subread data)
Sequencing, assembly and annotation of the genome of another genotype, HA412-HO, is presented in Supplementary Note 1.7
and it detected 92% of complete gene models (590 complete single copy and 291 duplicated
respectively) plus 10 additional fragmented gene models
Speciation events were dated to 38 million years ago (Ma) for sunflower–artichoke
100 Ma for sunflower–coffee and 118 Ma for sunflower–grape
Palaeoploidization events were dated to 122–164 Ma for WGT-γ
Gene Ontology enrichment tests were carried out with Blast2GO Pro (one-sided Fisher’s exact tests
Selective sweeps reveal candidate genes for adaptation to drought and salt tolerance in common sunflower
Improving plant breeding with exotic genetic libraries
Selection of wild and cultivated sunflower for resistance to a new broomrape race that overcomes resistance of the Or 5 gene
deserticola for improving oil content and quality in sunflower
The sunflower (Helianthus annuus L.) genome reflects a recent history of biased accumulation of transposable elements
Most Compositae (Asteraceae) are descendants of a paleohexaploid and all share a paleotetraploid ancestor with the Calyceraceae
Multiple paleopolyploidizations during the evolution of the Compositae reveal parallel patterns of duplicate gene retention after millions of years
and challenges in simulating impacts and informing adaptation
Prioritizing climate change adaptation needs for food security in 2030
Hybrid speciation accompanied by genomic reorganization in wild sunflowers
Turning heads: the biology of solar tracking in sunflower
Evolution and diversification of the CYC/TB1 gene family in Asteraceae—a comparative study in Gerbera (Mutisieae) and sunflower (Heliantheae)
Progress towards a reference genome for sunflower
Analysis of retrotransposon structural diversity uncovers properties and propensities in angiosperm genome evolution
The genome sequence of the outbreeding globe artichoke constructed de novo incorporating a phase-aware low-pass sequencing strategy of F1 progeny
The coffee genome provides insight into the convergent evolution of caffeine biosynthesis
The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla
FLOR-ID: an interactive database of flowering-time gene networks in Arabidopsis thaliana
Contributions of flowering time genes to sunflower domestication and improvement
Genome scans reveal candidate domestication and improvement genes in cultivated sunflower
as well as post-domestication introgression with wild relatives
Evidence of selection on fatty acid biosynthetic genes during the evolution of cultivated sunflower
Genetic analysis of phytosterol content in sunflower seeds
Genetic dissection of tocopherol and phytosterol in recombinant inbred lines of sunflower through quantitative trait locus analysis and the candidate gene approach
Roles of phosphatidate phosphatase enzymes in lipid metabolism
Involvement of phosphatidate phosphatase in the biosynthesis of triacylglycerols in Chlamydomonas reinhardtii
Plant genome sequencing — applications for crop improvement
Translational genomics for plant breeding with the genome sequence explosion
Validating genome-wide association candidates controlling quantitative variation in nodulation
Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules
Assembling large genomes with single-molecule sequencing and locality-sensitive hashing
finished microbial genome assemblies from long-read SMRT sequencing data
Genome annotation in plants and fungi: EuGene as a model platform
BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs
The Arabidopsis information resource (TAIR): improved gene annotation and new tools
ShortStack: comprehensive annotation and quantification of small RNA genes
The small RNA diversity from Medicago truncatula roots under biotic interactions evidences the environmental plasticity of the miRNAome
miRBase: annotating high confidence microRNAs using deep sequencing data
an efficient and flexible software for de novo detection of LTR retrotransposons
Fine-grained annotation and classification of de novo predicted LTR retrotransposons
GenomeTools: a comprehensive software library for efficient processing of structured genome annotations
PAML 4: phylogenetic analysis by maximum likelihood
Molecular demographic history of the annual sunflowers Helianthus annuus and H
petiolaris—large effective population sizes and rates of long-term gene flow
Improved criteria and comparative genomics tool provide new insights into grass paleogenomics
Inference of population structure using multilocus genotype data
Genome-wide midrange transcription profiles reveal expression level relationships in human tissue specification
edgeR: a Bioconductor package for differential expression analysis of digital gene expression data
VarScan: variant detection in massively parallel sequencing of individual and pooled samples
BEDTools: a flexible suite of utilities for comparing genomic features
Estimation of levels of gene flow from DNA sequence data
analysis and simulation tools for population genetics and genomics
QTL mapping of seed-quality traits in sunflower recombinant inbred lines under different water regimes
Molecular basis of the high-palmitic acid trait in sunflower seed oil
Mapping quantitative trait loci controlling oil content
oleic acid and linoleic acid content in sunflower (Helianthus annuus L.)
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Kuhn for sharing his expertise in PacBio sequencing and H
Witsenboer for his help with the production of the Fingerprint-based physical map; the Genotoul bioinformatics platform Toulouse Midi-Pyrenees for providing help and computing resources
the common services of the LIPM for their support
and Genome Quebec Innovation Centre and Canada’s Michael Smith Genome Science Centre for 454 and Illumina sequencing; M
Barker for their help and advice during the Genome Canada/Genome BC project; and D
Swanevelder for contributing to the sequencing of the sunflower association mapping populations; members of the International Consortium for Sunflower Genomics resources (2012–2015): Advanta
Pioneer and Syngenta companies and their sunflower project leaders; F
Bonnafous for the development of the statistical pipeline for GWAS and P
André for the coordination of flowering time phenotyping on the sunflower hybrid panel (GWAS)
This project was funded by the French National Research Agency (SUNYFUEL/ANR-07-GPLA-0022 and SUNRISE/ANR-11-BTBR-0005 projects)
the European Fund for Regional Development
the French Fund for Competitiveness Clusters (FUI)
Genome Canada and Genome BC’s Applied Genomics Research in Bioproducts or Crops (ABC) Competition
the NSF Plant Genome Program (DBI-0820451) and the International Consortium for Sunflower Genomics Resources
Grassa: These authors contributed equally to this work
Langlade: These authors jointly supervised this work
Department of Botany and Biodiversity Research Centre
Institute of Plant Sciences Paris-Saclay (IPS2)
Christine Lelandais-Brière & Martin Crespi
Department of Ecology and Evolutionary Biology
Centre National de Ressources Génomiques Végétales
Marie-Christine Le Paslier & Elodie Marquand
Cécile Donnadieu & Céline Vandecasteele
UMR Arche INRA/ENSAT F-31320 Castanet-Tolosan
developed the DNA-extraction protocol for PacBio sequencing
contributed to DNA/RNA sample collection and data production
built the physical map and integrated the physical and genetic maps
performed quality control of XRQ pseudomolecules
annotated protein-coding genes and miRNA (XRQ)
performed integrated analyses on flowering time and oil metabolism
performed analysis of sunflower ohnologues
contributed to the genome consortium coordination
designed experiments and coordinated the project
coordinated the sunflower genome consortium
The authors declare no competing financial interests
Van der Peer for their contribution to the peer review of this work
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
The x axis represents the age of insertions in millions of years
the y axis is the density of insertions at a given time point
the age distribution of each superfamily of subclass I of the Class II transposons (the terminal inverted repeat transposons)
the age distribution of LTR-RT superfamilies
dot plots of orthologues between the grape genome (y axis
as a representative of the n = 21 post-γ ancestor) and
the sunflower (1–6 chromosomal relationships inherited from WGT-1 and WGD-2)
artichoke (1–3 chromosomal relationships deriving from WGT-1)
coffee (1–1 chromosomal relationships illustrating the absence of a coffee-specific WGD
despite WGT-1) genomes and the lettuce genetic map (1–3 chromosomal relationships deriving from WGT-1)
dot plots of orthologues between the sunflower genome (y axis
n = 9 chromosomes) genomes with 1–1 chromosomal relationships
Histogram of the specificity index Tau in expressed genes
Box plot distribution of the specificity index Tau in 11 different organs
The different organs are represented with the following colours: Ray floret ovary
Violin plot of the specificity index Tau for transcription factors (TFs
Cumulative bar plot showing the organ distribution of specific genes (left)
transcription factors (middle) and lncRNA (right)
Simple arrows represent positive regulation and other arrows negative regulation
Curved lines between genes represent protein–protein complexes
Whole-metabolic network (3,821 reactions and 475 pathways)
Genes are coloured by expression levels in developing seeds
Co-expression network of oil metabolic pathway
Genes that co-localize with QTLs are coloured in orange
Sub-network with genes from b co-localizing with QTLs
Node size is proportional to Fst between lines cultivated for oil production and other domesticated lines
Genes with an Fst in the top 5% are coloured in dark orange
Mapping of candidate genes (orange genes from c) on the pathways of diacylglycerol and triacylglycerol biosynthesis
Mapping of candidate genes on the pathway of linoleate biosynthesis
Tree of a gene cluster including a candidate gene of the PAP2 superfamily
involved in the synthesis of fatty acid precursors (d)
This contains Supplementary Notes split into 10 sections
data and discussion (Genome Sequencing and Assembly
Paleogenomics and ancestry of the sunflower genome
Analysis of sunflower ohnologs and oil metabolism) and Supplementary References
This file contains tables A-K regarding location and annotation of miRNA
I-Intersection between phasiRNA clusters and miRNA targets
J- Clusters of mapping of 24 nucleotide sRNA
K – Intersection between genes and 24 nucleotides mapping clusters
This table describes paralogy relationships in the sunflower genome
This table describes orthology relationship between genes of sunflower and grape
This document contains figures of windows estimates of the amount and origin of introgression in the genomes assemblies of the XRQ and Ha412 genotypes (one figure per chromosome)
This file contains tables lists of organ specific transcription factors of the MYB and TCP families in 11 sunflower organs
This file contains tables of Gene Ontology categories enriched in response to hormones or stress treatments in sunflower roots and leaves
This file contains sunflower orthologs and in-paralogs of flowering time genes in Arabidopsis thaliana
This table contains a curated list of sunflower genes involved in seed oil metabolism
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Nicolas Langlade and colleagues report the genome sequence of the domesticated sunflower
a global oil crop that can maintain stable yields across a wide range of environmental conditions
Their comparative analyses provide insights into the evolutionary history of Asterids
They also analysed transcriptomic data from vegetative and floral organs
re-sequenced 80 domesticated lines and performed genome-wide association studies identifying 35 loci associated with flowering time
These resources will be useful in breeding programs as well as ecological and evolutionary studies
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While attribution of a single LSF episode to climate change is difficult
both the frequency and the severity of damage due to LSF may be amplified by the climate crisis
attempts to mitigate LSF damage and climate change impacts at both shorter and longer timescales may only exasperate the issues
From early April 2021, frost and freezing temperatures caused severe damage to crops including vineyards and fruit trees, oilseed rapes, potatoes and sugar beets across Europe. In France, about 80% of the country’s wine and fruit tree regions were affected (Box 1)
with the extent of the damage also substantial in Italy
which threatens livelihoods and food security
is worsened for producers already suffering due to COVID-19
Prices may be subject to local taxes which are calculated during checkout
Faust, E. & Herbold, J. Spring frost losses and climate change — not a contradiction in terms. Munich Re https://bit.ly/3x3UQXS (2018)
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a multi-partnership initiative that aims to produce knowledge and tools useful for the diversification of cropping systems based on oilseeds and pulses
with a twofold objective: an improved adaptation to climate change as well as agro-ecological and nutritional transition
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DOI: https://doi.org/10.1038/s41558-021-01090-x
Theoretical and Applied Climatology (2025)
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The Creole cattle from Guadeloupe (GUA) are well adapted to the tropical environment
Its admixed genome likely played an important role in such adaptation
we sought to detect genomic signatures of selection in the GUA genome
we sequenced 23 GUA individuals and combined our data with sequenced genomes of 99 animals representative of European
We detect 17,228,983 single nucleotide polymorphisms (SNPs) in the GUA genome
of patterns of genetic variation in this breed
We confirm the higher level of African and indicine ancestries
compared to the European ancestry and we highlight the African origin of indicine ancestry in the GUA genome
We identify five strong candidate regions showing an excess of indicine ancestry and consistently supported across the different detection methods
These regions encompass genes with adaptive roles in relation to immunity
We confirmed a previously identified horn-related gene
as a gene under strong selective pressure in the GUA population likely owing to human-driven (socio-cultural) pressure
Findings from this study provide insight into the genetic mechanisms associated with resilience traits in livestock
These studies benefited from ongoing advancements in genomic technology and the development of improved statistical and computational methods to identify signatures of selection in cattle genome
that is genomic regions that appear to be shaped by selection
Owing to its three-way admixture and its long-term isolation under extreme environmental conditions
the genome of the Creole cattle from Guadeloupe offers a unique opportunity to study admixture-enabled adaptation
A previous study provided a first insight into footprints of selection in GUA cattle using the Illumina BovineSNP50 chip assay7
To expand our understanding of the genomic architecture of local adaptation in this population
we leverage whole-genome sequence data from 23 GUA animals and genomes of 99 cattle individuals from various origins
Our objective was to use high-resolution genomic data and consistency of signals among different methods based on the excess of haplotype homozygosity
differences in allele frequencies and excess ⁄ deficiency of local ancestry to identify new
strong candidate regions under selection in the GUA genome
Genetic diversity and population structure and genetic diversity of Creole cattle from Guadeloupe (GUA)
Population names and locations are described in supplementary table S13 (supplementary material)
(a) Genome-wide distribution of nucleotide diversity (Pi) estimated in 1-Mb nonoverlapping window
AFT African taurine (b) Principal component analysis
CRIO Criollo (Colombian and venezuelan cattle breeds)
AMASI American-Australian and Asian indicine breeds (c) Results of admixture analysis for K 3–5
Manhattan plots showing the results of Extended Haplotype Homozygisty-based tests. (a) Rsb test AFT versus GUA cattle. (b) Rsb test EUT versus GUA cattle (c) Rsb test IND versus GUA cattle. (d) XP-EHH test AFT versus GUA cattle. (e) XP-EHH test EUT versus GUA cattle (f) XP-EHH test IND versus GUA cattle. (g) iHS test for GUA cattle. Horizontal dashed lines mark the significance threshold applied to detect the outlier SNPs (− log10 (p value) = 6).
Nucleotide diversity and Pairwise Fst values [EUT vs
GUA (green)] calculated for each 50-kb window around the candidate regions on chromosomes 2 and 5
(c) Pairwise Fst values between GUA and EUT on chromosome 2
(d) Pairwise Fst values between GUA and EUT on chromosome 5
(e) Pairwise Fst values between GUA and AFT on chromosome 2
(f) Pairwise Fst values between GUA and AFT on chromosome 5
We identified another gene in this region: DNA Helicase B (HELB) located 250 Kb upstream the highest signal
the three SNPs of highest significance (8.48 < − log(p value) < 10.4) are less than 80 Kb upstream of relaxin family peptide receptor 2 (RXFP2)
the cluster of the four most significant outlier SNPs is less than 100 Kb upstream the ASIP gene
This further confirms the uniqueness of this population with respect to the other Creole breeds even those found in neighbouring regions and which are expected to have a similar genetic history
estimating with high accuracy the origin of African indicine ancestry in the GUA genome would require using additional cattle populations from western Africa and the Sahel region
Selection for draught traits in the GUA population thus seems to have provoked responses in a diversity of pathways involving at least EIF4E2
Such selective pressure promoted the fixation of beneficial alleles from an indicine origin that allowed GUA individuals to cope with withstanding hours of high intensities of physical activity
we have generated for the first time a catalogue of genetic variants found in the Creole cattle from Guadeloupe
We were able to show that GUA adaptation to local environment occurred mainly through its indicine component
We also demonstrate that pathogenic environment
thermotolerance and physical stamina are important drivers of local adaptation in Creole cattle
our results provide clues for understanding the adaptive admixture in the Creole cattle from Guadeloupe thus contributing to the emerging picture of the genes and pathways associated with traits resilience in livestock species
Our study may represent a starting point for a targeted and sustainable genetic breeding improvement of Creole cattle
Blood collection was done according to good practices recommended for identification of sires for paternity checking in France
Semen was collected for the main purpose of insemination
according to relevant technical guidelines for semen collection and preparation
This study was approved by the scientific committee of the Metaprogramme SELGEN of INRA
which afforded a grant to the project TROCADERO
The study is in accordance with ARRIVE guidelines
Twenty-three Creole bulls representative of the INRA nucleus in Guadeloupe were selected for the purpose of this study
INRA experimental nucleus was created in 1980 from local animals chosen according to their phenotype
New local sires are regularly introduced in the nucleus as a part of a conservation program aiming to avoid loss of variation in the GUA population
Ten out of the twenty-three individuals of the present study are sons of founder sires of the experimental flock while the remaining thirteen animals are unrelated sires (based on genealogical records and sampling sites) chosen from the local stock of Guadeloupe before introduction to the INRA nucleus
The animals selected for the purpose of this study are among the most frequently used in artificial insemination
Genomic DNA was extracted from whole-blood and semen samples collected between 1995 and 2015
Four Colombian individuals belonging to the IMAGE project (H2020 project 677353 'Innovative Management of Animal GEnetic resources) were also included in the study
Paired-end libraries with insert size of 500 bp were constructed for each individual and sequenced using the HiSeq 3000 platform (Illumina) in the Genome et Transcriptome (GeT) GénoToul platform (Toulouse
We applied the same marker selection thresholds (maf = 0.1
p value HWE = 0.01 and minQ = 100) to build these two databases which resulted in 16,360,962 and 8,955,346 high-quality SNPs for the public and the 1000 Bull Genomes genotyping data
All three databases were combined into a single one containing 5,316,956 common snps
Nucleotide diversity (Π) at a whole-genome scale was estimated in sliding windows of 1 Mb using VCFtools considering the following breed groupings: AFI (N = 11) and AFT (N = 7) and the each of the following breeds separately: Aubrac (AUB; N = 8)
we selected 10 GUA individuals among the least related
This was done to have sample groups with similar size (for a matter of comparison)
Muturu and Ndama as proxies for AFT ancestry
the French and the Spanish breeds as proxies for EUT ancestry
ELAI uses a two-layer hidden Markov model to detect the structure of haplotypes for unrelated individuals
The algorithm models two scales of linkage disequilibrium (one within a group of haplotypes and one between groups) and provides a map
to descend from each one of the ancestral populations
ELAI analysis was conducted across 19 GUA genomes by setting the parameters -mg (number of generations) to 70
we considered the 17 populations with more than two individuals
We built a maximum likelihood tree using blocks of 30,000 SNPs with no migration events allowed
we built a phylogenetic tree of these populations and started adding migration events (modelled as edges) sequentially to the phylogenetic model
The migration edges were added until 99.79% of the variance in ancestry between populations was explained by the model
Runs of homozygosity (ROHs) were identified in sliding windows of 100 SNPs using PLINK and the 1,064,342 SNPs that passed quality control filtering
ROH were detected within the following five breed groupings: Creole cattle from Guadeloupe (GUA)
European Taurine (EUT) : IBER (represented by the four Spanish breeds)
COL (represented by the four Colombian breeds) and American-Australian and Asian Indicine (AMASI) : BRA
The following parameters were used to define a ROH: (1) homozyg-snp (minimum number of SNPs that a ROH is required to have): 100
(2) homozyg-density (required minimum density to consider a ROH): 50
(3) homozyg-gap (length in Kb between two SNPs in order to be considered in two different segments): 1000 (4) homozyg-window-het (number of heterozygous SNP allowed in a window): 3 (5) homozyg-window-missing (number of missing calls allowed in a window): 5
The –homozyg-group option implemented in PLINK was used to assess ROH islands shared among GUA individuals
These were defined as the homozygous segments shared by at least 30% of the samples
Imputeqc package has been designed to assess the imputation quality and/or to choose the model parameters for imputation
we found that K = 20 provided the best imputation quality (for 5% of masked data)
Considering that Rsb and XP-EHH values are normally distributed
a Z-test was applied to identify significant SNPs under selection
Two-sided p values were derived as pRsb = − log10[1 − 2|Φ(Rsb) − 0.5|] and pXP-EHH = − log10[1 − 2|Φ(XP-EHH) − 0.5|] where Φ (x) represents the Gaussian cumulative distribution function
the maximum allowed gap between two SNPs was set to 500 Kb
We used sliding nonoverlapping 500-Kb windows to perform selection signature detection
A window is classified as putatively under selection when it contains at least 5 markers exceeding the significance threshold of − log10 (p value) = 6
Positive XP-EHH and Rsb values indicate longer haplotypes in the target population (i.e
GUA) therefore suggesting that selection occurred in the GUA population
The software detects Site Frequency Spectrum (SFS) patterns generated by complete selective sweeps
We collected information through a literature search to discuss the biological implications of our findings with regard to the candidate genes located in the relevant genomic regions known to be involved in phenotypic variation of adaptive traits
The generated sequences for the 23 Creole cattle from Guadeloupe samples are available from the European Nucleotide Archive (ENA) with the Bioproject Accession Number PRJEB58555
Primitive andalusian livestock and their implications in the discovery of America
El hanado bovino ibérico en las Américas: 500 años después
Historique du peuplement bovin et de l’introduction de la tique Amblyomma variegatum dans les îles françaises des Antilles: Synthèse bibliographique
Predominant African-derived mtDNA in Caribbean and Brazilian Creole cattle is also found in Spanish cattle (Bos taurus)
Genetic footprints of Iberian cattle in America 500 years after the arrival of Columbus
Demography and rapid local adaptation shape Creole cattle genome diversity in the tropics
Footprints of selection in the ancestral admixture of a new world Creole cattle breed
Les ruminants domestiques de la Caraïbe: Le point sur les ressources génétiques et leur exploitation
Epidemiology of heartwater in Guadeloupe and in the Caribbean
Whole-genome resequencing reveals world-wide ancestry and adaptive introgression events of domesticated cattle in East Asia
Adaptive introgression from indicine cattle into white cattle breeds from Central Italy
The mosaic genome of indigenous African cattle as a unique genetic resource for African pastoralism
Genetic architecture and signatures of selection in the Caqueteño Creole (Colombian native cattle)
Mathematical model for studying genetic variation in terms of restriction endonucleases
A map of recent positive selection in the human genome
Role of transcriptional coregulator GRIP1 in the anti-inflammatory actions of glucocorticoids
An attempt to identify genetic markers of resistance or susceptibility to dermatophilosis in the zebu Brahman population of Martinique
Adaptive admixture in the West African bovine hybrid zone: Insight from the Borgou population
Population history and genetic adaptation of the Fulani nomads: Inferences from genome-wide data and the lactase persistence trait
Genome-wide detection and characterization of positive selection in human populations
A new approach for using genome scans to detect recent positive selection in the human genome
Structural basis of oligomerization in septin-like GTPase of immunity-associated protein 2 (GIMAP2)
NK cell activation and protection occur independently of natural killer T cells during Trypanosoma cruzi infection
Natural selection and infectious disease in human populations
Controlling the false-positive rate in multilocus genome scans for selection
Reconstituting the frequency spectrum of ascertained single-nucleotide polymorphism data
How do SNP ascertainment schemes and population demographics affect inferences about population history?
A composite of multiple signals distinguishes causal variants in regions of positive selection
Genetics of skin color variation in Europeans: Genome-wide association studies with functional follow-up
Molecular analysis of reverse mutations from nonagouti (a) to black-and-tan (a(t)) and white-bellied agouti (Aw) reveals alternative forms of agouti transcripts
Whole genome analysis of water buffalo and global cattle breeds highlights convergent signatures of domestication
Variants at the ASIP locus contribute to coat color darkening in Nellore cattle
Thermal consequences of colour and near-infrared reflectance
Selection signatures in tropical cattle are enriched for promoter and coding regions and reveal missense mutations in the damage response gene HELB
Molecular mechanisms of ultraviolet radiation-induced DNA damage and repair
The role of RXFP2 in mediating androgen-induced inguinoscrotal testis descent in LH receptor knockout mice
Whole-genome resequencing uncovers molecular signatures of natural and sexual selection in wild bighorn sheep
Whole-genome sequences of 89 Chinese sheep suggest role of RXFP2 in the development of unique horn phenotype as response to semi-feralization
The 1.78-kb insertion in the 3′-untranslated region of RXFP2 does not segregate with horn status in sheep breeds with variable horn status
Nasal heat exchange in the giraffe and other large mammals
De la compétition des boeuf-tirants et des charretiers en Guadeloupe
An oxygen-regulated switch in the protein synthesis machinery
Selection signatures of Fuzhong Buffalo based on whole-genome sequences
Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells
Mechanisms of mitochondrial respiratory adaptation
An integrative approach to the regulation of mitochondrial respiration during exercise: Focus on high-intensity exercise
Whole-genome sequencing of 234 bulls facilitates mapping of monogenic and complex traits in cattle
Trimmomatic: A flexible trimmer for Illumina sequence data
Fast and accurate short read alignment with Burrows-Wheeler transform
The sequence alignment/map format and SAMtools
A program for annotating and predicting the effects of single nucleotide polymorphisms
PLINK: A tool set for whole-genome association and population-based linkage analyses
adegenet: A R package for the multivariate analysis of genetic markers
Fast model-based estimation of ancestry in unrelated individuals
Distruct: A program for the graphical display of population structure
Genepop’007: A complete re-implementation of the genepop software for Windows and Linux
Detecting structure of haplotypes and local ancestry
Pickrell, J. K. & Pritchard, J. K. Inference of population splits and mixtures from genome-wide allele frequency data. PLoS Genet. https://doi.org/10.1038/npre.2012.6956.1 (2012)
rehh: An R package to detect footprints of selection in genome-wide SNP data from haplotype structure
A fast and flexible statistical model for large-scale population genotype data: Applications to inferring missing genotypes and haplotypic phase
imputeqc: An R package for assessing imputation quality of genotypes and optimizing imputation parameters
SweeD: Likelihood-based detection of selective sweeps in thousands of genomes
Small GTPase immunity-associated proteins mediate resistance to toxoplasma Gondii infection in Lewis rat
LNX1/LNX2 proteins: Functions in neuronal signalling and beyond
A polymorphism in the agouti signaling protein gene is associated with human pigmentation
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The authors wish to express their sincere gratitude to Didier Boichard
for allowing access to the raw sequence data of French samples (part of the 1000 Bull Genomes Project)
The first author is financially supported by FEDER AGROECODIV
The sequencing of the samples from Guadeloupe is financed by SELGEN TROCADERO
Sequencing data of the Columbian samples were provided by IMAGE (H2020 project 677353)
The Colombian Corporation for Agricultural Research for providing the Colombian samples
Laboratoire des Productions Animales et Fourragères
Institut National de la Recherche Agronomique de Tunisie
Ecole Nationale Vétérinaire de Toulouse (ENVT)
performed sequence alignment and variant discovery
performed population structure and selection signature analyses and wrote the manuscript
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DOI: https://doi.org/10.1038/s41598-023-38774-7
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Dreissenids are established model species for ecological and ecotoxicological studies
since they are sessile and filter feeder organisms and reflect in situ freshwater quality
Despite this strong interest for hydrosystem biomonitoring
we achieved full de novo assembly transcriptomes of digestive glands to gain insight into Dreissena polymorpha and D
Transcriptomes were obtained by Illumina RNA sequencing of seventy-nine organisms issued from fifteen populations inhabiting sites that exhibits multiple freshwater contamination levels and different hydrosystem topographies (open or closed systems)
Based on a recent de novo assembly algorithm
quality-checked and annotated transcriptomes
The power of the present study lies in the completeness of transcriptomes gathering multipopulational organisms sequencing and its full availability through an open access interface that gives a friendly and ready-to-use access to data
The use of such data for proteogenomic and targeted biological pathway investigations purpose is promising as they are first full transcriptomes for this two Dreissena species
Dreissena polymorpha • Dreissena rostriformis bugensis
Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.9934226
Their distribution and invasion dynamic is well documented
as well as ecological features of colonized ecosystems
Dreissenids thus become model species in ecology of biological invasion to identify pathway of invasion
and genetic mechanisms associated to colonization at various spatial and temporal scales
Top 10 fields treemap for Dreissena publications
Studies from several populations of the two species can pave the way for several new research possibilities
The most promising and useful ones are to make available a reference transcriptome for proteogenomic studies
to identify sequences to develop new sets of biomarkers
and to better understand acclimation mechanisms occuring during the invasion process and implied in tolerance to contamination
Samples were performed on 12 sites representative of french dreissenid populations diversity. Sites were chosen according to their variable contamination levels, chemicophysical quality and topography characteristics (see Supplementary Table 1)
Five bivalves were sampled by populations and digestive glands were in situ dissected and stored in RNAlater (Qiagen) at 4 °C
Total RNA extraction was done using RNeasy MinElute kit (Qiagen) according to the manufacturer’s protocol with slight modifications
tissus disruption was done first with a pestle and then with a beads beater with glass bead (200 µm) for 1 min
at max speed in 1 mL of RLT buffer supplemented with 40 mM DTT
Samples were centrifuged 3 min at 20,000 × g
Qiagen protocol was used for the next steps
Genomic DNA was removed by DNase digestion on column and total RNA was eluted in ultra pure water
RNA purity and quantity were assessed by OD measurements (OD 260 nm and OD ratio 260/280 and 260/230) and RNA integrity was checked using Bioanalyseur 2100 (Agilent
Samples were send to GeT-PlaGe core facility in dried-ice
RNAseq was performed at the GeT-PlaGe core facility
RNA-seq libraries have been prepared according to Illumina’s protocols using the Illumina TruSeq Stranded mRNA sample prep kit to analyze mRNA
The 79 individuals were sequenced separately on 8 different lanes of one flow cell
RNA were fragmented to generate double stranded cDNA and adaptators were ligated to be sequenced
Eleven cycles of PCR were applied to amplify libraries
Library quality was assessed using a Fragment Analyser and libraries were quantified by QPCR using the Kapa Library Quantification Kit
RNA-seq experiments have been performed on an Illumina HiSeq3000 using a paired-end read length of 2 × 150 pb with the Illumina HiSeq3000 sequencing kits
Further KEGG annotation were done with KAAS interface (KEGG Automatic Annotation Server) using contig sequences in fasta format
Biomarker explorations were done using the implemented BLAST in NGSpipeline interface
Biomarker sequences come from close related species (i.e
Candidates were selected by the user on score
the sequence with highest score was selected
The absence of degradation fragment in the 5S
Fast and Inter regions was a criteria of selection for sequencing
Top 5 best species hits for (A) Dreissena polymorpha and (B)
We present here the first whole de novo transcriptomes of the two invasive Dreissena polymorpha and D
rostriformis bugensis species who will be fully available in TSA repository
One of the strength of proposed transcriptome is the conditions in which it was obtained: by investigating individuals coming from several populations
we encompassed as much as possible the variability of the transcriptome induced by environmental conditions (pollution
with a mRNA set that is representative of this ecological heterogenity
Our ambitious approach also lead to identification of numerous sequence variants (insertion
deletion and single-nucleotide polymorphism)
since studied populations are established for a long time and may have been prone to mutation
this may lead to the development of biomarker based assays that
By providing Dreissena sequences through a user-friendly interface
we open the way to further explorations of Dreissenids molecular mechanisms by such biomarker assays development
primer design allowing targeted expression analysis or promising proteogenomics studies when coupling with mass spectrometry analysis
Parameters to involved softwares tools are described in the following paragraph
DRAP (De novo RNA-seq Assembly Pipeline): version 1.7, code available online at http://www.sigenae.org/drap/quick_start.html
BUSCO: version 3.0.2 using the metazoa OrthoDB (v
History of Dreissena research and the ICAIS gateway to aquatic invasions science
Sympatric Dreissena species in the Meuse River: towards a dominance shift from zebra to quagga mussels
Estimating the benthic population of Dreissena polymorpha and its impact in the lower Seine River
Patterns of biological invasions in french freshwater systems by non-indigenous macroinvertebrates
Update on the environmental and economic costs associated with alien-invasive species in the United States
Does zebra mussel (Dreissena polymorpha) represent the freshwater counterpart of Mytilus in ecotoxicological studies
Contaminant and nutrient element levels in soft tissues of zebra and quagga mussels from waters of southern Lake Ontario
Bioavailability of particulate metal to zebra mussels: biodynamic modelling shows that assimilation efficiencies are site specific
Biomarkers in zebra mussels (Dreissena polymorpha) for the assessment and monitoring of water quality of the St Lawrence River (Canada)
Separating natural from anthropogenic causes of impairment in zebra mussel (Dreissena polymorpha) populations living across a pollution gradient
One-year monitoring of core biomarker and digestive enzyme responses in transplanted zebra mussels (Dreissena polymorpha)
The use of biomarkers in biomonitoring: a 2-tier approach assessing the level of pollutant-induced stress syndrome in sentinel organisms
biogeography and systematics of Dreissena in the Balkans
Genetic variability and phylogeographical patterns of a nonindigenous species invasion: a comparison of exotic vs native zebra and quagga mussel populations
Using massive parallel sequencing for the development
and application of population genetics markers in the invasive bivalve zebra mussel (Dreissena polymorpha)
A phylogenetic backbone for bivalvia: an RNA-seq approach
Next-generation sequencing of Dreissena polymorpha transcriptome sheds light on its mitochondrial DNA
Molecular resolution of the family Dreissenidae (Mollusca: Bivalvia) with emphasis on Ponto-Caspian species
including first report of Mytilopsis leucophaeata in the Black Sea basin
Multilocus phylogeny of the zebra mussel family Dreissenidae (Mollusca: Bivalvia) reveals a fourth neotropical genus sister to all other genera
Comparative study of the effects of gadolinium chloride and gadolinium - based magnetic resonance imaging contrast agent on freshwater mussel
Identification of differentially expressed genes in Dreissena polymorpha exposed to contaminants
Compacting and correcting Trinity and Oases RNA-Seq de novo assemblies
Oases: robust de novo RNA-seq assembly across the dynamic range of expression levels
RNAmmer: consistent and rapid annotation of ribosomal RNA genes
InterProScan 5: genome-scale protein function classification
The Genome Analysis Toolkit: a MapReduce framework for analyzing next- generation DNA sequencing data
RNAbrowse: RNA-Seq de novo assembly results browser
NGSPipeline interface, http://ngspipelines.toulouse.inra.fr:9014 (2017)
Péden R. et al. Environmental transcriptomes of invasive dreissena: a move towards an upcoming model species. figshare, https://doi.org/10.6084/m9.figshare.c.4483856 (2019)
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This work was supported by the the BIOMICS program (Biomarkers and Omics in the freshwater Bivalve Dreissena polymorpha) of the French Agency for Biodiversity/National Agency for Water and Aquatic Environments (AFB/ONEMA) Authors would like to thanks the GeT-PlaGe for performing sequencing and Genotoul platform for transcriptome assembly
These authors contributed equally: Romain Péden
Mathématiques et Informatique Appliquées de Toulouse
performed sequencing and transcriptome assembly and annotation
The Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the metadata files associated with this article
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DOI: https://doi.org/10.1038/s41597-019-0252-x
Applied Microbiology and Biotechnology (2022)
Volume 10 - 2019 | https://doi.org/10.3389/fpls.2019.01755
Developing new cropping strategies (very early sowing
double cropping) to improve soybean production in Europe under climate change needs a good prediction of phenology under different temperature and photoperiod conditions
a simple phenology algorithm (SPA) was developed and parameterized for 10 contrasting soybean cultivars (maturity group 000 to II)
Two experiments were carried out at INRA Toulouse (France) for parameterization: 1) Phenological monitoring of plants in pots on an outdoor platform with 6 planting dates
2) Response of seed germination to temperature in controlled conditions
Multi-location field trials including 5 sites
and 10 cultivars were used to evaluate the SPA phenology predictions
respectively with significant differences among cultivars
The photoperiod sensitivity coefficient varied among cultivars when fixing Popt and Pcrt
optimal and critical photoperiods respectively
The parameterized algorithm showed an RMSE of less than 6 days for the prediction of crop cycle duration (i.e
cotyledons stage to physiological maturity) in the field trials including 75 data points
and beginning of grain filling (R5 stage) dates were satisfactorily predicted with RMSEs of 8.2 and 9.4 days respectively
Because SPA can be also parameterized using data from field experiments
it can be useful as a plant selection tool across environments
The algorithm can be readily applied to species other than soybean
and its incorporation into cropping systems models would enhance the assessment of the performance of crop cultivars under climate change scenarios
These new crop management systems using either early or late sowing and new cultivation areas will expose the crop to a wider range of thermal and photoperiodic conditions under higher latitudes (from 43° to 52° N)
which has the advantage of requiring only an incubator
It is also possible to use the natural variation of day length by testing several planting dates
The main difficulty encountered for extending soybean cropping to northern Europe and/or for modifying the sowing period is relative to the short-day behavior of the plant
it is important to understand the temperature-photoperiod interactions of current and future cultivars
The aim of this study was three-fold: (i) Develop a simplified phenotyping method for phenology in (semi-)controlled conditions (EXP1
(ii) parameterize a simple phenology algorithm (SPA) using EXP1 and EXP2
and (iii) evaluate SPA using field experiments (EXP3)
‘ES_Pallador,’ which belongs to the same MG as ‘Isidor,’ has a totally different type of growth
CV ‘Blancas’ has variable classification depending on location
being classified as MG I/II in Italy and II in South-West of France
Table 1 Characteristics of the 10 cultivars used in this study
which is fitted to experimental data to obtain the three cardinal temperatures (Equation 1)
The Beta function adjustment was performed by simultaneously optimizing the three function parameters (mu
A constraint was imposed on the T0 parameter so that it must be greater than or equal to 0 and Tmax were fixed according observations
The method used was the non-linear GRG with the goal of minimizing the RMSE between the simulated and the observed germination rate values
The 10 cultivars were randomized within each treatment-block combination
Hourly temperatures were recorded by an automatic weather station located within 150 m from the platform
Table 2 Summary of field experiments used for the validation of the simple phenology algorithm
PDDc,p was calculated for four development phases: VC–R7
The temperature function (Equation 3) was described by Wang and Engel (1998)
T: average temperature of the day (°C)
Tmax: cultivar-specific cardinal temperatures (°C)
The parameter α is calculated using Equation 4:
The photoperiod function f(P) was calculated as shown in Equation 5
Where Pcrt is the day length above which development rate is zero
Popt is the day length below which development rate is not limited
P is the day length including civil twilight
and S is the sensitivity coefficient of the cultivar to photoperiod (S=0 for highest sensitivity; >100 for no sensitivity)
Plant parameters needed in SPA are presented in Table 3
obtained from three methods of acquisition: experimental data
Table 3 Parameters used in Simple Phenology Algorithm (SPA)
Once all other parameters were determined (i.e. cardinal temperatures, Popt and Pcrt—Table 3)
PDDoptc,p and S were set by simultaneous optimization over the entire life cycle VC–R7
The optimization was performed with the Excel solver using the non-linear GRG method (Newton Global)
It was carried out in two steps: 1) simultaneous optimization of the minimum number of days for the VC–R7 phase (PDDoptc,p) and of the sensitivity to the photoperiod (S); 2) optimization of the minimum number of days for the R1–R5 and R5–R7 phases while maintaining the value of S as determined in (1)
The objective of the optimization was to minimize the RMSE between predicted and actual day of appearance of R7 for all planting dates of a given cultivar
and their interactions were identified by analysis of variance using a two-way ANOVA performed with R software (version 3.2.2; [R Core Team {2015} R: a language and environment for statistical computing
Comparison of means was done with post-hoc Student-Newman-Keuls (SNK) tests with a significance level of p < 0.05
The performance of SPA was evaluated with several indicators including RMSE (Root Mean Square Error), MBE (Mean Bias Error), RRMSE (Relative Root Mean Square Error), and EF (Efficiency). Equations of performance indicators are presented in Table 4
Table 4 Performance indicators used for the evaluation of SPA
The Yin model fitted well the observation data; these are are available in Data Sheet 1 (Supplementary Material). Determination coefficients R² ranked from 0.95 to 1. The germination rate progressively increased from the minimum temperature to the optimum temperature and then decreased. The maximum germination rates varied according to the cultivars (Figure 1)
Figure 1 Graphical display of the Yin adjustments for all cultivars at 50% of germination
Dots represent mean observed data and lines the adjusted Yin function
The average T0, Topt, Tmax of all the 10 cultivars were 2, 28.1, 40.6°C respectively. The lowest temperature tested was 3°C and having no point of reference we chose to set T0 to 2 °C. The final germination percentage was not significantly affected in the 6.5 to 40 °C temperature range under these experimental conditions (Table B—Supplementary Material)
Cultivar differences were significant for optimum and maximum temperatures (Table 5)
The optimum temperature was the most discriminating
and allowed to separate four significantly different groups at the p < 0.05 threshold
‘ES_Mentor’ is the cultivar which takes the best advantage of high temperatures (Topt = 32.1°C) while ‘Ecudor’ requires a lower temperature (28.1°C)
Tmax for ‘Ecudor’ was the highest of the cultivar group tested (42°C)
with a majority of cultivars being between 40.3 and 41°C
Table 5 Estimates (and standard errors [±]) of optimum (Topt) and maximum (Tmax) temperatures obtained by the Yin function for the germination of the 10 soybean cultivars studied
The different planting dates did not experience the same photoperiod and temperature conditions. For example, D1, D2, and D3 met a varying photoperiod, increasing at the beginning and decreasing at the end of the cycle. D4, D5, and D6 only met a decreasing photoperiod. The temperature and photoperiod conditions encountered during EXP2 are presented in Figure B (Supplementary Material)
Table 6 Analysis of variance of cycle duration from VC to R7 stage in calendar days (CD VC–R7) of 10 cultivars of soybean for five planting dates in outdoor pot experiment (EXP2)
Figure 2 Comparison of fitted (FIT) and observed (OBS) day of appearance of R7 stage
for algorithm parameterization on outdoor pot experiment data (EXP2)
Colors represent the four planting dates D2 to D5
Table 7 Parameters used for each phase. The temperatures T0, Topt, and Tmax were those determined during EXP2 (Table 5)
The parameterization of SPA made it possible to highlight the phases impacted by limiting temperatures or photoperiods for each cultivar. As an example, Figure 3 shows individually the effects of f(T) and f(P) and their multiplicative effects on the development of CV ‘Ecudor’ for planting date D3 on the outdoor platform
The photoperiod acts on development at a time when the temperature would not be limiting (f[T] ~ 1) around DOY 170
the value of f(P) being at its minimum at ~0.4
soybean development would be more impacted by temperature with a value of f(T) close to 0.4 and a f(P) of 0.8
Figure 3 Graphical representation of the temperature function f(T)
dotted blue line; photoperiod function f(P)
dashed yellow line; and their multiplicative effect f(T) × f(P)
with parameterization parameters of ‘Ecudor’ cultivar for planting date D3 (10th may)
The planting date as well as the stages R1
Sensitivity coefficient (S—Table 7) ranged from 1 for ‘ES_Pallador’ to 1.50 for ‘Blancas.’ However, this sensitivity was also impacted by Popt and Pcrt, which were determined according to the maturity group. Figure 4 shows the photoperiod function for all cultivars with this calibration
there is a trend for the response to photoperiod to decrease from late to early cultivars
Figure 4 Photoperiod function representation for all cultivars using the calibration parameters
Maturity groups of the cultivars are the following: 000: ‘Klaxon,’ ‘Sultana,’ ‘RGT_Shouna’; 00: ‘Sigalia,’ ‘ES_Mentor’; I: ‘ES_Pallador,’ ‘Isidor’; I/II: ‘Santana’; II: ‘Blancas,’ ‘Ecudor’
Predictions by phases gave variable levels of performance
but always lower than for the entire cycle
Performance indicators for each phase are reported in the table below the two graphs
VE–R1 phase tended to be underestimated for short phase length (before calendar day 160) while R1–R5 phase was the only stage overestimated by SPA
Figure 5 Observed (OBS) and predicted (SIM) days after planting for maturity (R7)
all cultivars combined (A) and for three successive phenological phases (B)
colors indicate years from 2013 to 2018 on data produced at INRA Auzeville
and R7 with simulations beginning respectively at VE
Line 1:1 is reported on both graphs A and B
Table below shows the indicators of performance of SPA during its field evaluation
The aim of this study was to develop and apply a phenology phenotyping method coupled with a simple phenology algorithm for soybean cultivars
Phenotyping was divided into two complementary protocols to determine a) cardinal temperatures (EXP1) and b) development response to temperature and photoperiod (EXP2)
Data produced in these experiments were used to parameterize SPA (Simple Phenology Algorithm)
which was evaluated with independent data from field experiments
photoperiod sensitivity of cultivars appears variable among maturity groups
two cultivars of the same maturity group can be classified into two different groups according to the SNK test when considering the cumulative thermal time needed to complete the cycle
Table 8 Thermal time accumulation from VC (unifoliate leaves) to R7 (beginning maturity stage)
we have applied it to the different phases of the cycle
This assumption was not refuted when applying the model to the different phases then comparing the resulting simulations to field observations
2007) use different cardinal temperatures depending on the phases of the cycle
It may be possible to improve phase prediction by doing this with SPA but this would need specific experiments in controlled conditions or very wide experimental field network to determine them
EXP2 did not allow soybean to be exposed to continuous optimal photoperiod and temperature conditions throughout the cycle
which would have made it possible to directly determine PDDoptc,p of each phase
Experiments in a growth chamber would allow collecting this information
but this requires the necessary equipment and such a long cycle (VC–R7) has never been tested in this type of experiments to our knowledge
These results show that it is possible to use a semi-controlled phenotyping method such as an automated outdoor platform to parameterize an algorithm that can be used to predict phenological stages in the field
This could be useful as a plant breeding tool
Because the algorithm has been developed based on a limited range of MGs
it is not possible to check its suitability for later MGs with our data
maturity groups tested correspond to most MGs grown in Europe
The optimization of the photoperiod sensitivity coefficient (S) made it possible to highlight differences in sensitivity within the same maturity group
This element must be taken into consideration by breeders when adapting cultivars to new management practices or new latitudes
To test if the approach could be simplified by replacing cultivar-specific Topt by soybean-specific Topt, we calculated an average value of Topt across the 10 cultivars then re-estimated the values of PDDoptc,p, and S parameters. The prediction quality of VC–R7 duration was compared at field level (75 data points) for the two sets of parameters, either cultivar- or soybean-specific (Table C—Supplementary Material)
−0.70) indicators performances were lessened when using soybean- instead of cultivar-specific Topt values respectively
Therefore using a cultivar-specific calibration in this study was justified
due to the slight decrease of the performance of SPA with average Topt
end-users could prefer adopt a soybean-specific Topt value for a direct and simpler application of the phenological algorithm
In this study we provide a path for soybean cultivar phenology × environment × crop management (sowing date) advice by: (1) developing a simple algorithm of phenology; and (2) proposing a simple method in semi-controlled conditions for phenotyping cultivar phenology and determining parameters for the algorithm
SPA allows users to quantify the effects of temperature and photoperiod on soybean phenology that affect the possible extension of the crop to northern Europe
It will also help to define new cultivation areas and management practices (i.e
early or late sowing) according to cultivar characteristics in the context of climate change
it is possible to parameterize SPA directly from phenology data produced in field experiments
enhancing the value of these experiments and assisting plant selection efforts
SPA can be parameterized for other crop species without modification
integration of SPA into cropping system models can enhance the performance of these models for assessing the adaptation of cultivars to climate change scenarios and changing management options
All datasets generated for this study are included in the article/Supplementary Material
and PM acquired the funding for the research project
The authors thank the Occitanie / Pyrénées-Méditerranée region and Terres Inovia for financial support of this research
The works presented are part of the “Sojamip” research project (http://www6.inra.fr/sojamip) involving various partners (Euralis Semences
We thank Toulouse INP for the invitation of Prof
COS as “Visiting Professor” at UMR AGIR
and Hugo Quintana for their technical assistance in carrying out the agronomic tests and Elie Maza for his expert advice in statistical analysis
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.01755/full#supplementary-material
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*Correspondence: Céline Schoving, Y2VsaW5lLnNjaG92aW5nQGlucmFlLmZy
WALTHAM, Mass., June 22, 2021 (GLOBE NEWSWIRE) -- Repligen Corporation (NASDAQ:RGEN)
a life sciences company focused on bioprocessing technology leadership
today announced that it has entered into an agreement to acquire Toulouse
a leading industrial expert in the development and manufacture of hollow fiber membranes and modules
Franc and the Polymem team join the Repligen family
The acquisition significantly expands our hollow fiber membrane and module production capabilities and adds core R&D
engineering and production expertise in hollow fiber technology for both industrial and bioprocessing markets
The business complements what we have already in place at our Rancho Dominguez site and establishes a world-class center of excellence in Europe to meet the accelerating demand for hollow fiber products and to serve our rapidly expanding global customer base.”
Polymem has remained true to our expertise in hollow fiber membrane development and manufacturing for industrial and life sciences applications
we now have the opportunity to focus on two market segments
This deal further extends our market reach
allowing us to boost our growth across both our historical Municipal/Industrial markets and the Bioprocessing market
We continue to increase capacity with the extension of our production site in Castanet-Tolosan
and we look forward to working together with Repligen to further penetrate our core markets.”
Jean-Michel Espenan and Franc Saux will stay on with the company in their current respective roles of President and Technical Director
The proposed acquisition is expected to close during Repligen’s third fiscal quarter of 2021
subject to the satisfaction of customary closing conditions
Repligen intends to use its cash on hand which totaled approximately $711 million at March 31
Additional financial details were not disclosed
Perella Weinberg Partners LP is acting as financial advisor and Goodwin Procter LLP is serving as legal counsel to Repligen
About Repligen CorporationRepligen Corporation is a global life sciences company that develops and commercializes highly innovative bioprocessing technologies and systems that increase efficiencies in the process of manufacturing biological drugs
We are inspiring advances in bioprocessing for the customers we serve; primarily biopharmaceutical drug developers and contract development and manufacturing organizations (CDMOs) worldwide
Our corporate headquarters are located in Waltham
with additional administrative and manufacturing operations worldwide
The majority of our manufacturing sites are located within the U.S
located outside of Toulouse in southwestern France
was established in 1997 by two engineers specializing in hollow fiber membrane technology
The company has grown into a team of 60 people
dedicated to the development and manufacture of hollow fiber membranes
These products are used in water treatment
liquid/gas transfer and bioprocessing applications
Polymem anticipated the demands of the worldwide municipal and industrial markets and is now recognized not only for its ability to develop innovative products
but also for the quality of its production of membranes
cartridges and modules for various applications and sectors
Forward-Looking StatementsThis press release contains forward-looking statements within the meaning of the federal securities laws
Investors are cautioned that statements in this press release which are not strictly historical statements including
express or implied statements or guidance regarding the expected results of the proposed acquisition of Polymem on Repligen’s future financial performance
including the accretive nature and the timing of the accretive nature of the acquisition
expected synergies following the acquisition of Polymem
the expected expansion of Repligen’s product lines
the timing of the closing of the acquisition and other statements identified by words like “believe,” “expect,” “may,” “will,” “should,” “seek,” or “could” and similar expressions
Such forward-looking statements are subject to a number of risks and uncertainties that could cause actual results to differ materially from those anticipated
risks associated with: the risk that the proposed acquisition may not be completed in a timely manner
change or other circumstance that could give rise to the termination of the acquisition; our ability to integrate Polymem’s business and personnel and to achieve expected synergies; our ability to maintain or expand Polymem’s historical sales; our ability to accurately forecast the acquisition
related restructuring costs and allocation of the purchase price
goodwill and other intangibles acquisition related and other asset adjustments; and other risks detailed in Repligen’s most recent Annual Report on Form 10-K on file with the Securities and Exchange Commission and the other reports that Repligen periodically files with the Securities and Exchange Commission
Actual results may differ materially from those Repligen contemplated by these forward-looking statements
you should not rely on any of these forward-looking statements
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any of these forward-looking statements to reflect a change in its views or events or circumstances
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Repligen Contact: Sondra S. NewmanGlobal Head of Investor Relations(781) 419-1881investors@repligen.com
Polymem S.A. Contact: Isabelle DucheminMarketing and Commercial Manager +33 (0) 786 008 625iduchemin@polymem.fr
Metrics details
Lakes play an important role in the global carbon (C) cycle by burying C in sediments and emitting CO2 and CH4 to the atmosphere
The strengths and control of these fundamentally different pathways are therefore of interest when assessing the continental C balance and its response to environmental change
based on new high-resolution estimates in combination with literature data
we show that annual emission:burial ratios are generally ten times higher in boreal compared to subarctic – arctic lakes
These results suggest major differences in lake C cycling between biomes
as lakes in warmer boreal regions emit more and store relatively less C than lakes in colder arctic regions
Such effects are of major importance for understanding climatic feedbacks on the continental C sink – source function at high latitudes
If predictions of global warming and northward expansion of the boreal biome are correct
it is likely that increasing C emissions from high latitude lakes will partly counteract the presumed increasing terrestrial C sink capacity at high latitudes
we combine new detailed measurements of annual C emission as well as annual C burial in six subarctic - arctic lakes in northern Sweden with literature data to compare these fluxes across biomes
Total annual C emissions (CO2 + CH4) from the investigated subarctic lakes ranged between 5 and 54 g C m−2 yr−1
the C emissions were dominated by CO2 which accounted for more than 90% of the total annual C emission in all lakes except one
where CO2 emission was low and CH4 accounted for 40% of the annual C emission
The dissolved CO2 and CH4 accumulated during winter under ice and released at ice break-up constituted between 7 to 80% of annual emissions
The burial of C varied between 5 and 25 g C m−2 yr-1
We were not able to detect any significant mass loss in any of the sediment samples after acidification
indicating negligible accumulation of inorganic C in the sediments of our sampled lakes
Linear relationships between paired C emissions and sediment burial in boreal (open circles) and subarctic – arctic (squares) lakes
with the regression 95% confidence intervals (in red shading)
our results thus reveal a long-term unforeseen climate feedback; regardless of whether terrestrial C sequestration is favored by the future climate or not
lakes’ direct and indirect response to an expansion of the boreal biome weakens the northern inland C sink
We therefore conclude that the understanding of lakes’ sink – source functions will be most essential when predicting the future C cycle at high latitudes
Areas of the lakes were obtained by digitalizing an orthophoto (1 m pixel resolution) using the software package Arc GIS 9.3.1 (ESRI
Lake volumes were determined from interpolations of integrated GPS and echo sounding depth measurements (m52i
U.S.) and shoreline points randomly chosen from the orthophoto given a depth value of 0 m
Altogether were 2733 points used for the interpolations divided on 6 lakes (between 115 to 1370 points per lake depending on the lake size)
Winter depth and volumes were determined by subtraction of the ice volumes covering the lakes
The interpolations were performed in the Arc GIS 9.3.1 geostatistical analysis package using the ordinary Kriging method
Maximum depths were confirmed by manual depths measurements
DOC was analyzed after filtration (0.45 μm sterile filter
Germany) and acidification (100 μL 20% HCl to 50 mL filtrate) by high temperature catalytic oxidation (HTCO) using a Shimadzu TOC-V CPH analyzer (Shimadzu Corporation
the calculated ebullition flux exceeded the diffusive flux
chamber concentrations were higher than the equilibrium concentration in water
fluxes were estimated by linear mass balance calculations
Only lakes with a maximum depth above 1.5 m were assumed to accumulate gases during the winter season since shallower lakes freeze solid during winters
divided between the six lakes depending on lake size and morphology
All sediment cores were sectioned directly in the field into 1 cm slices
transferred into polypropylene containers (4K 100
then transported back to the laboratory within the same day and stored frozen at −20 oC
ground by hand and homogenized prior to analysis
The isotope 209Po was used as a tracer for yield determination
Po sources were counted using Ortec (U.S.) ULTRA-AS Ion-Implanted-Silicon Charged-Particle Detectors (Model U-020-450-AS)
Excess 210Pb was assessed by subtracting the 210Pb activities at depth from the total 210Pb
which were not corrected for sediment focusing
are thus conservative estimates for most of the cores
we did not include C burial estimates based on the first top cm only
If multiple estimates from the same lake existed and the quality of work was considered equal
We express all our result as arithmetic means ± standard deviations
Large difference in carbon emission-burial balances between boreal and arctic lakes
Freshwater Methane Emissions Offset the Continental Carbon Sink
Global carbon dioxide emissions from inland waters
Long-term C accumulation and total C stocks in boreal lakes in northern Quebec
A large carbon pool and small sink in boreal Holocene lake sediments
doi: 10.1111/j.1365-2486.2004.00848.x (2004)
Holocene carbon burial by lakes in SW Greenland
doi: 10.1111/j.1365-2486.2009.01942.x (2009)
The global abundance and size distribution of lakes
Arctic lakes and streams as gas conduits to the atmosphere-implications for tundra carbon budgets
Temperature independence of carbon dioxide supersaturation in global lakes
Carbon evasion/accumulation ratio in boreal lakes is linked to nitrogen
Global climate-change and terrestrial net primary production
Cross-regional prediction of long-term trajectory of stream water DOC response to climate change
Links between terrestrial primary production and lake mineralization and CO2 emission in a climate gradient in subarctic Sweden
doi: 10.1007/s10021-10008-19127-10022 (2008)
Catchment productivity controls CO2 emissions from lakes
Net ecosystem production in clear-water and brown-water lakes
Temperature-controlled organic carbon mineralization in lake sediments
Reconciling the temperature dependence of respiration across timescales and ecosystem types
Light limitation of nutrient-poor lake ecosystems
Shifts in Lake N:P Stoichiometry and Nutrient Limitation Driven by Atmospheric Nitrogen Deposition
An analysis of the carbon balance of the Arctic Basin from 1997 to 2006
doi: 10.1111/j.1600-0889.2010.00497.x (2010)
Miller (eds.)) 996 (Cambridge University Press
Future changes in vegetation and ecosystem function of the Barents Region
A global meta-analysis of treeline response to climate warming
doi: 10.1111/j.1461-0248.2009.01355.x (2009)
Plot-scale evidence of tundra vegetation change and links to recent summer warming
Amplified carbon release from vast West Siberian peatlands by 2100
Latitudinal patterns of magnitude and interannual variability in net ecosystem exchange regulated by biological and environmental variables
doi: 10.1111/j.1365-2486.2009.01870.x (2009)
Boreal carbon loss due to poleward shift in low-carbon ecosystems
Riverine coupling of biogeochemical cycles between land
What determines the current presence or absence of permafrost in the Tornetrask region
climatic changes and net carbon sequestration in a North-Scandinavian subarctic mire over 30 years
Direct and continuous measurement of dissolved carbon dioxide in freshwater aquatic systems-method and applications
Methane emissions from lakes: Dependence of lake characteristics
two regional assessments and a global estimate
High emission of carbon dioxide and methane during ice thaw in high latitude lakes
Integrating carbon emissions from lakes and streams in a subarctic catchment
Quantifying the relative importance of lake emissions in the carbon budget of a subarctic catchment
Pelagic and benthic net production of dissolved inorganic carbon in an unproductive subarctic lake
Atmospheric exchange of carbon dioxide in a low-wind oligotrophic lake measured by the addition of SF6
Quantification of C uptake in subarctic birch forest after setback by an extreme insect outbreak
Acid fumigation of soils to remove carbonates prior to total organic carbon or carbon-13 isotopic analysis
Geochronology with 210Pb in radioactive dating
Application of lead - 210 to sedimentation studies
Pb-210 and Po-210 analysis in sediments and soils by microwave acid digestion
Carbon and nitrogen loss rates during aging of lake sediment: Changes over 27 years studied in varved lake sediment
The origin of lead in the organic horizon of tundra soils: Atmospheric deposition
plant translocation from the mineral soil or soil mineral mixing
doi: 10.1016/j.scitotenv.2011.07.005 (2011)
The tundra-taiga interface and its dynamics: concepts and applications
Carbon mineralization and oxygen dynamics in sediments with deep oxygen penetration
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Westin for field and laboratory assistance
we thank the Abisko Scientific Research Station (Sweden) for providing climatologically data and T
Heliasz for sharing wind data from Stordalen
Krab for their linguistic comments on the manuscript and S
Sobek for feedback on an early version of the manuscript
This study was financially supported by the Swedish Research Council (dnr
Department of Environmental Science and Analytical Chemistry (ACES)
Department of Ecology and Environmental Science
Department of Thematic Studies–Environmental Change
designed the study and formed the conceptual idea
The gas flux measurements were led by E.J.L
The sediment coring was carried out by E.J.L.
The CO2 flux calculations were performed by E.J.L
The statistical analyses were performed by E.J.L
wrote most of the manuscript with major contributions from J.K.A.
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Metrics details
RNA-Seq enables the generation of extensive transcriptome information providing the capability to characterize transcripts (including alternative isoforms and polymorphism)
to quantify expression and to identify differential regulation in a single experiment
Our aim in this study was to take advantage of using RNA-Seq high-throughput technology to provide a comprehensive transcriptome profiling of the sheep lactating mammary gland
Eight ewes of two dairy sheep breeds with differences in milk production traits were used in this experiment (four Churra and four Assaf ewes)
Milk samples from these animals were collected on days 10
120 and 150 after lambing to cover the various physiological stages of the mammary gland across the complete lactation
RNA samples were extracted from milk somatic cells
The RNA-Seq dataset was generated using an Illumina HiSeq 2000 sequencer
The information reported here will be useful to understand the biology of lactation in sheep
providing also an opportunity to characterize their different patterns on milk production aptitude
time series design • strain comparison design • transcription profiling design
selectively maintained organism • time measurement datum
Machine-accessible metadata file describing the reported data (ISA-Tab format)
The knowledge of the transcriptome profiling of the lactating mammary gland is of special interest since it allows the characterization of the genes implicated in the biology of lactation and the physiological and metabolic changes occurring in the mammary gland during this period
in dairy livestock the knowledge of the transcripts expressed in lactating mammary gland enhances our understanding on the genes underlying dairy traits
Considering a generic sheep lactation curve (provided in the top of the figure)
represented with green arrows in the graphic
were established to cover the different physiological stages of the mammary gland across the lactation curve
Milk samples were collected on days 10 (D10)
A total of eight healthy Assaf and Churra sheep were selected to be included in the experiment
four Assaf and four Churra ewes were milked to obtain milk somatic cells as source of RNA
Based on the quality scores of the extracted RNA samples for each breed
a total of 30 samples were sequenced: samples from four animals for the time points D10
whereas three biological replicates were sequenced for D120
The dataset reported in this data descriptor may be helpful for future studies examining the biology of sheep lactation
we used our own pilot RNA-Seq data obtained from MSCs from four sheep per breed
To estimate the power based on our pilot dataset we set the following parameters: a cost per replicate of 50 US Dollars (USD)
a cost per million reads aligned to genes of 150 USD
a read depth between 10 and 40 millions of reads
a maximum cost of the experiment of 100,000 USD
a 50% of differential expressed genes detected with a fold change of 2 and a P-value of 0.01 and a minimum of 30% of genes with at least 50% of maximum power
The D120 and D150 sampling points correspond to the end of the normalized lactation in Churra and Assaf
whereas for Churra D120 is close to the final lactation point
for Assaf this time point corresponds to a transition stage from the lactation peak to the final lactation point (D150)
For each sampled animal and lactation point
at least four milk samples of 50 ml were collected; two of them were obtained on the exact sampling day whereas two additional samples were collected the previous or the following day to ensure RNA source material for each desired sampling
All protocols involving animals were approved by the Animal Welfare Committee of the University of Leon
following proceedings described in Spanish and EU legislations (Law 32/2007
The animals used in this study were handled in strict accordance with good clinical practices and all efforts were made to minimize suffering
To ensure RNA purification of high yield and quality
we used the following protocol during the sampling process
the collection milk containers were cleaned with RNaseZap (Ambion
udder cleaning was performed with special care: first
the udders were cleaned with water and soap; then
they were disinfected with povidone iodine; and finally the nipples were cleaned with RNAseZap (Ambion
Milk samples were collected from both mammary glands
A sterile gauze was used to cover the collection container during milk collection to minimize the risk of sample contamination
After collection the milk was transferred to 50 ml RNAse-free tubes
Samples were maintained at 4 °C during their transport from the farm to the laboratory where they were immediately processed
and in the presence of a final concentration of 0.5 mM of EDTA to eliminate casein and fat globules
a fatty layer frequently appeared on the top of the tube
a sterile pipette tip was introduced to separate this fatty layer from the tube walls
the cell pellet was washed in 10 ml of PBS (pH 7.2) with 0.5 mM EDTA and centrifuged at 540×g in 15 ml RNAse free sterile tubes for 10 min at 4 °C
The last step was repeated until the fatty layer was minimized (usually twice)
it was resuspended in 500 μl of Trizol (Invitrogen
the homogenized sample was incubated for 15 min at room temperature to permit the complete dissociation of the nucleoprotein complex
the sample was shaken vigorously by hand for 15 s
incubated 15 min at room temperature and centrifuged at 12,000×g for 15 min at 4 °C
the upper aqueous phase of the sample was taken and placed in a new tube where 250 μ of isopropanol were added
The sample was then incubated for ten minutes at room temperature and centrifuged at 12 000×g for 15 min at 4 °C
The RNA pellet was washed with 0.5 ml of ethanol
the sample was vortexed briefly and the tube was centrifuged at 7,500×g for 5 min at 4 °C
the sample was dried for seven minutes at room temperature
To elute the sample 150 μl of DEPC water with DNAse (0.2 μl in 100 μl) was added and then
The Fastq files generated were deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE74825
Gene abundances were normalized by library and gene length by calculating Fragments Per Kilobase Of Exon Per Million Fragments Mapped (FPKM) using the Ensembl annotated genes (Oar_v3.1) as a reference
The output file from the quantification of transcripts by Sigcufflinks is also deposited in the Gene Expression Omnibus (GEO) under the same accession number GSE74825
It contains all the genes identified in the assembly and the raw counts per gene for each sample
Millions of reads aligned to genes per replicate are represented in the X-axis
whereas the number of replicates in each condition is represented in the Y-axis
The percentage of genes detected with a 2x Fold Change are coloured as indicated in the bar placed at the right of the grid
Experiments that do not conform the fixed constraints are filled as indicated in the legend
Total RNA integrity was assessed by the RNA Integrity Number (RIN) algorithm calculated by the Agilent Bioanalyzer software. The Agilent Bioanalyzer RIN scores are listed in Table 2
All the total RNA samples used for this RNA-seq study had a RIN score above 7 showing the high integrity of the samples used
A total of 30 RNA libraries were sequenced to a depth between 23–46 million paired-end reads among which about 88.10% of the reads mapped to unique locations in the ovine genome assembly (Oar_v3.1) (Table 2)
No contamination was found in the alignment against the Escherichia coli genome
FPKM values are represented in the X-axis
whereas the gene names are indicated in the Y-axis
A colour code is used to represent the four time points studied: day 10 (D10)
Heatmap display of supervised hierarchical clustering of the DEGs identified across the four considered time points of the sheep lactation (D10
The genes are displayed in rows and the normalized counts per sample are displayed in columns
Each column represents a sample; sample names indicate the corresponding breed (A=Assaf; C=Churra) and day of sampling (D10
A colour code indicates up-regulated (orange) and down-regulated (blue) expression levels
Comprehensive RNA-Seq profiling to evaluate lactating sheep mammary gland transcriptome
Data 3:160051 doi: 10.1038/sdata.2016.51 (2016)
RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays
RNA-Sequencing: A tool to explore new frontiers in animal genetics
Transcriptional profiling of bovine milk using RNA sequencing
Sequencing the transcriptome of milk production: milk trumps mammary tissue
RNA sequencing of the human milk fat layer transcriptome reveals distinct gene expression profiles at three stages of lactation
Transcriptome analysis of the mammary gland from GH transgenic goats during involution
Transcriptional profiling of mammary gland in Holstein cows with extremely different milk protein and fat percentage using RNA sequencing
Functional development of the adult ovine mammary gland--insights from gene expression profiling
The influence of breed on the organoleptic characteristics of Zamorano sheep’s raw milk cheese and its assessment by instrumental analysis
Role of somatic cells on dairy processes and products: a review
Regulation of cell number in the mammary gland by controlling the exfoliation process in milk in ruminants
Evolution des types cellulaires du lait de brebis (race Churra) en fonction des dénombrements cellulaires totaux pendant la traite mécanique et manuelle
Comparison of five different RNA sources to examine the lactating bovine mammary gland transcriptome using RNA-Sequencing
Comparative analysis of bovine milk and mammary gland transcriptome using RNA-Seq (No
Paper presented at 9th World congress on genetics applied to livestock production
Heritability of test day somatic cell counts and its relationship with milk yield and protein percentage in dairy ewes
Potential uses of milk epithelial cells: a review
Characterization and Comparative Analysis of the Milk Transcriptome in Two Dairy Sheep Breeds using RNA Sequencing
Somatic cells of goat and sheep milk: Analytical
Diurnal variation in the somatic cell count of ewe milk
and electrical conductivity among various fractions of ewe’s milk
Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation
Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2
TopHat2: accurate alignment of transcriptomes in the presence of insertions
HTSeq - A Python framework to work with high-throughput sequencing data
Babelomics 5.0: functional interpretation for new generations of genomic data
WEB-based GEne SeT AnaLysis Toolkit (WebGestalt): update 2013
Suárez-Vega, A., Gutiérrez-Gil, B., Klopp, C., Tosser-Klopp, G., & Arranz, J. J. Gene Expression Omnibus GSE74825 (2015)
Download references
This work is included in the framework of the project AGL2012-34437 funded by the Spanish Ministry of Economy and Competitiveness (MINECO)
is funded through the Spanish ‘Ramón y Cajal’ Program (RYC-2012-10230) from the MINECO
Beatriz Gutiérrez-Gil & Juan-José Arranz
UR875 Biométrie et Intelligence Artificielle
Conceived and designed the experiments: J.J.A
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DOI: https://doi.org/10.1038/sdata.2016.51
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Organic farming is promoted to reduce environmental impacts of agriculture
but surprisingly little is known about its effects at the farm level
Here we report the effects of organic farming on species diversity at the field
farm and regional levels by sampling plants
spiders and bees in 1470 fields of 205 randomly selected organic and nonorganic farms in twelve European and African regions
10.5% higher in organic than nonorganic production fields
with highest gains in intensive arable fields (around +45%)
Gains to species richness are partly caused by higher organism abundance and are common in plants and bees but intermittent in earthworms and spiders
Average gains are marginal +4.6% at the farm and +3.1% at the regional level
targeted measures are therefore needed to fulfil the commitment of organic farming to benefit farmland biodiversity
Significant differences within regions (U-test) at 0.05
wind shelter or as part of an agri-environmental scheme
were grassy or shrubby strips along field or water edges
Organic farming gains/losses (OFG, ±s.d.) to species richness in the regionally most frequent production habitats (a), in the most frequent nonproduction habitats (b) and on total species richness per farm (c), for the four taxonomic groups of plants, earthworms, spiders and bees in the twelve regions shown in Fig. 1a
X axes are log-scaled to equalize distances on both sides of parity
Significant differences within regions (U-test) at 0.05 and 0.01 are indicated by * and **
Average regional gains to species richness in production habitats were positively correlated to regional average N input per ha (Spearman’s ρ=0.68
In contrast to production habitats, organic farming did not alter species richness in nonproduction habitats (−3.6% (NS) for plants, +13.4% (NS) for earthworms, −7.1% (NS) for spiders, +9.1% (NS) for bees and −0.7% (NS) overall; Fig. 2b)
Similar habitat richness in all farms resulted in higher floral but lower faunal species richness on organic than nonorganic Venetian farms
Habitat composition was taken into account in the resampling procedure
which highlights a continuous decrease in the positive effects of organic farming on plant and bee species richness as more farm habitats are sampled
Such fading from field to farm may be explained by two processes: the regional pool of farmland species may be limited and simply attained faster on organic farms
or additional species in organic production habitats are ubiquitous
invading more easily from boundaries into fields and contribute little to the total species richness per farm
Ubiquity of species appears to be more likely than limited pools as the individual farms contained
only 27% (±6.8% s.d.) of all plant species and 24% (±13.2%) of all bee species found in the region
We further calculated the occurrence of each plant and bee species relative to all samples in a region as a measure of species rarity
but did not find organic farming effects on species rarity
This suggests that the higher species richness in organic production fields is mostly due to common species
which contribute relatively little to total farm species richness because they are frequently found in other habitats of each farm
it shows that a higher abundance of individuals is likely the most important effect of organic farming on species richness
organic farming is not significantly increasing the number of species present in a given number of individuals but sustains a higher number of individuals in a given sampling unit
Despite substantial variation between taxonomic groups and regions
the majority of the average effects of organic farming on species diversity demonstrate a positive tendency
This is true for most of the nonsignificant effects on species richness
abundance and evenness in productive fields and at farm and regional level
organic farming tends to sustain species diversity to a higher degree than nonorganic farming by allowing more individuals to survive in a given unit of agricultural habitat
Our study highlights that only by means of such targeted measures it is possible to accommodate the dual objectives of food production and biodiversity conservation on farmland
reserve random selections were used to complete the set
the total number of farms studied was limited to 12 due to sampling time constraints caused by the short growing season and the complex habitat structure
only three nonorganic horticultural farms within the study region agreed to participate in the study
only seven organic farms were available for investigation and in Obwalden
a nonorganic farmer ceased participation during the study
farms had to be selected from three separate vine areas because there were not enough organic farmers within one single area
organic and nonorganic farms were selected in pairs because they were located along a geographical and intensity gradient that made it difficult to get an unbiased subset by random sampling
Counts of mechanical operations included field cultivation
Counts of pesticide applications included natural pesticides
mechanical operations and pesticide applications on fields were totalled and the area-weighted averages per farm were calculated
Gathering of management information in African countries involved more uncertainty than in Europe
especially in multiple cropping systems and the characterization of organic fertilizers
This method is based on a generic system of habitat definitions
which characterize individual habitats with respect to their ecological features
include categories specifically related to farming areas
the method has been adapted with refined GHC definitions to deal with the specific characteristics of farm holdings
The most important adaptation was the division of the annual crop GHC into four subcategories
namely summer or winter-sown non-entomophilic annuals
entomophilic and/or bee-attracting annuals and perennials
the three dominant plant species were recorded and allowed for comparisons within the regionally most frequent crops
Plant species in selected plots of areal habitats were recorded in squares of 10 m × 10 m
which were by definition less than 5 m wide
plant species were recorded in a rectangular strip of 1 m × 10 m
a soil core of 30 cm × 30 cm × 20 cm depth was excavated
and a single person hand-sorted the earthworms from the soil for a duration of 20 min
five subsamples were collected for 30 s within a sample ring of 0.357 m internal diameter haphazardly pre-placed on the target vegetation within each plot
Subsamples were immediately transferred to a cool-box
As a taxonomic catalogue of spiders is lacking in the Kayunga region
either identified in the field or immediately transferred into a kill jar
Domesticated bees were counted in the field but not captured
Each plot was surveyed three times during the growing season
but specific timing depended on local conditions
Organism abundance and species richness at the field level was calculated by summing all individuals and species per plot
Data points without or with only one sampled species were omitted from the evaluation of evenness
as no meaningful values could be calculated
Presented values therefore still reflect differences in the frequency of plots where 0 and 1 individual was sampled
we relied on mixed-effects models for assessing the impact of organic farming
farming effects on each metric of species diversity (S) were calculated for each taxonomic group over all the 12 regions
For organism abundance and species richness
mixed-effects models analogous to equation (2) but with a Gaussian error structure were estimated
The number of samples had no effect at the field level and was omitted from these models
The most parsimonious model was finally used for inference on the overall organic farming gain to species richness
For each evaluated measure of species richness and abundance (S)
Random effects b1 to b4 are normally distributed with mean 0 and variance σ2
Term b5ij is a random effect of taxonomic group k within farm ij
To account for the nestedness of the observations of the four taxonomic groups within farm ij
For individual regions and taxonomic groups
organic farming gains (OFGs) and losses were calculated as percent difference of organic farms (OFs) relative to nonorganic farms (NOFs)
where is the mean in organic and nonorganic farms in each region
The standard deviation60 of the OFG is
is the standard deviation and n is the number of observations in each group
Organic farming gains and losses across regions and taxonomic groups were calculated based on coefficients estimated from mixed-effect models (equations 2 and 3)
the expected effect of organic farming is eβ0+β1/eβ0=eβ1 and hence
This resulted in random sequences of habitats predominantly starting with those habitats with high areal proportions
We then calculated the accumulation of species richness along each sequence and
the mean accumulation of species richness per farm
we fitted mixed-effects models for each taxonomic group at each number of sampled habitats using equation (2) and calculated organic farming gains using equation (6)
Gains to species diversity in organically farmed fields are not propagated at the farm level
Biodiversity conservation and the millennium development goals
Global biodiversity: indicators of recent declines
Changing drivers of deforestation and new opportunities for conservation
What conservationists need to know about farming
biodiversity conservation and the future of agricultural intensification
How effective are European agri-environment schemes in conserving and promoting biodiversity
High Nature Value Farming in Europe Verlag Regionalkultur (2012)
The IFOAM Norms for Organic Production and Processing International Federation of Organic Agriculture Movements (2012)
Policy for organic farming: Rationale and concepts
Agroecosystem management and nutritional quality of plant foods: the case of organic fruits and vegetables
Environmental impact of different agricultural management practices: conventional versus organic agriculture
Comparing organic farming and land sparing: optimizing yield and butterfly populations at a landscape scale
Reconciling food production and biodiversity conservation: land sharing and land sparing compared
The effects of organic agriculture on biodiversity and abundance: a meta-analysis
Benefits of organic farming to biodiversity vary among taxa
Beta diversity at different spatial scales: plant communities in organic and conventional agriculture
Scale matters: the impact of organic farming on biodiversity at different spatial scales
Mixed effects of organic farming and landscape complexity on farmland biodiversity and biological control potential across Europe
Food production versus biodiversity: comparing organic and conventional agriculture
Land-use intensity and the effects of organic farming on biodiversity: a hierarchical meta-analysis
Using bioindicators based on biodiversity to assess landscape sustainability
Conserving and promoting evenness: organic farming and fire-based wildland management as case studies
Farmers’ perceptions of biodiversity: lessons from a discourse-based deliberative valuation study
Does conservation on farmland contribute to halting the biodiversity decline
The spatial scale mismatch between ecological processes and agricultural management: do difficulties come from underlying theoretical frameworks
Mixed biodiversity benefits of agri-environment schemes in five European countries
agricultural management and surrounding landscape in European arable fields
Patterns and trends in nitrogen use and nitrogen recovery efficiency in world agriculture
Farmland biodiversity: is habitat heterogeneity the key
Ecological cross compliance promotes farmland biodiversity in Switzerland
Indicators for biodiversity in agricultural landscapes: a pan-European study
Land use and management effects on carbon and nitrogen in Mediterranean Cambisols
Organic farming benefits local plant diversity in vineyard farms located in intensive agricultural landscapes
The role of earthworms for assessment of sustainability and as bioindicators
Quantifying biodiversity: procedures and pitfalls in the measurement and comparison of species richness
Landscape-moderated biodiversity effects of agri-environmental management: a meta-analysis
Assessment of the strategies of organic fruit production and fruit drying in Uganda
A standardized procedure for surveillance and monitoring European habitats and provision of spatial data
Manual for Habitat and Vegetation Surveillance and Monitoring: Temperate
Mediterranean and Desert Biomes Alterra Report 2154 (2011)
Biodiversity in Organic and Low-Input Farming Systems: Handbook for Recording Key Indicators Alterra Report 2308 (2012)
Earthworm collection from agricultural fields: comparisons of selected expellants in presence/absence of hand-sorting
Differential effects of landscape and management on diversity and density of ground-dwelling farmland spiders
Studies on methods of censusing the numbers of bees (Hymenoptera
Measuring bee diversity in different European habitats and biogeographical regions
The nonconcept of species diversity: a critique and alternative parameters
R Development Core Team. R: A Language and Environment for Statistical Computing. (R Foundation for Statistical Computing, (2013) at http://www.R-project.org
Estimating species richness using the jackknife procedure
and comparing incidence-based species accumulation curves
Why we (usually) don’t have to worry about multiple comparisons
Variance partitioning in multilevel logistic models that exhibit overdispersion
Mixed Effects Models and Extensions in Ecology With R Springer (2009)
Data Analysis Using Regression and Multilevel/Hierarchical Models Cambridge University Press (2006)
The meta-analysis of response ratios in experimental ecology
Modern Applied Statistics with S Springer (2002)
rworldmap: A new R package for mapping global data
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This work was funded by the European Union through FP7 project BioBio (Indicators for biodiversity in organic and low-input farming systems; www.biobio-indicator.org; Agreement Nr
by the Austrian Ministry for Science and Research
and by the Lendület program of the Hungarian Academy of Sciences
We thank the farmers for access to land and for information on land management
Institute of Evolutionary Biology & Environmental Sciences
University of Natural Resources & Life Sciences
Institut National de Recherche en Génie Rural
Institute of Environmental & Landscape Management
Lendület Ecosystem Services Research Group
András Báldi & Anikó Kovács-Hostyánszki
Institut Méditerranéen de Biodiversité et d’Ecologie marine et continentale
Institut de Ciència i Tecnologia Ambientals
Bellaterra (Cerdanyola del Vallès - Barcelona)
coordinated field sampling and data processing in the respective study region; M.K.S
wrote the paper with input from all the authors
Supplementary Figures 1-8 and Supplementary Tables 1-3 (PDF 225 kb)
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While the extent and impact of horizontal transfers in prokaryotes are widely acknowledged
their importance to the eukaryotic kingdom is unclear and thought by many to be anecdotal
Here we report multiple recent transfers of a huge genomic island between Penicillium spp
Sequencing of the two leading filamentous fungi used in cheese making
and comparison with the penicillin producer P
rubens reveals a 575 kb long genomic island in P
roqueforti—called Wallaby—present as identical fragments at non-homologous loci in P
Wallaby is detected in Penicillium collections exclusively in strains from food environments
Wallaby encompasses about 250 predicted genes
some of which are probably involved in competition with microorganisms
The occurrence of multiple recent eukaryotic transfers in the food environment provides strong evidence for the importance of this understudied and probably underestimated phenomenon in eukaryotes
the lack of specific evolutionary trends in reported cases of lateral gene transfer in eukaryotes has led to the view of ancient
sporadic and isolated events with relatively little global impact on eukaryotic kingdoms
rather than a more frequently and widely occurring phenomenon
The frequency and importance of eukaryote-to-eukaryote gene transfer may
despite the importance of the filamentous fungi used in cheese making
no genome sequence has yet been published for any of these species
The availability of these first two genome sequences will therefore provide a useful resource for improving our knowledge of edible cheese moulds and for comparative genomics
present-day horizontal transfers of a very large (over 500 kb) genomic island between several cheese fungi
This genomic island harbours about 250 genes
some of which are probably involved in competition with other microorganisms
Beyond the potential conceptual and applied implications of recurrent horizontal transfers occurring in food
this finding indicates that horizontal gene transfer (HGT) may be more widespread and important than previously thought in eukaryotes
as shown by the N50 metric and the number of scaffolds
roqueforti has been experimentally validated and further improved (see below)
The structure of the horizontally transferred genomic island is compared between P. roqueforti, P. rubens and P. camemberti. The sequence shown at the top corresponds to the Wallaby locus in P. roqueforti FM164, that is, from positions 1,487,035 to 2,061,670.
(a) A strain bearing Wallaby isolated from cheese
isolated from an environment other than dairy products
This figure displays a count of the number of reads at each single base position
The y axis scale represents the number of reads counted for every position
sequence identity in Wallaby fragments with RIP footprints dropped to 90–97% between P
roqueforti and the other two Penicillium sequences
but this concerned exclusively RIP C:G to T:A substitutions
This is consistent with the occurrence of RIP after the transfer event in P
The region flanking Wallaby in P. roqueforti may be a hotspot for DNA insertions, as other unrelated fragments appear to be inserted at this locus in other Penicillium genomes (Fig. 4). However, no footprints of duplication or transposable elements were found around the Wallaby insertion points for which flanking regions could be identified.
and those for which homology could be detected in this region in other species are linked and blue
White predicted genes have no detectable similarity to sequences in this region from other species
rubens Wallaby regions are fragmented and located in non-homologous regions and are therefore not shown on this figure
Penicillium species associated with the cheese environment are indicated in purple
Colours in the pseudotable indicate positive amplification for the three primer pairs (red
respectively) for single-copy predicted genes in Wallaby
some of the annotated genes were predicted to be involved in the regulation of conidiation (spore production) or in antimicrobial activities
suggesting functional advantages for Wallaby-bearing strains associated with competition in the cheese
suggests that it may confer adaptation associated with competition with other microorganisms
Our analysis of additional sequenced genomes revealed that these dispersed elements were specific to P
rubens and that horizontal transfers occurred in addition to possible gene family expansions
this finding indicates that horizontal gene transfer can occur within the food chain
The frequency of this phenomenon should be investigated more thoroughly
agricultural and biotechnological practices
We added 1.15 g solid caesium chloride and 4 μl of DAPI solution (1 mg ml−1) per ml of DNA solution (final density: 1.65 g ml−1)
The DNA/CsCl/DAPI solution was centrifuged in a Quick-Seal centrifuge tube (part #342412
Two bands of DNA were visible under UV light
the upper band corresponding to mitochondrial DNA) was collected with a syringe (1.2 × 40 mm needle); DAPI was extracted with isoamyl alcohol saturated with CsCl (1.15 g ml−1) and the DNA solution was dialyzed for 4 days against 10 mM TRIS
1 mM EDTA (pH 8.0) with twice-daily replacement of the dialysis solution
SOAPdenovo was run (kmer values 43–67) to generate contigs
Velvet was then run with combined raw reads and SOAPdenovo contigs (parameters ‘-cov_cutoff 5 -min_contig_lgth 100 -max_divergence 0.05 -long_mult_cutoff 1 -exp_cov auto’)
The range of kmer values for Velvet was 41–57
The short-reads assembly with the maximum N50 value was used as input for the scaffolding process (SOAPprepare and SOAPdenovo)
maximum N50 values were obtained with a kmer value of 37 and a minimum of six links between contigs (parameter (default=5) pair_num_cutoff=6)
a kmer value of 27 and a minimum of 14 links gave the highest N50 value
InterPro was used to identify protein domains and families
roqueforti FM164 draft genome was assembled before molecular combing into 73 scaffolds of over 2 kb
camemberti FM 013 draft genome spans 34 Mb
We analysed all 241 terverticillate Penicillium strains from the public collection of the MNHN
We also analysed the LUBEM-Brest collection encompassing 76 P
These isolates were obtained from blue cheeses from 14 countries
The numbers after the ‘F’ correspond to individual cheeses
morphologically different strains were treated as different strains
labelled by a number following that identifying the cheese (for example
F17.1 and F17.2 are two strains from cheese 17)
Single-spore isolation was systematically performed by the dilution method
after growth for 3–5 days at 25 °C on malt agar
The ‘F’ strains were also obtained by spore dilution
Genomic DNA was extracted from fresh mycelium of the isolates listed in Supplementary Tables S2–S4
Mycelium was obtained after 3–5 days on malt agar for Penicillium
Scopulariopsis and Fusarium strains and on a confidential medium provided by starter producers for Sporendonema casei
The Qiagen DNeasy Plant Mini Kit (Qiagen Crawley
Amplifications were performed with 30 cycles of 30 s at 95 °C
30 s at 58 °C for the three Wallaby primers
the thermal regime was 35 cycles of 30 s at 94 °C
All PCR programs had a final 5 min extension step at 72 °C
PCR products were purified and sequenced by Genoscope (Évry
roqueforti conidial suspension was plated on M3 medium (0.25 g l−1 KH2PO4
25 mg l−1 chloramphenicol in seven Petri dishes and incubated 4 days at 25 °C
Conidia were harvested and resuspended in 20 ml M3 medium
Two 50 ml M3 medium liquid cultures were inoculated with the conidial suspension
Germination occurred after 18 h of incubation at 25 °C
The germinating conidia were harvested by centrifugation (10 min
Dried mycelium (1 g) was suspended in the protoplast isolation solution (400 mg Filtrozym (Laffort)
20 mg bovine serum albumin in 10 ml of TRF1 solution (1.2 M MgSO4
The suspension was incubated at 30 °C for 120–150 min
we transferred the 10 ml suspension to a 30 ml glass tube and overlaid it with 10 ml TRF2 (0.6 M sorbitol
After centrifugation for 10 min at 2869g at room temperature
the protoplasts formed a layer at the interface
The protoplast layer was removed and washed with an equivalent volume of TRF3 (1 M sorbitol
The protoplast pellet was suspended in 1 ml TRF4 solution (1 M sorbitol
Molecular combing experiments involve embedding cells into an agarose plug
releasing DNA into solution by digesting this plug
stretching the DNA on a coverslip with a molecular combing system and performing fluorescent in situ hybridization experiments on the combed DNA
roqueforti FM164 protoplast prepared as described above were embedded in agarose plugs
Protoplasts were lysed overnight by incubation in a 1% sarkosyl
proteinase K (2 mg ml−1) in 0.5 M EDTA pH 8.0 at 50 °C
Agarose plugs were then washed in 10 mM Tris
Genomic DNA was stained by incubating the plug in a 40 mM Tris
The combing solution containing the genomic DNA was obtained by melting agarose at 68 °C for 20 min and subsequently digesting agarose molecules using β-agarase (42 °C
DNA fibres were combed on combicoverslips using the molecular combing system (both from Genomic Vision
The PCR amplicons for probe production were purified and used directly as templates for the labelling reaction
rather than after subcloning (primer sequences available on request)
Probes were labelled by random priming and revealed using Alexa 594 (red)
Alexa 350/AMCA (blue) conjugated antibody sandwiches
The combed DNA was counterstained after the fluorochrome detection step
by incubating the coverslip in 30 μl of a 1:1,000 YoYo-1 solution in milli-Q water for 30 s and then washing three times
we concatenated the data sets to obtain a single phylogenetic tree based on 363 bp
Branch support was determined from a bootstrap analysis of 1,000 re-sampled data sets
Correlations between Z-scores of tetranucleotide composition were assessed using jspecies version 1.2.161
Multiple recent horizontal transfers of a large genomic region in cheese making fungi
Eukaryote-to-eukaryote gene transfer events revealed by the genome sequence of the wine yeast Saccharomyces cerevisiae EC1118
The evolutionary imprint of domestication on genome variation and function of the filamentous fungus Aspergillus oryzae
Functional and ecological impacts of horizontal gene transfer in eukaryotes
Gene transfer and diversification of microbial eukaryotes
Evolutionary implications of horizontal gene transfer
Patterns of prokaryotic lateral gene transfers affecting parasitic microbial eukaryotes
Emergence of a new disease as a result of interspecific virulence gene transfer
An analysis of the phylogenetic distribution of the pea pathogenicity genes of Nectria haematococca MPVI supports the hypothesis of their origin by horizontal transfer and uncovers a potentially new pathogen of garden pea: Neocosmospora boniensis
Horizontal gene and chromosome transfer in plant pathogenic fungi affecting host range
Horizontal transfer of a large and highly toxic secondary metabolic gene cluster between fungi
Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium
Widespread lateral gene transfer from intracellular bacteria to multicellular eukaryotes
Widespread impact of horizontal gene transfer on plant colonization of land
Horizontally transferred fungal carotenoid genes in the two-spotted spider mite Tetranychus urticae
Rapid disease emergence through horizontal gene transfer between eukaryotes
Introduction to food-and airborne fungi pp389 ((2000)
Fungi food spoilage pp519 (Springer (2009)
Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum
Genome sequence of the necrotrophic fungus Penicillium digitatum
Single DNA molecule analysis: applications of molecular combing
Repeat induced point mutation in two asexual fungi
Aspergillus niger and Penicillium chrysogenum
Sex in cheese: evidence for sexuality in the fungus Penicillium roqueforti
Characterization of the Penicillium chrysogenum antifungal protein PAF
The paf gene product modulates asexual development in Penicillium chrysogenum
In silico characterization and molecular evolutionary analysis of a novel superfamily of fungal effector proteins
a composite chitinase and tRNase killer toxin from yeast
The limitations of draft assemblies for understanding prokaryotic adaptation and evolution
Limitations of next-generation genome sequence assembly
Role of horizontal gene transfer in the evolution of fungi
Heterokaryosis as a cause of culture rundown in Penicillium
Conidial anastomosis tubes in filamentous fungi
Population genomic sequencing of Coccidioides fungi reveals recent hybridization and transposon control
Population genomics and local adaptation in wild isolates of a model microbial eukaryote
Horizontal transfer of a nitrate assimilation gene cluster and ecological transitions in fungi: a phylogenetic study
Extraction of DNA from milligram amounts of fresh
inPlant Molecular Biology Manual (eds Gelvin S.
Schilperoot R.) A6,1–10Kluwer Academic Publishers (1988)
Velvet: algorithms for de novo short read assembly using de Bruijn graphs
EuGène: an eukaryotic gene finder that combines several sources of evidence
Genome annotation in plants and fungi: EuGène as a model platform
Combined evidence annotation of transposable elements in genome sequences
A taxonomic and ecological overview of cheese fungi
Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes
Microsatellite loci to recognize species for the cheese starter and contaminating strains associated with cheese manufacturing
Dynamic molecular combing: stretching the whole human genome for high-resolution studies
A diagnostic genetic test for the physical mapping of germline rearrangements in the susceptibility breast cancer genes BRCA1 and BRCA2
DNA replication origin interference increases the spacing between initiation events in human cells
BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT
K TREEFINDER: a powerful graphical analysis environment for molecular phylogenetics
RIP: the evolutionary cost of genome defense
RIPCAL: a tool for alignment-based analysis of repeat-induced point mutations in fungal genomic sequences
Download references
We thank Ricardo Rodriguez de la Vega for invaluable help with the genomic analyses
We thank Marco van den Berg for sharing raw data of the Penicillium chrysogenum Wisconsin 54-1255 strain (recently renamed Prubens)
Kevin Cheeseman and Jeanne Ropars: These authors contributed equally to the work
Laboratoire des Interactions Plantes-Microorganismes (LIPM)
Institut de Génétique et Microbiologie UMR8621
13 ruelle d’Aigrefoin 78470 St Rémy-lès-Chevreuse
helped with experiments and/or provided strains or data
Supplementary Figures S1-S7 and Supplementary Tables S1-S4 (PDF 894 kb)
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Volume 2 - 2020 | https://doi.org/10.3389/fagro.2020.591494
From bacterial quorum sensing to the signals of bees
communication is the basis of biotic interactions
more than two organisms can take part in the speeches
resulting in a complex network of cross-talks
Recent advances in plant-microbe interactions research have shown that communication
the soil close to the root surface) provides a specific microhabitat where complex interactions occur
The complex environment that makes up the rhizosphere can select for certain microbial populations
rhizobia have emerged as an important component of the rhizospheric microbiome
The aim of this review is to explore the components of such a rhizospheric Talk Show in the frame of the rhizobium-legume interactions
This symbiosis is a complex process that involves several signals that can be shaped by plant rhizospheric exudates and microbiome composition
The relationship established by rhizobia with other rhizospheric organisms
together with the influence of the environmental factors
results in their beneficial role on host plant health
and organisms that influence the place of rhizobia in the rhizosphere
The focus is on the most recent approaches for the study and subsequent exploitation of the diversity of the organisms
the study of plant-microbes communication and evolution is fundamental to develop highly efficient inoculants able to reduce the use of fertilizers in agriculture
In a particular environment, individuals can communicate and interact with multiple partners, and the nature of interaction can determine variable costs and benefits to the partner, as a biological market (Werner et al., 2015)
One of the most fascinating environments where complex biological interactions occur is the rhizosphere
a large number of signals can be exchanged involving the plant itself
This all take place in a high-density environmental niche
communication is the result of chemical responses of cells to signatory molecules coming from other cells
These signals affect both the metabolism and transcription of genes activating several regulatory mechanisms
Actors and interactions in the rhizosphere
Inter-kingdom and intra-kingdom communication involving plants and microbes in the rhizosphere: the consistent role of rhizobia
The most known plant-microbe dialogue on the rhizosphere scene, which determine direct and indirect advantages to the partners, was properly addressed as early as 1904 when Hiltner described the symbiotic interaction among legumes and rhizobia (Hartmann et al., 2008)
This symbiosis is a highly specific process in which the genetic and chemical communication signals are strictly plant-bacterium specific
rhizobia positively influence the host's growth thanks to the nitrogen fixation process and at the same time can benefit from the nutrients provided by the plant
This review provides an outline of the most studied intra- and inter-kingdom communication strategies in the rhizobium-legume cross talk
defining the most relevant mechanisms of rhizospheric communication
We conceived the exchange of signals between plants and microorganisms as an intricate Talk Show discussion
in which a large variety of dialogues can take place
regulated and coordinated by a defined line-up
The plant acts as a mediator in the rhizospheric stage, consistently contributing to the balance of soil microbiome composition (Hartmann et al., 2009). On the other hand, the soil microbial community actively participates in the composition of the rhizospheric environment, taking part in degradation and production of organic compounds, for its own benefit (Paterson et al., 2007)
Furthermore, water deficiency, salinity, heavy metals, acidity, as well as low nutrients levels are all abiotic factor that frequently can interfere with the role of rhizobia in the rhizosphere scene (Fagorzi et al., 2018; Bellabarba et al., 2019)
In addition, recent data have revealed that under identical soil conditions, the plant genotype, through its phenotypic features, can filter and modulate the microbial community structure and function, as well as the diversity of root associated bacteria (el Zahar Haichar et al., 2008; Berg and Smalla, 2009) carrying out a partner choice in rhizobia mutualism (Simms and Taylor, 2002)
Increasing field studies including large-scale rhizosphere samplings are now emerging with the aim to better understand the rhizospheric microbiome rules
the preservation and on the importance of communication occurring among the plants
the abiotic environmental factors (positive or negative)
together with the communication among plants and rhizobial/non-rhizobial species are able to influence the rhizosphere Talk Show
giving order to the existing messy scenario
The rhizobial QS is based on the LuxR – LuxI type regulatory system. It is dependent on the threshold level of AHL which allows to induce expression of specific target genes (Veliz-Vallejos et al., 2020)
A wide spectrum of other physiological traits plays an important role in the rhizosphere by contributing to the complex phenomenon of nodulation competitiveness (Triplett and Sadowsky, 1992). These traits include swarming motility, type III secretion system, plasmid transfer, cell division, metabolism and transport (Calatrava-Morales et al., 2018)
It is of note that their occurrence varies among species
Contrarily, in some cases, soil microbial communities can improve the infection capabilities of particular rhizobia and their communication with the host plant (Mehboob et al., 2013). The recent work by Miao et al. (2018) highlighted that strains of Rhizobium fabae are able to improve Rhizobium etli nodulation capabilities through a particular intra-species QS mechanism
Although there are many combinations of bacteria co-inoculation that have been explored for the improvement of rhizobia-legumes symbiosis
there is still the need for advanced comprehensive research in their communication systems
a selection of the most appropriate and performing panels of PGP strains
might be used in the near future as inoculants replacing fertilizers by a more environmentally friendly agricultural practice
A large part of the rhizobia-host communication strategies in the rhizosphere influences the highly regulated and ongoing rhizobial interactions in the root tissue. In compatible legume cultivars, after the establishment in the rhizosphere, rhizobia detect plant-derived flavonoids inducing nod genes, which are involved in Nod factors (lipochitooligosaccharides) production (Poole et al., 2018)
Besides positive effects, fungi can act as antagonistic plant pathogens, which rhizobia can counteract as biocontrol agents for multiple plant species (Deshwal et al., 2013). Furthermore, it was demonstrated that the multiple inoculations of fungi, PGP bacteria can improve symbiotic behavior and plant nutrients availability, as was shown in semi-arid and alkaline soils (Requena et al., 1997; Abd-Alla et al., 2014)
The current understanding of the rhizosphere is highlighting the complexity of the communication strategies taking place in this model environmental scenario
Increasing evidence is pointing out how in this scenario rhizobia occupy an important place that extends to non-legume plants
This niche is a consequence of the influence of several factors (biotic and abiotic)
at different levels (intra- and inter-kingdom) and different degrees (competition and cooperation)
The rhizosphere scene can be considered as a highly suitable model for the application of a system biology study approach, including the large number of plants and microorganisms sequenced genomes, the studies on partners metabolic functionalities (Korenblum et al., 2020) and on the transcriptomic changes related to different partners interaction (Pathan et al., 2020)
It is expected that future studies will continue to explore the selective forces that shape rhizosphere microbiome further elucidate the potential of the communication among the different rhizospheric partners
All authors listed have made a substantial
direct and intellectual contribution to the work
This review was performed at the LIPM belonging to the Laboratoire d'Excellence (LABEX) entitled TULIP (ANR-10-LABX-41) and in the Laboratory of Microbiology
Department of Agricultural and Food Science
Lenie Dijkshoorn for the critical review and the English grammar editing of the manuscript
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Citation: Checcucci A and Marchetti M (2020) The Rhizosphere Talk Show: The Rhizobia on Stage
Received: 04 August 2020; Accepted: 16 November 2020; Published: 02 December 2020
Copyright © 2020 Checcucci and Marchetti. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY)
*Correspondence: Alice Checcucci, YWxpY2UuY2hlY2N1Y2NpMkB1bmliby5pdA==
Microbe-Microbe and Microbe-Host Interactions
Metrics details
Soil bacteria known as rhizobia are able to establish an endosymbiosis with legumes that takes place in neoformed nodules in which intracellularly hosted bacteria fix nitrogen
Intracellular accommodation that facilitates nutrient exchange between the two partners and protects bacteria from plant defense reactions has been a major evolutionary step towards mutualism
Yet the forces that drove the selection of the late event of intracellular infection during rhizobium evolution are unknown
we took advantage of the previous conversion of the plant pathogen Ralstonia solanacearum into a legume-nodulating bacterium that infected nodules only extracellularly
We experimentally evolved this draft rhizobium into intracellular endosymbionts using serial cycles of legume-bacterium cocultures
The three derived lineages rapidly gained intracellular infection capacity
revealing that the legume is a highly selective environment for the evolution of this trait
we identified in each lineage a mutation responsible for the extracellular–intracellular transition
All three mutations target virulence regulators
strongly suggesting that several virulence-associated functions interfere with intracellular infection
We provide evidence that the adaptive mutations were selected for their positive effect on nodulation
we showed that inactivation of the type three secretion system of R
solanacearum that initially allowed the ancestral draft rhizobium to nodulate
was also required to permit intracellular infection
suggesting a similar checkpoint for bacterial invasion at the early nodulation/root infection and late nodule cell entry levels
We discuss our findings with respect to the spread and maintenance of intracellular infection in rhizobial lineages during evolutionary times
Intracellular accommodation of rhizobia was an evolutionary quantum leap in the rhizobium–legume symbiosis as it boosted metabolic exchanges between the two partners while shielding internalized bacteria
Which evolutionary forces drove the selection and maintenance of intracellular accommodation
a relatively late event in the symbiotic process
We took advantage of this latter biological material to specifically explore the dynamics and genetics of acquisition of intracellular infection
we report on the experimental evolution of CBM356 into nodule intracellular endosymbionts using serial plant bacterial coculture cycles
We identified new adaptive mutations for intracellular infection that were genetically and functionally different from hrpG inactivation
and contingent to the initial T3SS− nodulation-conferring mutation
This allowed us to investigate the relationships between nodulation and intracellular infection acquisition
Antibiotics were used at the following concentrations (in micrograms per milliliter): spectinomycin 40
The ancestral strain CBM356 grown overnight in peptone broth rich medium supplemented with trimethoprim was inoculated to three sets of 30 M
Bacteria (107) per tube were used as inoculum
all nodules from each set of 30 plants were pooled
sterilized with 2.6% sodium hypochlorite during 15 min and crushed
Ten percent of the nodule crush was used to inoculate the same day a new set of 30 plants
Three independent and parallel lineages of 16 cycles of inoculation-isolation of nodule bacteria were generated in this way
The use of 30 plantlets per cycle may minimize the effect of plant individual variability
serial dilutions of each nodule crush were plated and one clone was randomly selected from the highest dilution and purified
the rest of the nodule crushes and a 48-h culture of bacteria from an aliquot of nodule crushes were stored at −80 °C
When the total number of recovered nodule bacteria was below 105
106–107 bacteria from the 48-h culture were added per tube of plants 3 days after inoculation with the nodule crush
The number of infected cells per nodule section was estimated on the largest section of each nodule
nodules were collected from 10 tubes of plants
plants were coinoculated with 104/104 (ratio 1/1) or 104/106 (ratio 1/100) cells of two different strains
About 100 nodules were collected and sterilized 21 days after inoculation
Surface-sterilized nodules were individually crushed and plated on selective medium for nodulation competitiveness analysis (estimated by the number of nodules formed by each clone)
For in planta fitness analysis (estimated by the number of each clone in the whole-nodule population)
all surface-sterilized nodules were pooled
three tubes containing two plants were coinoculated with 105/105 cells of two different clones (inoculum ratio 1/1)
each root was transferred into 10 ml of sterile water and strongly vortexed
were diluted and plated on selective medium
The remaining plant culture medium was centrifuged 15 min at 4000 r.p.m.
Two independent experiments were performed
inoculum ratios were verified by plating serial dilutions of inocula on selective medium
All statistical analyses were performed using the R software
Multiple comparisons to assess differences in fitness and number of infected cells were performed using the non-parametric statistical test of Kruskal–Wallis with a P-value threshold of 0.05
Comparisons were done among evolved clones belonging to the same lineage and the corresponding reconstructed mutants taken either the last extracellular infective clone or the first intracellular infective clone as reference
Box-plots represent the distribution of the number of bacteria per nodule recovered at 21 dpi
The central rectangle spans the first quartile to the third quartile (that is
the bold segment inside the rectangle shows the median
unfilled circles indicate suspected outliers
whiskers above and below the box show either the locations of the minimum and maximum in the absence of suspected outlying data or 1.5 × IQR if an outlier is present
Data correspond to 6–12 measures obtained from 2 to 4 independent experiments for each strain
Single stars indicate significant differences with the last extracellular clones
and double stars indicate significant differences with the first intracellular infective clones
N12 or S9 (P<0.05; multiple comparison test after Kruskal–Wallis)
We thus identified three shifts in infection capacity that occurred with different efficiencies in the M
9th and 12th cycles of the evolution experiment
while effects symbolized by blue lines were evidenced in this study
The thickness of the line symbolizes the strength of the interference
the hrpB transcription and the post-transcriptional modification of HrpG are induced
Intracellular infection occurs with either a hrpG or a double T3SS and prh cascade mutant
activation of the 184 HrpG-controlled genes via post-transcriptional modification of HrpG may not be sufficient to block intracellular infection
DMI2 and the DMI3 interacting protein IPD3
which are part of the plant signaling pathway induced by rhizobial Nod factors
are involved in nodule and IT formation and have a role in symbiosome formation
vsrA had a weak effect on intracellular infection
Effect of the prhI p112hrpG and vsrA mutations on the nodulation kinetics and infection phenotype of Ralstonia chimera
(a) Nodulation kinetics of the single mutants CBM124 prhI
Values are from one representative experiment
c) Toluidine blue staining of 1 μm ultrathin sections of 14-day-old M
pudica nodules induced by CBM124 prhI (b) and CBM124 p112hrpG (c)
(d) Nodulation kinetics of double hrcS vsrA
hrcS prhI and hrcV p112hrpG mutants of CBM124
HrcS and HrcV are structural components of the T3SS pilus
In coinoculation experiments (Table 3) prhI and p112hrpG mutations had a huge effect on the in planta fitness of the strains that could not be explained solely by their impact on nodulation competitiveness indicating that their very quick fixation in the lineages originated from their impact on both nodulation and in planta bacterial multiplication
The vsrA mutation, which had a weak effect on intracellular infection, also had a weak effect on nodulation (Figure 4d and Table 3)
confirming the correlation between nodulation and intracellular infection capacity levels observed with the prhI and p112hrpG mutants
How recipient genomes rewire to express adaptive traits that operate profound changes in their lifestyle is largely unknown
The rhizobium–legume symbiosis is an excellent biological system to address this issue
encoding functions such as plant cell-wall degradation
virulence determinants or phytohormone production
Which HrpG-controlled functions negatively impact on intracellular infection remains to be elucidated
our work showed that T3SS can block both root hair and intracellular infection on the same host plant
nodule cell invasion however appeared more stringent than nodulation
as the former requires the inactivation of the T3SS and of unknown T3SS-independent functions
while the latter occurs —although poorly— in a T3SS single mutant
any rhizospheric bacterium has a very remote chance to invade the root if it is not highly competitive for nodulation/initial infection
The coupling of the bacterial abilities to nodulate and infect nodules intracellularly may thus have favored the spread and maintenance of intracellular infection capacity in nodulating bacteria and
have generated a positive selection pressure on nodulation genes by increasing bacterial fitness in planta
This may explain why rhizobia inducing intercellularly invaded nodules have never been described
Our work provides support to similar requirements at both root hair and nodule cell entry levels that we speculate to have favoured the coevolution of bacterial capacity to enter root hairs and nodule cells in the course of the legume–rhizobium evolution
A bacterial sensor of plant cell contact controls the transcriptional induction of Ralstonia solanacearum pathogenicity genes
Genome sequence of the beta-rhizobium Cupriavidus taiwanensis and comparative genomics of rhizobia
Burkholderia species are ancient symbionts of legumes
Transposon mutagenesis of Pseudomonas solanacearum—isolation of Tn5 induced avirulent mutants
two new components of the plant signal-dependent regulatory cascade controlled by PrhA in Ralstonia solanacearum
Next-generation sequencing as a tool to study microbial evolution
a plant receptor essential for symbiosome formation
Interaction of Rhizobium fredii USDA257 and nodulation mutants derived from it with the agronomically improved soybean cultivar McCall
by the beta-proteobacterium Ralstonia taiwanensis
Rhizobial plasmids that cause impaired symbiotic nitrogen fixation and enhanced host invasion
Ancestral genome sizes specify the minimum rate of lateral gene transfer during prokaryote evolution
Symbiotic use of pathogenic strategies: rhizobial protein secretion systems
Analysis of infection thread development using Gfp- and DsRed-expressing Sinorhizobium meliloti
Infection and invasion of roots by symbiotic
nitrogen-fixing rhizobia during nodulation of temperate legumes
Molecular traits controlling host range and adaptation to plants in Ralstonia solanacearum
Evidence that the hrpB gene encodes a positive regulator of pathogenicity genes from Pseudomonas solanacearum
Lipo-chitooligosaccharide signaling in endosymbiotic plant-microbe interactions
A complex network regulates expression of eps and other virulence genes of Pseudomonas solanacearum
Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium loti strain R7A VirB/D4 type IV secretion system
Intracellular plant microbe associations: secretory pathways and the formation of perimicrobial compartments
Formation of organelle-like N2-fixing symbiosomes in legume root nodules is controlled by DMI2
The molecular network governing nodule organogenesis and infection in the model legume Lotus japonicus
Experimental evolution of a plant pathogen into a legume symbiont
Cupriavidus taiwanensis bacteroids in Mimosa pudica indeterminate nodules are not terminally differentiated
Establishing nitrogen-fixing symbiosis with legumes: how many rhizobium recipes
A motN mutant of Ralstonia solanacearum is hypermotile and has reduced virulence
Eukaryotic control on bacterial cell cycle and differentiation in the Rhizobium-legume symbiosis
Using artificial systems to explore the ecology and evolution of symbioses
Genes lost and genes found: evolution of bacterial pathogenesis and symbiosis
IPD3 controls the formation of nitrogen-fixing symbiosomes in pea and Medicago spp
Parallel changes in global protein profiles during long-term experimental evolution in Escherichia coli
a transcriptional regulator responding to growth conditions
is involved in the control of the type III secretion system regulon in Ralstonia solanacearum
Trends and barriers to lateral gene transfer in prokaryotes
Secreted proteins from Ralstonia solanacearum: a hundred tricks to kill a plant
A LuxRI-family regulatory system controls excision and transfer of the Mesorhizobium loti strain R7A symbiosis island by activating expression of two conserved hypothetical genes
Excision and transfer of the Mesorhizobium loti R7A symbiosis island requires an integrase IntS
a novel recombination directionality factor RdfS
Nitrogen-fixing nodules with Ensifer adhaerens harboring Rhizobium tropici symbiotic plasmids
Evolutionary transitions in bacterial symbiosis
Genome sequence of the plant pathogen Ralstonia solanacearum
a second two-component sensor regulating virulence genes of Pseudomonas solanacearum
The global virulence regulators VsrAD and PhcA control secondary metabolism in the plant pathogen Ralstonia solanacearum
Ecology drives a global network of gene exchange connecting the human microbiome
Evolving ideas of legume evolution and diversity: a taxonomic perspective on the occurrence of nodulation
Evolution of rhizobia by acquisition of a 500-kb symbiosis island that integrates into a phe-tRNA gene
The glutamine synthetases of rhizobia: phylogenetics and evolutionary implications
Integrated regulation of the type III secretion system and other virulence determinants in Ralstonia solanacearum
R gene-controlled host specificity in the legume-rhizobia symbiosis
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We are grateful to E Rocha and J Cullimore for comments and M Marchetti for help in cytological work
This work is part of the ‘Laboratoire d’Excellence’ (LABEX) entitled TULIP (ANR -10-LABX-41) and is supported by grants from SPE INRA and ANR-08-BLAN-0295-01
Roche GS FLX 454 sequencing was performed at the PlaGe Platform of the Toulouse Genopole
SHG and LT were supported by a fellowship from the China Scholarship Council and ANR-08-BLAN-0295-01
Catherine Masson-Boivin & Delphine Capela
CNRS-UMR 8030 and Commissariat à l’Energie Atomique CEA/DSV/IG/Genoscope LABGeM
Stéphane Cruveiller & Claudine Médigue
Supplementary Information accompanies this paper on The ISME Journal website
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DOI: https://doi.org/10.1038/ismej.2013.24
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and compare them to each other and to the potato genome (Solanum tuberosum)
The two tomato genomes show only 0.6% nucleotide divergence and signs of recent admixture
but show more than 8% divergence from potato
with nine large and several smaller inversions
tomato and potato small RNAs map predominantly to gene-rich chromosomal regions
The Solanum lineage has experienced two consecutive genome triplications: one that is ancient and shared with rosids
These triplications set the stage for the neofunctionalization of genes controlling fruit characteristics
The exact number of small inversions is difficult to determine due to the lack of orientation of most potato scaffolds
a, Xyloglucan transglucosylase/hydrolases (XTHs) differentially expressed between mature green and ripe fruits (Supplementary Information section 5.7)
These XTH genes and many others are expressed in ripening fruits and are linked with the Solanum triplication
marked with a red circle on the phylogenetic tree
Red lines on the tree denote paralogues derived from the Solanum triplication
Developmentally regulated accumulation of sRNAs mapping to the promoter region of a fruit-regulated cell wall gene (pectin acetylesterase
Variation of abundance of sRNAs (left) and messenger RNA expression levels from the corresponding gene (right) over a tomato fruit developmental series (T1
The promoter regions are grouped in 100-nucleotide windows
For each window the size class distribution of sRNAs is shown (red
The height of the box corresponding to the first time point shows the cumulative sRNA abundance in log scale
The height of the following boxes is proportional to the log offset fold change (offset = 20) relative to the first time point
The expression profile of the mRNA is shown in log2 scale
The horizontal black line represents 1 kb of the promoter region
0 to 12 represent arbitrary units of gene expression
lycopersicum is inactivated by a premature stop codon
allelic variation in a subset of SP family genes has played a major role in the generation of both shared and species-specific variation in solanaceous agricultural traits
Introgression lines exist for seven wild tomato species
The genome sequences presented here and the availability of millions of SNPs will allow breeders to revisit this rich trait reservoir and identify domestication genes
providing biological knowledge and empowering biodiversity-based breeding
Taxonomy of tomatoes: a revision of wild tomatoes (Solanum section Lycopersicon) and their outgroup relatives in sections Juglandifolia and Lycopersicoides
Comparison of plant DNA contents determined by Feulgen microspectrophotometry and laser flow cytometry
Analysis of the genome sequence of the flowering plant Arabidopsis thaliana
The Sorghum bicolor genome and the diversification of grasses
Tomato genome is comprised largely of fast-evolving
Characterization of the tomato (Lycopersicon esculentum) genome using in vitro and in situ DNA reassociation
Genome sequence and analysis of the tuber crop potato
Close split of sorghum and maize genome progenitors
A clarified position for Solanum lycopersicum var
cerasiforme in the evolutionary history of tomatoes (solanaceae)
Phylogenetic analysis of 83 plastid genes further resolves the early diversification of eudicots
Introduction to the Darwin special issue: the abominable mystery
Genetics and control of tomato fruit ripening and quality attributes
The linkage between cell wall metabolism and fruit softening: looking to the future
Profiling of short RNAs during fleshy fruit development reveals stage-specific sRNAome expression patterns
FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis
Control of flowering and storage organ formation in potato by FLOWERING LOCUS T
The tomato FT ortholog triggers systemic signals that regulate growth and flowering and substitute for diverse environmental stimuli
The flowering gene SINGLE FLOWER TRUSS drives heterosis for yield in tomato
The SELF-PRUNING gene of tomato regulates vegetative to reproductive switching of sympodial meristems and is the ortholog of CEN and TFL1
Hybridization between Lycopersicon esculentum and Solanum pennellii: phylogenetic and cytogenetic significance
Relaxed selection among duplicate floral regulatory genes in Lamiales
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This work was supported by: Argentina: INTA and CONICET
Belgium: Flemish Institute for Biotechnology and Ghent University
China: The State Key Laboratory of Plant Genomics
Institute of Genetics and Developmental Biology
Chinese Academy of Sciences; Ministry of Science and Technology (2006AA10A116
2007DFB30080) Ministry of Agriculture (‘948’ Program: 2007-Z5); National Natural Science Foundation (36171319); Postdoctoral Science Foundation (20070420446)
European Union: FP6 Integrated Project EU-SOL PL 016214
France: Institute National de la Recherche Agronomique and Agence Nationale de la Recherche
Government of India; Indian Council of Agricultural Research
ItaLyco and GenoPOM projects); Ministry of Agriculture (Agronanotech and Biomassval projects); FILAS foundation; ENEA; CNR-ENEA project L
Japan: Kazusa DNA Research Institute Foundation and National Institute of Vegetable and Tea Science
Korea: KRIBB Basic Research Fund and Crop Functional Genomics Research Center (CFGC)
Netherlands: Centre for BioSystems Genomics
Netherlands Organization for Scientific Research
Spain: Fundación Genoma España; Cajamar; FEPEX; Fundación Séneca; ICIA; IFAPA; Fundación Manrique de Lara; Instituto Nacional de Bioinformatica
UK: BBSRC grant BB/C509731/1; DEFRA; SEERAD
USA: NSF (DBI-0116076; DBI-0421634; DBI-0606595; IOS-0923312; DBI-0820612; DBI-0605659; DEB-0316614; DBI 0849896 and MCB 1021718); USDA (2007-02773 and 2007-35300-19739); USDA-ARS
We acknowledge the Potato Genome Sequencing Consortium for sharing data before publication; potato RNA-Seq data was provided by C
Buell from the NSF-funded Potato Genome Sequence and Annotation project; tomato RNA-Seq data by the USDA-funded SolCAP project
Maloof; the Amplicon Express team for BAC pooling services; construction of the Whole Genome Profiling (WGP) physical map was supported by EnzaZaden
owns patents and patent applications covering its AFLP and Whole Genome Profiling technologies; AFLP and Keygene are registered trademarks of Keygene N.V
The following individuals are also acknowledged for their contribution to the work described: J
Zabeau is thanked for his support and encouragement and S
collection of wild germplasm and the distribution of seed and knowledge
Shusei Sato (Principal Investigator) & Satoshi Tabata (Principal Investigator)
Beijing Academy of Agriculture and Forestry Sciences
Danlin Fan & Bin Han (Principal Investigator)
Boyce Thompson Institute for Plant Research
Zhangjun Fei (Principal Investigator) & James J
Centro Nacional de Análisis Genómico (CNAG)
Key Laboratory of Horticultural Crops Genetic Improvement of Ministry of Agriculture
Sino-Dutch Joint Lab of Horticultural Genomics Technology
Sanwen Huang (Principal Investigator) & Yongchen Du (Principal Investigator)
State Key Laboratory of Plant Genomics and National Centre for Plant Gene Research
Zhukuan Cheng (Principal Investigator) & Jianru Zuo (Principal Investigator)
Ying Wang (Principal Investigator) & Ying Wang (Principal Investigator)
State Key Laboratory of Plant Cell and Chromosome Engineering and National Center for Plant Gene Research
Hongqing Ling (Principal Investigator) & Hongqing Ling (Principal Investigator)
Laboratory of Molecular and Developmental Biology and National Center for Plant Gene Research
Yongbiao Xue (Principal Investigator) & Yongbiao Xue (Principal Investigator)
Richard McCombie (Principal Investigator) & Zachary B
Stack (Principal Investigator) & Stephen M
Tanksley (Principal Investigator) & James J
Department of Plant Biotechnology and Bioinformatics
Yves Van de Peer (Principal Investigator) & Yves Van de Peer (Principal Investigator)
Heilongjiang Academy of Agricultural Sciences
Institute for Bioinformatics and Systems Biology (MIPS)
Helmholtz Center for Health and Environment
Klaus Mayer (Principal Investigator) & Klaus Mayer (Principal Investigator)
National Key Laboratory of Crop Genetic Improvement
Bishop (Principal Investigator) & Sarah Butcher (Principal Investigator)
Nagendra Kumar Singh (Principal Investigator) & Nagendra Kumar Singh (Principal Investigator)
UR1052 Génétique et amélioration des fruits et légumes
Unité de Biométrie et d'Intelligence Artificielle UR 875
Céline Noirot & Thomas Schiex (Principal Investigator)
Institut National Polytechnique de Toulouse - ENSAT
Mondher Bouzayen (Principal Investigator) & Mondher Bouzayen (Principal Investigator)
Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV)
Antonio Granell (Principal Investigator) & Antonio Granell (Principal Investigator)
Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”
Universidad de Malaga - Consejo Superior de Investigaciones Cientificas (IHSM-UMA-CSIC)
Fernando Carrari (Principal Investigator) & Fernando Carrari (Principal Investigator)
Gianluca De Bellis (Principal Investigator)
Clelia Peano & Gianluca De Bellis (Principal Investigator)
Silvana Grandillo & Pasquale Termolino
Giovanni Giuliano (Principal Investigator) & Giovanni Giuliano (Principal Investigator)
Piazza Martiri della Libertà 33 - 56127 Pisa
Glenn Bryan (Principal Investigator) & Glenn Bryan (Principal Investigator)
van Eijk (Principal Investigator) & Roeland C
Plant Systems Engineering Research Center
Korea Research Institute of Bioscience and Biotechnology
Max Planck Institute for Plant Breeding Research
Department of Plant Science and Plant Pathology
NARO Institute of Vegetable and Tea Science
Satomi Negoro & Hiroyuki Fukuoka (Principal Investigator)
National Institute of Plant Genome Research
Debasis Chattopadhyay (Principal Investigator) & Debasis Chattopadhyay (Principal Investigator)
van Ham (Principal Investigator) & Roeland C
Department of Plant Science and Plant Genomics and Breeding Institute
Seon-In Yeom & Doil Choi (Principal Investigator)
Jane Rogers (Principal Investigator) & Jane Rogers (Principal Investigator)
United States Department of Agriculture - Agricultural Research Service
Instituto de Hortofruticultura Subtropical y Mediterranea
Departamento de Biologia Molecular y Bioquimica
BIO-5 Institute for Collaborative Research
Rod Wing (Principal Investigator) & Rod Wing (Principal Investigator)
Institute of Crop Science and Resource Conservation
Heiko Schoof (Principal Investigator) & Heiko Schoof (Principal Investigator)
Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology
Khurana (Principal Investigator) & Akhilesh K
Tamas Dalmay (Principal Investigator) & Tamas Dalmay (Principal Investigator)
Department of Biology and the UF Genetics Institute
Cancer and Genetics Research Complex 2033 Mowry Road
Paterson (Principal Investigator) & Xiyin Wang (Principal Investigator)
Center for Genomics and Computational Biology
Xiyin Wang (Principal Investigator) & Xiyin Wang (Principal Investigator)
University of Naples “Federico II” Department of Soil
Environmental and Animal Production Sciences
Luigi Frusciante (Principal Investigator) & Luigi Frusciante (Principal Investigator)
Seymour (Principal Investigator) & Graham B
Roe (Principal Investigator) & Bruce A
Giorgio Valle (Principal Investigator) & Giorgio Valle (Principal Investigator)
University of Tennessee Health Science Center
Department of Agriculture and Environmental Sciences
de Jong (Principal Investigator) & Hans H
Klein Lankhorst (Principal Investigator) & René M
For full details of author contributions, please see the Supplementary Information
The author declare no competing financial interests
Supplementary Figures 1-56 and additional references –see Contents for details
This zipped excel file contains Supplementary Tables 1-78
This zipped archive contains Supplementary HTM Tables 1-269
The tables can be opened by any web browser and contain additional text that can be visualized by placing the mouse over the image
This article is distributed under the terms of the Creative Commons Attribution-Non-Commercial-Share Alike licence (http://creativecommons.org/licenses/by-nc-sa/3.0/)
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Medicago truncatula is a long-established model for the study of legume biology
Here we describe the draft sequence of the M
truncatula euchromatin based on a recently completed BAC assembly supplemented with Illumina shotgun sequence
A whole-genome duplication (WGD) approximately 58 Myr ago had a major role in shaping the M
truncatula genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation
truncatula genome experienced higher levels of rearrangement than two other sequenced legumes
truncatula is a close relative of alfalfa (Medicago sativa)
a widely cultivated crop with limited genomics tools and complex autotetraploid genetics
truncatula genome sequence provides significant opportunities to expand alfalfa’s genomic toolbox
Another 146 unfinished BACs/BAC pools that cannot be placed on the optical map contribute 17.3 Mb
Regions not represented in pseudomolecules or unanchored BACs were captured through assembly of approximately 40× coverage Illumina sequencing
yielding 104.2 Mb of additional unique sequence
the Illumina sequence is expected to lie predominantly within the boundaries of pseudomolecules (see below)
On the basis of expressed sequence tag alignments
the combined data sets capture ∼94% of expressed genes
providing a highly informative platform for analysing the euchromatin of M
truncatula pseudomolecules with other sequenced plant genomes to learn more about shared synteny and genome duplication history
Homologous gene pairs were identified for all pairwise comparisons between M. truncatula, G. max, L. japonicus and V. vinifera genomes. Syntenic regions associated with the ancestral WGD events were identified by visually inspection of corresponding dot-plots. The large Mt5–Mt8 synteny block (yellow) was found to have two syntenic regions in L. japonicus (red), four syntenic regions in G. max (blue) and three in V. vinifera (green).
Higher rates of mutation and greater levels of rearrangement in M
truncatula following the papilionoid duplication may have been driven by factors including short generation times
high selfing rates or small effective population sizes
although these characteristics are not unique to M
truncatula genome to ask whether the 58-Myr-ago WGD might have had a role in the evolution of rhizobial nodulation in M
the 58-Myr-ago WGD seems to have led to sub-functionalization of an ancestral gene participating in both interactions
resulting in two homeologous genes that each performs just one of the original functions
Nodule-enhanced expression was even higher in WGD-derived transcription factors (11 out of 142 or 7.7%)
although this enrichment did not reach statistical significance (P = 0.113)
ERN1 is found within this group of WGD-retained
the 58-Myr-ago WGD would have resulted in additional genes
ERN1 and the transcription factors described above
that went on to become specialized for nodule-related functions in the Papilionoideae
Our results indicate that the WGD early in papilionoid evolution allowed the emergence of critical components in Nod factor signalling and contributed to the complexity of rhizobial nodulation observed in this clade
the WGD seems to have had a crucial role in the success of papilionoid legumes
Rosid radiation and the rapid rise of angiosperm-dominated forests
Evolutionary rates analysis of Leguminosae implicates a rapid diversification of lineages during the tertiary
Integration of the FISH pachytene and genetic maps of Medicago truncatula
Analysis of the genome sequence of the flowering plant Arabidopsis thaliana
International Rice Genome Sequencing Project
Unraveling ancient hexaploidy through multiply-aligned angiosperm gene maps
Placing paleopolyploidy in relation to taxon divergence: a phylogenetic analysis in legumes using 39 gene families
Polyploidy did not predate the evolution of nodulation in all legumes
Genome sequence of the palaeopolyploid soybean
The evolutionary fate and consequences of duplicate genes
Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms
Legume diversity and evolution in a phylogenetic context
provide predictable drive to increase morphological complexity
Coordinating nodule morphogenesis with rhizobial infection in legumes
The Medicago truncatula lysine motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes
An ERF transcription factor in Medicago truncatula that is essential for Nod factor signal transduction
LysM-type mycorrhizal receptor recruited for rhizobium symbiosis in nonlegume Parasponia
Following tetraploidy in an Arabidopsis ancestor
genes were removed preferentially from one homeolog leaving clusters enriched in dose-sensitive genes
Evolution of signal transduction in intracellular symbiosis
Expression of genes encoding late nodulins characterized by a putative signal peptide and conserved cysteine residues is reduced in ineffective pea nodules
Plant peptides govern terminal differentiation of bacteria in symbiosis
Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis
Recent duplications dominate NBS-encoding gene expansion in two woody species
Genome-wide identification of NBS genes in japonica rice reveals significant expansion of divergent non-TIR NBS-LRR genes
induces expression of Rhizobium meliloti nodulation genes
Relevance of seed polysaccharides and flavonoids for the classification of the leguminosae: a chemotaxonomic approach
Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis
Venturing beyond beans and peas: what can we learn from Chamaecrista
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from The Noble Foundation and NSF-PGRP 0321460
from FP6 EU project GLIP/Grain Legumes FOOD-CT-2004-506223; to G.E.D.O
from ANR project SEQMEDIC 2006-01122; to R.G
from the Dutch Science Organization VIDI 864.06.007
from the Belgian Federal Science Policy Office IUAP P6/25
Institute for the Promotion of Innovation by Science and Technology in Flanders and Ghent University (MRP N2N); to D.R.C
Gouzy from ‘Laboratoire d'Excellence’ (LABEX) TULIP (ANR-10-LABX-41)
We also acknowledge technical support from the University of Minnesota Supercomputer Institute and thank Y
Nam for a BamHI BAC library used by Genoscope
Harrison for supplying myc infected and control root tissues used to make small RNA libraries
Oldroyd: These authors contributed equally to this work
Departments of Plant Pathology and Plant Biology
Laboratoire des Intéractions Plantes-Microorganismes (LIPM)
USDA-ARS Corn Insects and Crop Genetics Research Unit
Department of Genetics and Developmental Biology
MIPS/Institute for Bioinformatics and Systems Biology
Department of Plant & Soil Sciences and Delaware Biotechnology Institute
Laboratory for Molecular and Computational Genomics
Centre National de Ressources Génomiques Végétales (CNRGV)
Department of Computer & Information Sciences
Max Planck Institute for Plant Breeding Research
International Institute for Tropical Agriculture
National Institute of Agricultural Biotechnology
Unité de Biométrie et d'Intelligence Artificielle (UBIA)
The file contains Supplementary Figures 1-18 with legends
Supplementary Discussion and additional references
The data shows gene models in Mt3.5 with significant sequence homology to organellar sequence
The data shows Mt gene pairs in Mt3.5 derived from the ~58 Mya WGD
identity with known nodulation/symbiosis genes
highlighted with cases where one or both paralogs shows maximal expression in nodules
The data shows transcription factors (TFs) in Mt3.5 and corresponding RNA-seq expression data
Table includes also entries for predicted splice variants
The data shows duplicate TF gene pairs predicted to be derived from the ~58 Mya WGD
The data shows graphic (.pdf) and .ph guide tree files for all 37 gene families used in the analysis of WGT and WGD events
The data shows nodule cysteine-rich proteins in Mt3.5 pseudomolecules
The data shows Mt genes related to chalcone
P450 or saponin metabolism in pseudomolecules
The data shows Mt small RNAs with >85% similarities to annotated At miRNAs in miRBase v15
provided the original author and source are credited
This licence does not permit commercial exploitation
and derivative works must be licensed under the same or similar licence
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Legumes are unusual among plants in that they can carry out endosymbiotic nitrogen fixation with rhizobial bacteria
The genome of Medicago truncatula (also known as barrel medic or barrel clover)
a well-established model for the study of legume biology
truncatula has undergone several rounds of whole-genome duplication
and that the duplication that took place approximately 58 million years ago played an important part in the evolution of endosymbiotic nitrogen fixation
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This study presents a dynamic characterization of the sheep milk transcriptome aiming at achieving a better understanding of the sheep lactating mammary gland
Transcriptome sequencing (RNA-seq) was performed on total RNA extracted from milk somatic cells from ewes on days 10
The experiment was performed in Spanish Churra and Assaf breeds
which differ in their milk production traits
Nearly 67% of the annotated genes in the reference genome (Oar_v3.1) were expressed in ovine milk somatic cells
For the two breeds and across the four lactation stages studied
the most highly expressed genes encoded caseins and whey proteins
We detected 573 differentially expressed genes (DEGs) across lactation points
Upregulated GO terms at late lactation stages were linked mainly to developmental processes linked to extracellular matrix remodeling
A total of 256 annotated DEGs were detected in the Assaf and Churra comparison
Some genes selectively upregulated in the Churra breed grouped under the endopeptidase and channel activity GO terms
These genes could be related to the higher cheese yield of this breed
this study provides the first integrated overview on sheep milk gene expression
The knowledge of gene expression involved in lactation informs the basic study of mammary gland biology
morphogenesis and metabolic activity as well as enhances our understanding of milk composition in relation to the production of milk-based newborn formulas
The aim of this research was to gain a better understanding of the sheep lactating mammary gland and to compare the mammary gland transcriptome of two sheep breeds with different dairy production characteristics
we performed RNA-seq analysis of MSCs in these two dairy sheep breeds at four different lactation time points with the aim of characterizing the expression pattern of the principal stages of lactation
the successful isolation and generation of global gene expression data from ovine MSCs is reported for the first time
The dynamic characterization of milk sheep expression profile and differences between the milk transcriptomes of Assaf and Churra breeds are described
The results described herein provide a significant advance in our knowledge of sheep lactating mammary gland gene expression and valuable information for future studies
A total of eight healthy sheep were selected to be included in the experiment
The animals belong to the commercial farm of the University of León
These sheep were kept in free stall housing
fed with the same rations and had no water restrictions
The lambing by these sheep was between the November 11
The sampling points were established to cover the different physiological stages of the mammary gland across the complete lactation
D10 after lambing is the first day of lactation that is considered totally free of colostrum; it is also the day considered in the normalized lactation for both breeds starts
D50 is a time point close to the lactation peak
The sampling days D120 and D150 correspond to final normalized lactation in Churra and Assaf
whereas for Churra D120 is close to final lactation point
for Assaf this time point corresponds to a transition stage form the lactation peak to the final lactation point
1201/2005 and Council Directive 2010/63/EU)
All animals used in this study were handled in strict accordance with good clinical practices and all efforts were made to minimize suffering
the collecting milk containers were cleaned with RNaseZap (Ambion
Sterile gauze was used to cover the collecting container during milk collection
The milk was passed from the collecting container to RNAse-free 50 ml tubes after collection
Samples were maintained at 4 °C during their transport from the farm to the laboratory
The cells were pelleted by centrifugation at 540 g in 50 ml RNAse free sterile tubes for 10 minutes at 4 °C in the presence of a final concentration of 0.5 mM of EDTA
a fatty layer was usually placed in the top of the tube that was removed using a sterile pipette tip
the cell pellet was washed in PBS (pH 7.2) with 0.5 mM EDTA and centrifuged at 540 g in 15 ml RNAse free sterile tubes for 10 minutes at 4 °C
The last step was repeated until the fatty layer was minimized
RNA extraction continued following a standard Trizol protocol (Invitrogen
The Agilent 2100 Bioanalyzer device (Agilent Technologies
USA) was used to assess the integrity of the RNA
The RNA integrity value (RIN) of the samples ranged between 7.1 and 9
Paired-end libraries with fragments of 300 bp were prepared using the True-Seq RNA-Seq sample preparation Kit v2 (Illumina
The fragments were sequenced on an Illumina Hi-Seq 2000 sequencer (Fasteris SA
according to the manufacturer’s instructions at CNAG (Centro Nacional de Análisis Genómico
To compare the expression levels of genes across samples, raw counts for the genes and transcripts were obtained using SigCufflinks (available at http://www.sigenae.org)
SigCufflinks is a modified version of the cufflinks code that provides raw read counts per gene and transcript
by using the sorted bam file from the alignment and the reference gtf file created in the assembly
The “–G” option of sigcufflinks was used to guide the alignment but excluding new genes
The DESeq2 package performs independent filtering
genes that have very low counts are not likely to see significant differences due to high dispersion
The edgeR package has no function for independent filtering
To choose a cut-off to eliminate the lowest expressed genes
we plotted a histogram on R with the log10 (effmax + 1)
thereby maintaining those genes for which the distribution becomes normal and eliminating the genes with less than 15 counts
RNA-Seq read counts were modeled by a generalized linear model considering the experimental design
with two breed groups (Churra and Assaf) and four lactation time points per group (D10
The model for both programs includes breed and day factors and the interaction day x breed
After discarding a possible interaction between these two factors
we performed the following analyses with edgeR and DESeq2
with the aim of providing a dynamic expression profile of the mammary gland across lactation
differential expression analyses were performed for all the possible time point pairs
considering the Churra and Assaf datasets jointly
to assess the impact of the breed factor on the global expression pattern of sheep lactation
a differential expression analysis between Churra and Assaf
DESeq2 and edgeR perform pairwise comparisons between two or more groups using parametric tests where read-counts follow a negative binomial distribution with a gene-specific dispersion parameter
These packages mainly differ in the estimation of the dispersion parameter and the type of normalization they follow
DESeq2 bases the estimation of the dispersion on calculated mean–variance relationships in the data set provided
while edgeR assumes a common dispersion for all the genes
These programs normalize the read count per gene based on total gene depth per individual
Our choice of these two methods was based on the literature evidence for their robustness
the DEGs were defined as those genes that had and an absolute log2-fold change >1.5 and an adjusted p-value (padj-value) < 0.05
whereas edgeR DEGs were those that had a log2-fold change >1.5 and an adjusted FDR (false discovery rate) <0.05
genes identified as DEGs by the two packages were defined as core DEGs and were subjected to subsequent functional analyses
The web-based Gene Set Analysis Toolkit (WebGestalt18) was used to perform a Gene-Ontology (GO) enrichment analysis
Enriched terms were considered statistically significant when padj-value was <0.05 and a minimum of six genes were grouped for each significant term
The GO terms were categorized in three major functional groups: biological process
Bar graph with the highly expressed genes in milk somatic cells at 10
whereas the gene names are indicated in Y-axis
A color code is used to represent the four time points studied
(a) Top-10 highly expressed genes in Assaf
(b) Top-10 highly expressed genes in Churra
The α-lactoalbumin is a major milk whey protein involved in the synthesis of lactose
which is responsible for drawing water into the milk
The higher milk yields of Assaf as compared to Churra could be due
to higher expression levels of the LALBA gene in this breed
Venn diagrams showing the number of differential expressed genes (DEG) across lactation (considering the four time points studied) in sheep.
For all the time point pair comparisons considered and considering the two breed datasets jointly, the number of DEG identified by the edgeR analysis (red ellipse), the DESeq2 analysis (green ellipse) or both programs (intersection) is indicated.
Comparison of differentially expressed genes (DEGs) detected with DESeq2 and edgeR in the D10 vs
(a) Comparative graphical representation of the logFC values resulting from the differential expression analysis performed for the D10 vs
D150 contrast with the edgeR package (X-axis) and the DESeq2 package (Y-axis)
a color code is used to distinguish the following categories: logFC values of the genes exclusively detected as DEGs by edgeR (red); logFC values of the genes exclusively detected as DEGs by DESeq2 (green); logFC values of the genes detected as DEGs by both packages (yellow); logFC values of the genes not identified as DEG by any software (black)
For the genes detected as DEGs by both packages (colored in yellow) the expression levels (logFC values) measured by the two packages showed a high and linear correlation (r2 = 0.99)
Expression profile of the five differentially expressed genes (DEGs) showing the largest fold-changes across lactation
The counts normalized by sequencing depth with the DESeq2 package (y-axis) are represented against the lactation stages studied in the present work (x-axis)
For each gene at each stage of lactation the point indicates the mean of counts over all samples at this stage and error bars show the standard error of the mean
In the across lactation study, the significant GO enrichment terms (padj < 0.05), for the annotated DEGs were categorized into 140 functional groups (Supplementary Table S2)
across the three GO established categories: “biological processes” (106 terms)
“cellular component” (30 terms) and “molecular function” (4 terms)
the most significant terms of these three categories were associated to DEGs identified in the D10 vs D150 comparison
Schematic representation of the most representative enrichment GO terms highlighted by our functional analysis in relation to the DEG identified across lactation
The four lactation time points considered are indicated with colored boxes: day 10 (D10
The different arrows correspond to a specific time-point comparison and are linked to a summary box where the number of annotated upregulated DEGs in the two time-points compared are shown
The most representative GO terms associated to the identified DEGs are also indicated
Note that if no significant results were identified in the GO enrichment analysis the upregulated genes at the corresponding stage are indicated
we have to take into account that dairy cattle have a longer lactation (normalized to 305 days in Holstein) than sheep (normalized to 150 days in Assaf and 120 days in Churra)
In the comparison between the Churra and Assaf breeds
whereas 774 genes were identified as DEGs with the DESeq2 package
were found in common as DEGs from both packages
A total of 172 annotated genes were upregulated in the Churra breed and 84 annotated genes were upregulated in the Assaf breed
In the comparison between breeds, the significant (padj < 0.05) GO enrichment terms for the annotated DEGs were categorized into 46 functional groups (Supplementary Table S3)
Twenty terms were significantly enriched in relation to the “biological processes” category
The most significantly enriched GO terms within this dataset were immune response (GO: 0006955) and immune system process (GO:0002376)
which were associated to upregulated genes in the Assaf breed
In the “cellular component” GO term category
11 terms were significantly enriched; the most significant ones were related to extracellular region (GO: 0005576) and extracellular region part (GO:0044421) also associated to Assaf upregulated genes
The rest of significantly enriched terms (11) belonged to the “molecular function” category
the most significant ones were receptor binding (GO:0005102) related to Assaf DEGs and three terms related to serine hydrolase (GO:0017171) and endopeptidase activities (GO:0008236
which could be explained by the action of immune related peptides in cheese manufacturing
The differential expression observed between Churra and Assaf breeds for these groups of genes (kallikrein
ion channel related genes) seems noteworthy when considering the differences in milk volume and physicochemical properties influencing renneting and ripening processes
this study represents the first integrated overview on the dynamic expression profile of the milk sheep transcriptome
this study allowed us to compare the milk transcriptome of two dairy sheep breeds
showing that the differences in the gene expression profiles
could serve to identify candidate genes explaining the known differences in production characteristics (milk yield and milk composition) that exist between these two breeds
This work may provide fundamental information for future studies on specific pathways involved in lactation as well as the functional annotation of novel genes detected
Gene expression in the human mammary epithelium during lactation: the milk fat globule transcriptome
present and future perspectives of small ruminant dairy research
The Awassi and Assaf breeds in Spain and Portugal (Paper S14.2)
Paper presented at 57th Annual Meeting of the EAAP
International Journal of Dairy Technology 63
RNA-Seq: a revolutionary tool for transcriptomics
The Sequence Alignment/Map format and SAMtools
Mapping and quantifying mammalian transcriptomes by RNA-Seq
Characterization and comparative analyses of muscle transcriptomes in Dorper and small-tailed Han sheep using RNA-Seq technique
A comprehensive evaluation of normalization methods for Illumina high-throughput RNA sequencing data analysis
Milk Production in Dairy Sheep Feeding and Nutrition (ed
GWA analysis for milk production traits in dairy sheep and genetic support for a QTN influencing milk protein percentage in the LALBA gene
Structure and expression of goat GLYCAM1 gene: lactogenic-dependent expression in ruminant mammary gland and interspecies conservation of the proximal promoter
Demonstration of in vivo mammogenic and lactogenic effects of recombinant ovine placental lactogen and mammogenic effect of recombinant ovine GH in ewes during artificial induction of lactation
Perspective– FcRn transports albumin: relevance to immunology and medicine
Association of polymorphisms of beta-2-microglobulin gene (β2m) with milk IgG1 content in Chinese Holstein dairy cows
Perspectives on immunoglobulins in colostrum and milk
Role of progesterone and glucocorticoids in the transcription of the beta-casein and 28-S ribosomal genes in the rabbit mammary gland
Stress-associated endoplasmic reticulum protein 1 (SERP1)/Ribosome-associated membrane protein 4 (RAMP4) stabilizes membrane proteins during stress and facilitates subsequent glycosylation
Short communication: Endoplasmic reticulum stress gene network expression in bovine mammary tissue during the lactation cycle
A functional genomics approach to evaluate candidate genes located in a QTL interval for milk production traits on BTA6
Targeted inhibition of osteopontin expression in the mammary gland causes abnormal morphogenesis and lactation deficiency
Osteopontin: an early innate immune marker of Escherichia coli mastitis harbors genetic polymorphisms with possible links with resistance to mastitis
Modulation of clusterin gene expression in the rat mammary gland during pregnancy
Indicators of inflammation in the diagnosis of mastitis
Identification in milk of a serum amyloid A peptide chemoattractant for B lymphoblasts
Transcriptome analysis of mammary tissues reveals complex patterns of transporter gene expression during pregnancy and lactation
Extracellular matrix composition reveals complex and dynamic stromal-epithelial interactions in the mammary gland
The role of collagen-derived proteolytic fragments in angiogenesis
Proteinases of the mammary gland: developmental regulation in vivo and vectorial secretion in culture
Cloning and characterization of FAM13A1–a gene near a milk protein QTL on BTA6: evidence for population-wide linkage disequilibrium in Israeli Holsteins
PPARGC1A genes and somatic cell count of Jersey cows—preliminary study
IL-19 and IL-20: two novel cytokines with importance in inflammatory diseases
Interleukin-20 promotes angiogenesis in a direct and indirect manner
Vascular remodelling during the normal and malignant life cycle of the mammary gland
prolactin signalling and involution pathways explain considerable variation in bovine milk production and milk composition
Endoplasmic reticulum lumenal proteins of rat mammary gland
Potential involvement in lipid droplet assembly during lactation
Estudio del perfil transcripcional de genes implicados en el metabolismo de los ácidos grasos de la leche en dos razas ovinas in XVI Jornadas sobre Producción Animal
Asociación Interprofesional para el Desarrollo Agrario) 462-464 (INO Reproducciones S.A.
Factors of variation influencing bulk tank somatic cell count in dairy sheep
Contribution of proteolytic activity associated with somatic cells in milk to cheese ripening
International Journal of Dairy Technology 57
Distribution of 15 human kallikreins in tissues and biological fluids
Stress down regulates milk yield in cows by plasmin induced beta-casein product that blocks K + channels on the apical membranes
Transport of milk constituents by the mammary gland
Physico-chemical characteristics of goat and sheep milk
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BGG is funded through the Spanish “Ramón y Cajal” Programme (RYC-2012-10230) from the MINECO
ASV is funded by an FPU contract from Spanish Ministry of Education
Beatriz Gutiérrez-Gil & Juan José Arranz
Christèle Robert-Granie & Gwenola Tosser-Klopp
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Journal of Animal Science and Biotechnology (2020)
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An Addendum to this article was published on 28 March 2019
One of the most efficient plant resistance reactions to pathogen attack is the hypersensitive response
a form of programmed cell death at infection sites
The Arabidopsis transcription factor MYB30 is a positive regulator of hypersensitive cell death responses
Here we show that MIEL1 (MYB30-Interacting E3 Ligase1)
an Arabidopsis RING-type E3 ubiquitin ligase that interacts with and ubiquitinates MYB30
leads to MYB30 proteasomal degradation and downregulation of its transcriptional activity
MIEL1 attenuates cell death and defence through degradation of MYB30
repression of MIEL1 expression removes this negative regulation allowing sufficient MYB30 accumulation in the inoculated zone to trigger the hypersensitive response and restrict pathogen growth
Our work underlines the important role played by ubiquitination to control the hypersensitive response and highlights the sophisticated fine-tuning of plant responses to pathogen attack
this work emphasizes the importance of protein modification by ubiquitination during the regulation of transcriptional responses to stress in eukaryotic cells
The sharp boundary of the HR suggests the existence of efficient mechanisms that control cell death and survival
the regulatory mechanisms of HR development and restriction remain largely unknown
All these data underline MYB30 as an important regulator of plant defenses
we describe a new MYB30-interacting partner encoding a RING E3 Ub ligase protein that we named MIEL1 (MYB30-Interacting E3 Ligase1)
We demonstrate that MIEL1 is an active E3 ligase that interacts with MYB30 in the plant cell nucleus
MIEL1 ubiquitinates MYB30 thereby leading to its degradation by the 26S proteasome
Arabidopsis miel1 mutant plants display enhanced HR and resistance after inoculation with avirulent bacteria
These phenotypes are MYB30-dependent and correlate with downregulation of MYB30 target gene expression
modulation of MIEL1 expression provides an efficient regulatory mechanism to (i) attenuate the activation of defence and related cell death responses in the absence of the pathogen and (ii) promote MYB30 accumulation and triggering of the HR/defence at the site of inoculation
this work underlines the importance of transcription regulation by ubiquitination during the establishment of plant defence reactions
which is a shared feature in eukaryotic cells in response to a variety of stress cues
no protein interaction could be detected in yeast coexpressing MYB30ΔAD and an additional Zinc Finger RING C3H2C3-type protein that we called RINGc (RING control; At1g61620)
these data demonstrate the specificity of the interaction between MIEL1 and MYB30 in yeast
Confocal images of epidermal cells of Arabidopsis seedlings 72 h after Agrobacterium-mediated transient expression of the indicated constructs
The interaction (+) between MYB30 and RING proteins as determined by Y2H and FRET-FLIM is indicated
His-tagged MIEL1 was tested for E3 Ub ligase activity in presence or absence of yeast E1
anti-His and anti-myc antibodies were used to detect ubiquitinated proteins
An integral RING catalytic domain is required to detect MIEL1-mediated ubiquitination that is not observed when using MIEL1m
these results demonstrate that (i) MIEL1 is a functional E3 Ub ligase able to ubiquitinate MYB30 in vitro and that (ii) MIEL1 enzymatic activity depends on the structure of the RING domain
(a) Arabidopsis wild-type Col-0 and transgenic plants overexpressing TAP-tagged MYB30 (MYB30OE) were treated with the proteasome inhibitor MG132 (+) or the solvent DMSO (−)
densitometry values for MYB30-TAP were calculated from six independent experiments
ubiquitinated proteins were purified using the TUBEs resin
Agarose resin was used to detect non-specific protein binding
Western blot with anti-Ub and anti-TAP antibodies detects ubiquitinated proteins and TAP-tagged MYB30
(a) FLAG-tagged MYB30 or TAP-tagged MYB123 were expressed in N
densitometry values for MYB30-FLAG and MYB123-TAP were calculated from six independent experiments and are shown at the bottom of each western blot
(b) Fluorimetric GUS assays in leaf discs 36 hpi of the following constructs: (1) KCS1p:GUS reporter alone or with (2) MYB30-FLAG; (3)MYB30-FLAG+MIEL1-HA; (4) MYB30-FLAG+MIEL1m-HA; and (5) MYB30-FLAG+RINGc-HA
Statistical significance according to a Student’s t-test P-value <10−5 is indicated by letters
(c) Western blot showing expression of MYB30-FLAG and HA-tagged RING-type protein constructs
confirming that MIEL1-mediated suppression of MYB30 accumulation and transcriptional activity require an intact RING domain in MIEL1
(a) Symptoms developed by the indicated Arabidopsis lines 64 hpi with Pst AvrRpm1 (2 × 106cfu ml−1)
Pictures are representative of three independent experiments in which five plants of each line were infiltrated
(b) Quantification of cell death by measuring electrolyte leakage before (white bars) and 24 h after inoculation (grey bars) of the indicated lines with Pst AvrRpm1 (5 × 106 cfu ml−1)
Cell death values are related to the value displayed by wild-type Col-0 plants
Statistical differences using multiple factor analysis of variance (ANOVA) (P<10−4) are indicated by letters
(c) Growth of Pst AvrRpm1 in the indicated Arabidopsis lines
Bacterial growth 0 (white bars) and 3 days (grey bars) was measured after inoculation (5 × 105 cfu ml−1)
Mean bacterial densities were calculated from six independent experiments with six individual plants (four leaves per plant)
Statistical differences according to a Multiple Factor ANOVA test (P<0.01) are indicated by letters
(d) Expression analysis of the MYB30 target genes KCS1 and FDH in the indicated Arabidopsis lines after inoculation with Pst AvrRpm1 (5 × 107 cfu ml−1)
Expression values of the individual genes were normalized using SAND family and β-tubulin4 as internal standards
Statistical differences according to a Student’s t-test P-value <0.05 are indicated by letters
demonstrating that miel1 mutant phenotypes are causally related to the loss of function of MIEL1
Finally, as MYB30OE plants, the miel1 null mutant line displayed higher expression of MYB30 VLCFA-related target genes, KCS1 and FDH, as compared with Col-0 wild-type plants following bacterial inoculation (Fig. 5d)
These data confirm that MIEL1 negatively regulates Arabidopsis defence through repression of MYB30 transcriptional activity
Col-0 plants were inoculated with Pst AvrRpm1 (5 × 107 cfu ml−1)
Leaf samples were harvested from areas both inside and immediately neighbouring the infiltrated zone at the indicated time points
Relative expression of MIEL1 (a) and MYB30 (b) in the inoculated (solid line) or in the adjacent (dashed line) zone was determined by Q-RT-PCR
Expression values were normalized using SAND family and β-tubulin4 genes as internal standards
values were calculated from three experiments with three replicates
all these findings strongly suggest that epitope-tagging of the proteins does not affect their function
our finding that MIEL1 regulates defence-related MYB30 transcriptional activation (i) highlights the crucial role played by ubiquitination in the transcriptional control of plant defence responses and (ii) provides a first description of a RING E3 Ub ligase that directly controls the transcriptional activation of the cell death associated to plant disease resistance
The fact that miel1 mutant plants do not show spreading HR after pathogen challenge suggests that additional factors (AtsPLA2-α and likely other regulators) control restriction of the HR to the inoculated zone
This hypothesis should be investigated in the future
negative regulatory mechanisms to control the HR thus provides an efficient means to temporally and spatially regulate the activation of programmed cell death and illustrates the sophisticated fine-tuning of plant responses to pathogen attack
An AtMYB30 version deleted from its C-terminal activation domain (amino acids 1–234) was used as bait for screening 2 × 106 independent transformants exhibiting His auxotrophy on selective plates
Details of cloning procedures are provided as Supplementary Information
overnight bacterial cultures of Agrobacterium tumefaciens strain C58C1 expressing the protein of interest were harvested by centrifugation
Cells were resuspended in induction buffer (10 mM MgCl2
and 150 μM acetosyringone) to an OD600 of 0.5
cells were infiltrated into leaves of 4-week-old N
leaf discs used for experiments were harvested and processed
or frozen immediately in liquid nitrogen and stored at –80 °C
Transient expression of proteins in Arabidopsis seedlings was performed as described32
tumefaciens overnight cultures were collected and resuspended at an OD600 of 1 in 2 ml of 5% sucrose
Infiltration was performed by covering the seedlings with the Agrobacterium solution and by applying vacuum (10 mm Hg) twice for 1 min
Excess infiltration medium was subsequently removed and the plates transferred to a culture room for 3 days
Arabidopsis 4-week-old plants were kept at high humidity 12 h before inoculation and injected with a bacterial suspension of Pst AvrRpm1 or Pst AvrPphB at the indicated bacterial densities using a blunt syringe on the abaxial side of the leaves
For determination of in planta bacterial growth
leaves samples were harvested 0 and 3 days postinoculation and ground on sterile water
A predetermined dilution for each sample was plated on King’s B medium and incubated at 28 °C for 2 days
Data were submitted to a statistical analysis using Statgraphics Centurion XV.II Professional Software (Statpoint Technologies Inc.
Normality of residues was verified by the Kolmogorov-Smirmov test
The effect of the genotype was tested by multiple factor analysis of variance
100 μM MG132 (Sigma-Aldrich) or the solvent DMSO (control) was infiltrated in N
Leaves were harvested 12 h after treatment
10-day-old seedlings were vacuum-infiltrated for 2 min with MG132 or DMSO and incubated for 4 h before harvesting
CFP and YFPv fluorescence was analysed with a confocal laser scanning microscope (TCS SP2-SE; Leica) using a × 63 water immersion objective lens (numerical aperture 1.20; PL APO)
CFP fluorescence was excited with the 458 nm ray line of the argon laser and recorded in one of the confocal channels in the 465–520 nm emission range
YFP fluorescence was excited with the 514-nm line ray of the argon laser and detected in the range between 520 and 575 nm
Images were acquired in the sequential mode (20 Z plains per stack of images; 0.5 μm per Z plain) using Leica LCS software (version 2.61)
Fluorescence lifetime of the donor was experimentally measured in the presence and absence of the acceptor
FRET efficiency (E) was calculated by comparing the lifetime of the donor in the presence (τDA) or absence (τD) of the acceptor: E=1−(τDA)/(τD)
Statistical comparisons between control (donor) and assay (donor+acceptor) lifetime values were performed by Student t-test
FRET-FLIM measurements were performed using a FLIM system coupled to a streak camera
The light source (λ=439 nm) was a pulsed diode laser working at 2 MHz (Hamamatsu Photonics
All images were acquired with a × 60 oil immersion lens (Plan Apo 1.4 numerical aperture
IR) mounted on an inverted microscope (Eclipse TE2000E
The fluorescence emission was directed back out into the detection unit through a band pass filter
The FLIM unit was composed of a streak camera (Streakscope C4334
Japan) coupled to a fast and high sensitivity CCD camera (model C8800-53C
average fluorescence decay profiles were plotted and lifetimes were estimated by fitting data with tri-exponential function using a non-linear least-squares estimation procedure
MIEL1-6His and MIEL1m-6His proteins were overexpressed and purified from E
coli Rosetta cell cultures using standard protocols
For in vitro ubiquitination assays reaction mixtures (20 μl) contained 0.5 μg of yeast (Saccharomyces cerevisiae) E1 enzyme (Sigma-Aldrich)
0.5 μg of purified Arabidopsis E2 enzyme UBC8
0.5 μg of purified His-tagged MIEL1 protein as the E3 Ub ligase
and 1 μg of ubiquitin (Sigma-Aldrich) in 25 mM Tris-HCl
Reactions were incubated at 30 °C for 2 h and stopped by adding Laemmli buffer and boiling for 10 min
Protein extracts were prepared in 50 mM Tris-HCl
and 1% plant protease inhibitor cocktail (Sigma-Aldrich) and centrifuged at 14,000 g for 10 min at 4 °C
Protein concentration in the supernatant was determined with the Bradford protein assay kit (Bio-Rad) using BSA as a standard
the deubiquitination inhibitor PR-619 (Tebu-bio) was added into the extraction buffer
Supernatants were pre-cleared for 30 min at 4 °C with agarose beads (Tebu-bio)
Samples were split into two fresh tubes and used either for TUBEs (Tandem Ub-Binding Entities
Tebu-bio) or agarose beads (control) pull downs
Pull downed proteins were eluted by boiling for 10 min in Laemmli buffer
Antibodies used for western blotting were anti-PAP soluble complex-HRP (Sigma
mouse monoclonal anti-Ub (PAD1) (Santa Cruz Biotechnology
1:2,000) and goat anti-mouse IgG-HRP (Santa Cruz
Proteins were visualized using the Immobilon kit (Millipore) under standard conditions
Densitometry of proteins was calculated using the Image J software
Thirty-six hours after transient expression of proteins in N
plant tissue was ground in liquid nitrogen
pH 7; 10 mM β-mercaptoethanol; 10 mM NaEDTA; 0.1% Sarcosyl; 0.1% Triton X-100)
One microgram of total protein in supernatants was used to fluorimetrically measure enzymatic GUS activities using 4-methylumbelliferyl-β-D-glucuronide as substrate in a microtiter fluorimeter (FL600
Standard curves were prepared with a range of increasing concentrations of 4-methylumbelliferone (Sigma-Aldrich)
values were calculated from the results of eight independent experiments (2–4 replicates/experiment)
Four leaf discs (6 mm diameter) were harvested 24 hpi
washed and incubated at room temperature in 5 ml of distilled water before measuring conductivity
Four independent experiments were performed with three plants (four leaves per plant)
Relative expression was calculated as the ΔCp between each gene and the average of internal controls (SAND family (At2g28390) and β-tubulin 4 (At5g44340))
Average ΔCp was calculated from three experimental replicates and related to the value of each gene at time 0
The ubiquitin-26S proteasome system at the nexus of plant biology
The HECT ubiquitin-protein ligase (UPL) family in Arabidopsis: UPL3 has a specific role in trichome development
Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis
The diversity of plant U-box E3 ubiquitin ligases: from upstream activators to downstream target substrates
The E3 ubiquitin ligase gene family in plants: regulation by degradation
The Arabidopsis TT2 gene encodes an R2R3-MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed
Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities
Hormone crosstalk in plant disease and defense: more than just JASMONATE-SALICYLATE antagonism
Direct ubiquitination of pattern recognition receptor FLS2 attenuates plant innate immunity
Stability of plant immune-receptor resistance proteins is controlled by SKP1-CULLIN1-F-box (SCF)-mediated protein degradation
The F-box protein CPR1/CPR30 negatively regulates R protein SNC1 accumulation
Rice XA21 binding protein 3 is a ubiquitin ligase required for full Xa21-mediated disease resistance
cDNA-AFLP reveals a striking overlap in race-specific resistance and wound response gene expression profiles
The transcriptional innate immune response to flg22
Interplay and overlap with Avr gene-dependent defense responses and bacterial pathogenesis
Loss-of-function mutations in chitin responsive genes show increased susceptibility to the powdery mildew pathogen Erysiphe cichoracearum
Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation
The Arabidopsis Botrytis Susceptible1 Interactor defines a subclass of RING E3 ligases that regulate pathogen and stress responses
Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings
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We thank Céline Remblière for help with plant transformation and Laurent Deslandes for kindly providing RINGc constructs and helpful discussions
We also thank Juan Carlos del Pozo for the kind gift of the UBC8 clone and Sylvain Raffaele for critical reading of the manuscript
funded by a grant from the Centre National de la Recherche Scientifique (Bourse Docteur-Ingénieur) and the French Ministry of National Education and Research
This work was supported by a French ANR-Jeunes Chercheurs grant (ANR JC08_324792) to S.R
part of the Laboratoire d’Excellence (LABEX) entitled TULIP (ANR-10-LABX-41)
Present address: Present address: Department of Plant Biology and Ecology
Present address: Present address: The Sainsbury Laboratory
Supplementary Table S1 and Supplementary Methods (PDF 1022 kb)
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we were interested by a new actinomycete strain belonging to the genus Streptosporangium and producing a new compound (R2) identified as a new angucyclinone
structure elucidation and antimicrobial activities of compound R2
Sg3 was cultivated at 30 °C for 9 days in Erlenmeyer flasks (500 ml) containing 100 ml of ISP2 broth (yeast extract 4 g
The cultures (8 l) were centrifuged and filtered to remove mycelium
The culture filtrate was extracted with an equal volume of n-butanol to generate a crude extract (1.9 g)
The latter was fractionated by size exclusion chromatography on Sephadex LH-20 (75% MeOH in H2O)
was of red color and exhibited an antibacterial activity
It was subjected to semipreparative reversed-phase HPLC using an Interchim UP5ODB column (250 × 7.8 mm) (Interchim
France) and developed using a continuous grade from 20 to 75% MeOH in H2O (UV detection at 220 nm)
The major peak 2 with the main antibacterial activity was subjected to repeated HPLC to yield 3.5 mg of compound R2
Compound R2 was obtained as a red powder (optical rotation [α]D21 +56 (c 0.11
The ESI-MS spectrum contained an ion peak at m/z 475.1 [M–H]−
and its molecular formula was determined by HRESI-MS analysis as C26H19O9 (calcd 475.43 for (M–H)−
The UV absorption maximum at 535 nm was because of the red color and suggested a quinone chromophore
2949 and 1721 cm−1 in the IR spectrum of R2 were characteristic of hydroxy
The structure of R2 was determined to be a benzo[a]naphtacene-2-carboxylic acid,4,7,11,12-tetrahydro-1,5,6,9,14-pentahydroxy-8,13-dioxo-3-methyl-10-ethyl
The minimum inhibitory concentrations (MICs) of R2 were determined by a conventional agar dilution method using nutrient agar medium
Compound R2 showed strong activity against the Gram-positive bacteria Micrococcus luteus ATCC 9314 and Bacillus subtilis ATCC 6633 (MICs=0.5 and 1 μg ml−1
and moderate activity against Staphylococcus aureus CIP 7625 (MIC=10 μg ml−1)
A weak activity was observed against Listeria monocytogenes CIP 82110 and Mycobacterium smegmatis ATCC 607 (MICs=40 and 50 μg ml−1
Compound R2 showed no activity (MICs>100 μg ml−1) against the Gram-negative bacteria (Escherichia coli ATCC 10536 and Pseudomonas fluorescens number 412)
the fungi (Mucor ramannianus NRRL 1829 and Aspergillus carbonarius number M333) and the yeasts (Saccharomyces cerevisiae ATCC 4226 and Candida albicans number 224)
Sg3 produces a new angucyclinone that displayed antibacterial activities
and biosynthetic studies of an emerging new class of antibiotics
and C: a novel family of antifungal antibiotics
Antifungal and antiviral activities of benanomycins and their analogues
new endothelin converting enzyme inhibitors isolated from Streptosporangium roseum No
physico-chemical properties and biological activities
a new inhibitor of Ca2+ and calmodulin-dependent cyclic nucleotide phosphodiesterase from Streptomyces californicus
source d'actinomycètes rares producteurs d'antibiotiques
Isolation and partial characterization of pigment-like antibiotics produced by a new strain of Streptosporangium isolated from an Algerian soil
Biosynthesis of the pradimicin family of antibiotics
isolation and structure determination of metabolites associated with the pradimicins biosynthesis
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A part of this work was supported by the grant from ‘Comité d'Evaluation et de Prospective de Coopération Inter-universitaire Franco-Algérienne’ (Programme Hubert Curien Tassili)
Laboratoire de Recherche sur les Produits Bioactifs et la Valorisation de la Biomasse
Abdelghani Zitouni & Nasserdine Sabaou
LCC (Laboratoire de Chimie de Coordination)
Département ‘Bioprocédés & Systèmes Microbiens’
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Applied Microbiology and Biotechnology (2016)
World Journal of Microbiology and Biotechnology (2012)
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Plant-parasitic nematodes are major agricultural pests worldwide and novel approaches to control them are sorely needed
We report the draft genome sequence of the root-knot nematode Meloidogyne incognita
Most of the assembled sequence of this asexually reproducing nematode
exists in pairs of homologous but divergent segments
This suggests that ancient allelic regions in M
incognita are evolving toward effective haploidy
The number and diversity of plant cell wall–degrading enzymes in M
incognita is unprecedented in any animal for which a genome sequence is available
and may derive from multiple horizontal gene transfers from bacterial sources
Our results provide insights into the adaptations required by metazoans to successfully parasitize immunocompetent plants
and open the way for discovering new antiparasitic strategies
Plant-parasitic nematodes are responsible for global agricultural losses amounting to an estimated $157 billion annually
Although chemical nematicides are the most reliable means of controlling root-knot nematodes
they are increasingly being withdrawn owing to their toxicity to humans and the environment
Novel and specific targets are thus needed to develop new strategies against these pests
Infective second-stage juveniles (J2) penetrate the root and migrate between cells to reach the plant vascular cylinder
The stylet (arrowhead) connected to the esophagus is used to pierce plant cell walls
to release esophageal secretions and to take up nutrients
Each J2 induces the dedifferentiation of five to seven root cells into multinucleate and hypertrophied feeding cells (*)
These giant cells supply nutrients to the nematode (N)
The nematode becomes sedentary and goes through three molts (J3
males develop and migrate out of the roots
it is believed that they play no role in reproduction
The pear-shaped female produces eggs that are released on the root surface
Embryogenesis within the egg is followed by the first molt
incognita presented here provides insights into the adaptations required by metazoans to successfully parasitize and counter defenses of immunocompetent plants
The five largest supercontigs are shown with plots of gene density (orange curve), conservation with C. elegans at amino acid level (green curve) and EST density (pink curve). Blue lines represent most similar matches at the protein level between each predicted gene on these five supercontigs and 70 matching supercontigs.
Exons are represented by red boxes and are linked together to form genes (arrows indicate the direction of transcription)
Highly diverged allelic genes are linked together using blue boxes
Gene order is well conserved between the two allelic-like regions
with only minor differences in predicted gene structure
Percentages of sequence identity at the protein level between the two allelic-like regions are indicated
We identified gene products that might be involved in parasitic interaction
particularly those that might modify plant cell walls
which most closely resemble bacterial enzymes
Chorismate mutase is a key enzyme in biosynthesis of aromatic amino acids and related products
incognita may subvert host tyrosine-dependant lignification or defense responses
these genes suggest a critical role of HGT events in the evolution of plant parasitism within root-knot nematodes
These secreted proteins of as-yet-unknown function are likely targets for novel intervention strategies
G-protein coupled receptors (GPCRs) and neuropeptides encompass some of the gene products most extensively involved in core physiological
Thus the expansion of SupNRs started before the Brugia-Meloidogyne-Caenorhabditis split and has proceeded independently in C
highlight the huge biodiversity in the phylum Nematoda
incognita may account for its parasitic lifestyle and lead to the development of new control strategies directed against plant-parasitic nematodes
(a) Comparison of the RNAi pathway genes of C
A gray background indicates that at least one homologous gene was found in M
and a white background indicates that no homologous gene was found in M
A yellow background indicates 148 nematode-only gene clusters
incognita genome is the first one sequenced and assembled for a strictly parthenogenetic species
we expect that its comparison with sexual nematode genomes will shed light on mechanisms leading to its peculiar structure
Functional divergence between ancient alleles of genes involved in the host-parasite interface could explain the extremely wide host range and geographic distribution of this polyphagous nematode
incognita revealed a suite of plant cell wall–degrading enzymes
which has no equivalent in any animal studied to date
The striking similarity of these enzymes to bacterial homologs suggests that these genes were acquired by multiple HGT events
Just as many instances of bacterial HGT involve sets of genes implicated in adaptations to new hosts or food sources
incognita involve genes with potential roles in interactions with hosts
The alternative hypothesis—that these genes were acquired vertically from a common ancestor of bacteria and nematodes and lost in most eukaryote lineages—appears less parsimonious
incognita-restricted secreted proteins or lineage-specific expansions and/or reductions that may play roles in host-parasite interaction
proteomic analysis and high throughput RNAi strategies are in progress and will lead to a deeper understanding of the processes by which a nematode causes plant disease
Combining such knowledge with functional genomic data from the model host plant A
thaliana should provide new insights into the intimate molecular dialog governing plant-nematode interactions and allow the further development of target-specific strategies to limit crop damage
animal- and plant-parasitic nematode genomes should provide new insights into parasitism and niche adaptation
We used the M. incognita strain 'Morelos' from the root-knot nematode collection held at INRA (Institut National de la Recherche Agronomique) Sophia Antipolis, France. Nematode eggs were collected in a sterile manner from tomato roots and checked for the presence of plant material contaminants. DNA was extracted as described in Supplementary Methods
The assembled genome was searched for repetitive and non-coding elements. Scaffolds were aligned to determine pairs and triplets of allelic-like regions. Gene positions along scaffolds were used to predict clusters of genes forming putative operons (Supplementary Methods
The 9,538 contigs resulting from the Meloidogyne incognita genome assembly and annotation were deposited in the EMBL/Genbank/DDBJ databases under accession numbers CABB01000001–CABB01009538
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and J.T.J.) received funding from the Scottish Government
This work benefited from links funded via COST Action 872
are also funded by the EU Cascade Network of Excellence and the integrated project Crescendo
We thank Philippe Lecomte for critical reading of the manuscript and all our collaborators from the “Plant-Nematode interaction” team of INRA Sophia Antipolis for technical help and support
Laboratoire Interactions Plantes Micro-organismes
UMR 6098 CNRS and Universités d'Aix-Marseille I & II
UR1164 Unité de Recherche en Génomique et Informatique (URGI)
Institut de Génomique Fonctionnelle de Lyon
Institut Fédératif 128 Biosciences Gerland
Dipartimento di Biochimica e Biologia Molecolare “E
Plateforme Bioinformatique du Génopole Toulouse Midi-Pyrénées
Unité de Biométrie et d'Intelligence Artificielle UR875
wrote the manuscript with input from J.T.J
for bioinformatics of allelism characterization
supervision and coordination of the sequencing
for global protein set comparative analysis
Proteome expert annotation was as follows: P.M.C.
for candidate nematode parasitism and pioneer genes
for protection against plant defenses and immune system
for RNAi pathway and lethal RNAi phenotypes
This article is distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike license (http://creativecommons.org/licenses/by-nc-sa/3.0/)
This license does not permit commercial exploitation
and derivative works must be licensed under the same or similar license
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Nakeeyat in a photograph with former France Ambassador to Ghana
Known for her charisma and role in the projection of the unique cultures in Ghana
is set to make an appearance at the maiden edition of Kente festival in France
The event is scheduled to take place at the Salle Du Lac
Organised by the Ghana Association in Toulouse
the Kente festival in France which comes off on December 23rd this year
aims to recognise and honour the remarkable artistic contributions made by Ghana in the Francophone world
It also seeks to project the iconic visual representation of wearing Kente with emphasis on its history
“Kente is exported as one of the key symbols of African heritage and pride in African ancestry throughout the diaspora
This year is the perfect time to celebrate the existence of kente and its benefits in Ghana as well as other parts of the world as well,” the organisers said
expresses the organisers’ excitement in extending an invitation to Nakeeyat and her team
who over the years have gained popularity due to the projection of the country’s cultures on the global stage
“This letter serves as an official invitation
as we wholeheartedly count on your unwavering support and collaboration for the Kente festival
Your presence and active participation will undoubtedly elevate the event to new heights of excellence
We are confident that your valuable contributions will not only inspire Ghanaian artists but also foster a stronger bond between Ghana and the Francophone world,” it added
joins the likes of the newly elected president of the Musicians Union of Ghana (MUSIGA)
Eno Barony among other high-profile personalities billed to grace the occasion