Volume 2 - 2022 | https://doi.org/10.3389/finsc.2022.1079756 This article is part of the Research TopicFocus on Popillia japonicaView all 11 articles is native to Japan and became established in North America in the early twentieth century first in Italy in 2014 and then in Switzerland in 2017 Metropolitan France is at the forefront of the Japanese beetle threat due to its geographical proximity to the European populations established in the Piedmont An express pest risk analysis for metropolitan France was therefore conducted The most likely pathways for entry include (i) natural dispersion (ii) trades of plant products with adherent soil and (iii) hitchhiking behaviour resulting from natural spread as well as human activities Given the absence of significant limiting factors japonica in France will likely be as important as in its current geographic distribution Although several sources of uncertainty were highlighted throughout the evaluation none of them has significant impact on the conclusions of the present express pest risk analysis japonica are recommended and include surveillance with pheromone traps and control via integrated pest management strategies most efforts should be concentrated on eradication measures while P japonica is still in the early stages of invasion yet these sightings were considered as incursions without establishment japonica in France as well as its potential impacts recommendations for surveillance and management measures in the event of an outbreak were formulated insofar as they favour the survival and reproduction of P We focused on these main hosts in the following pest risk analysis Figure 1 Phylogeny of the main and secondary host plants of P. japonica generated by the V.Phylomaker package (18). A total of 131 host plants, belonging to 39 families, are classified as “main” (17). This figure is also available as Supplementary Material to allow for magnification as well as their capacity of transfer to host plants cultivated in France temperature is favourable and many host plants are available irrigation practices increase the likelihood of establishment in the less rainy areas of the Mediterranean region the likelihood of establishment in protected conditions (e.g greenhouses) is considered low with a moderate level of uncertainty This is due to several points: (1) the facilities concerned are generally small and subject to various pest management methods (2) there have been no recent reports of P japonica populations seem unlikely to be overlooked during regular inspections by growers in indoor conditions Figure 2 Species richness in main host plants of P japonica in each department (NUTS3 region) of metropolitan France as no barriers to spread within the metropolitan French territory have been identified Within the French area at risk of establishment the magnitude of impact is also considered high with a low uncertainty This conclusion is mainly supported by (1) the importance of main host plants in terms of area (2) the absence of currently deployed cultural practices that would significantly reduce the impact of P japonica and (3) chemical control is mainly based on a single family of products (pyrethroids) The main point of uncertainty concerns the level of susceptibility of French varieties of the main host plants to P japonica for the threatened French metropolitan area is considered high with a low uncertainty This risk is therefore considered as unacceptable and justifies the recommendation of management measures an efficient surveillance strategy is needed to ensure the early detection and to allow the rapid implementation of eradication measures Using semiochemical-based traps is recommended along the border with infested countries to detect natural spread as well as near key entry points and transport networks (e.g and unloading areas) to take the risk of hitchhiking behaviour into account Pheromone traps should be positioned in preferred habitats such as grasslands vineyards and fruit crops fields located in the close vicinity of entry points visual inspections of the aerial parts of the main host plants of P japonica are recommended in these areas along the borders with infested countries including for instance nurserymen and garden owners The use of pheromone-baited traps is the most reliable monitoring method as it is selective and effective Compared to visual inspection of aerial parts trapping ensures permanent coverage of the area to be monitored with a higher level of sensitivity Since the infested area is a dynamic surface changing with each discovery of an infestation point the boundaries of the demarcated area (infested area + buffer zone) would be updated accordingly many chemical molecules used in the USA to control P japonica are banned in the European Union (notably neonicotinoids) restricting the available arsenal essentially to the family of pyrethroids soil and growing media as well as plant wastes originating from the infested area should be prohibited The same should apply to plants originating from the buffer zone These actions must be carried out within a short period to increase the chances of eradication the containment strategy is both time consuming and has since it would at most slow down the spread of P the containment strategy involves suppression of P japonica populations within the infested area by chemical control whose effectiveness is reduced in case of heavy infestations Pest risk analyses commissioned at a national level bring out limitations to the precise evaluation of the threat associated with invasive pests and identify opportunities to control their impact we addressed specifically the invasive Japanese beetle and its risk to metropolitan France We assessed the phytosanitary risks in terms of pathways for entry and actionable recommendations to mitigate this risk we highlight a few points that we believe deserve further attention it is crucial to identify and quantify the movements of goods and people from infested areas to susceptible regions the statistical office of the European Union provides extensive information on flows within well-defined pathways notably those listed in “pathways for entry” specific custom codes do not allow discriminating goods of particular interest regarding the biology and ecology of P making it difficult to capture only relevant information the accessibility and visibility of data describing the connectivity of means of transport deserve to be improved the road traffic during the period of beetle activity the list of cars and trucks stop locations would be valuable to better assess the entry routes of the pest The biology and ecology of P. japonica have been extensively studied (see for example 1, 41–44). However, further knowledge would be useful, such as the attraction radius of pheromone traps for P. japonica. Simple methods exist to estimate this radius (45) useful information to optimise the density of trapping networks A better understanding of the role of fruit odours on adult feeding attractiveness would also be helpful Knowing the correlations between the level of susceptibility of host plants and the degree of defoliation should help in assessing the impact on crop yield It would also be interesting to develop innovative and environmentally sound control strategies that are in line with the European Union commitment to reduce the use of pesticides such as the use of biological control agents we believe that biological invasions could be better regulated by promoting international scientific and technical collaboration to harmonise management practices and regulations This review was conducted by the expert working group “Popillia japonica” of the French Agency for Food and Occupational Health and Safety (ANSES) All authors have contributed equally to the risk assessment presented in this document SP wrote the first draft and all other authors have provided significant inputs into the text All authors contributed to the article and approved the submitted version Environmental and Occupational Health and Safety (ANSES) that funded the Pest Risk Analysis conducted by the expert working group “Popillia japonica” 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 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/finsc.2022.1079756/full#supplementary-material Google Scholar 2. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Sylvain Poggi, c3lsdmFpbi5wb2dnaUBpbnJhZS5mcg== Disclaimer: 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 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish Metrics details The potato cyst nematode Globodera rostochiensis originates from the Andean Mountain region in South America and has unintentionally been introduced to all inhabited continents Several studies have examined the population genetic structure of this pest in various countries by using microsatellite markers merging microsatellite data produced from different laboratories is challenging and can introduce uncertainty when interpreting the results To overcome this challenge and to explore invasion routes of this pest rostochiensis populations from all continents the highest genetic diversity was observed in the South American populations the European populations showed an intermediate level of genetic diversity and the remaining populations were the less diverse This confirmed pre-existing knowledge such as a first introduction event from South America to Europe but the less diverse populations could originate either from South America or from Europe STRUCTURE genetic clustering output indicated that North America and Asia have experienced at least two introduction events Comparing different evolutionary scenarios the Approximate Bayesian Computation analysis showed that Europe served as a secondary distribution centre for the invasion of G rostochiensis into all other continents (North America When a newly sampled population was found genetically distant from previous ones it can be challenging to distinguish the true genetic divergence from effects resulting from technical differences between laboratories the number of microsatellite markers used varied among the studies mentioned above To generate an accurate and extensive dataset on a global scale rostochiensis populations were collected from different parts of the world and all populations were genotyped from DNA extraction to allele-size reading allele sizes were identified using the automatic calling and binning procedure and completed by a manual examination by only one reader This study aimed to explore the genetic features rostochiensis invasion worldwide using 22 populations from North and South America rostochiensis populations that were genotyped (i.e. among the 793 individuals without missing data) Gp118 and Gp135) to 16 (for Gr90) alleles per locus Forty private alleles (alleles present in only one population) were detected Regarding the deviation from the random mating hypothesis, 20 populations were at the Hardy–Weinberg equilibrium (i.e., FIS not significantly different to zero). Only one Kenyan population, HAR2, and the Jap-Ho population showed strong heterozygote deficits, with significant positive values of FIS: 0.277 and 0.667, respectively (Table 1) Matrix of pairwise FST between the 22 Globodera rostochiensis populations Populations were separated according to the different groups constituted for the ABC analyses ns indicates non-significant differentiation between two populations Bayesian clustering analysis (STRUCTURE) of the 793 Globodera rostochiensis individuals from the 22 populations collected in different continents Assignment probabilities of individuals are presented for K = 3 the most likely number of clusters statistically determined using ΔK method (bottom-left graph) each vertical line represents an individual for which the genetic assignment is partitioned into three clusters represented by three colors (cluster 1 in green and vertical white lines separate each of the 22 populations The map at the top shows the geographical location of the 22 G rostochiensis populations and their membership proportion of clusters The four population divergence scenarios tested by the Approximate Bayesian Computation (ABC) approach using each target population (Canada Since an invasive population generally starts with a few individuals an initial size reduction (noted here ‘N pop X b’ for each target population and ‘N EU b’ for population Europe) was added 0 indicates the present day and t on top of the bar indicates the oldest split bd illustrates the duration of the bottleneck period A ghost population is a possible unsampled source population We concluded that the adequacy of the model-posterior combination sufficed to correctly explain the observed dataset 22 populations of the potato cyst nematode G rostochiensis originating from all inhabited continents Our results confirmed the South American origin for this species and its initial introduction into Europe our analyses provided support for the hypothesis that populations from North America Asia and Oceania most likely originated from Europe This suggests that Europe served as a bridgehead in the worldwide invasion history of G Future research is needed to explain this particular genetic feature of G which suggests the possibility of a significant cost of consanguineous mating or capacity of the females of this species to preferentially mate with non-sibling males This discovery led the authors to suggest that G rostochiensis was introduced to Japan from Peru suggested that various transportation means might have occurred in Japan Since we have only examined one Japanese population here further research is necessary to investigate the genetic diversity among different G rostochiensis populations from the same geographical area (Indonesia Kenya and Australia) suggested that in each country these populations recently diverged from their common ancestor (a recent introduction event) or that gene flow still remain strong between these populations in each country The latter is less probable due to the low dispersal abilities of nematodes and the strict regulatory measures applied in many countries Kenya and Australia occurred more recently compared to that in Europe we cannot determine the timing of introduction events for the populations Ama Leb-Be and Jap-Ho populations since we have genotyped only one population in these countries the different dates of first detection also suggested that the introduction was more recent than the initial one in Europe ABC analyses could have allowed us to estimate the number of generations since the introduction event due to the variations in the collection times of different populations and the variable number of generations per year for G rostochiensis based on environmental conditions we were unable to perform this type of estimation accurately we cannot rule out the possibility that in some cases the introduction occurred before the implementation of quarantine measures in the country each introduction of an exotic pathogen is not always successful due to the absence of suitable establishment conditions as the frequency of introduction events increases the likelihood of getting the right conditions also rises As growers worldwide seek for the best cultivars to compete globally the international transport of seed tubers is expected to increase Importing material from approved suppliers with processes to minimize pests and diseases incidence in seed tuber fields could be an effective way to further reduce the risk of new introductions in addition to the quarantine status and sole proof of pest absence in the field of origin of the plant material Microsatellite-based genotyping of Globodera rostochiensis populations from all continents allowed for a reconstruction of invasion routes at a worldwide scale This collaborative population genetic study confirms that this plant-parasitic nematode species evolved in the Andean region of South America and was first introduced into Europe Genetically less diverse populations sampled in North America which served as a secondary distribution hub for the worldwide invasion of G These insights are not only essential to prevent further introductions of genetically diverse populations but also to enable the development of effective control strategies Control methods proven successful within the European secondary hub could be extrapolated to effectively manage G rostochiensis populations in other global regions stemming from this hub the remaining five new populations of the dataset included: a Lebanese population (Leb-Be) a North American population from New York state (US) and a Japanese population from Hokkaido (Jap-Ho) PCR multiplex were performed on a 96-well Thermal Cycler (Applied Biosystems) PCR products were then diluted to 1:40 in sterile water and 3 μL of this dilution was mixed with 0.05 μL of GeneScan 500 LIZ Size Standard (Applied Biosystems) and 5 μL of formamide (Applied Biosystems) Genotyping was performed on ABI 3730XL sequencer (Applied Biosystems) at the Gentyane INRAE platform Allele sizes were identified using the automatic calling and binning procedure of GeneMapper® v 5.0 (Thermo Fisher Scientific) and completed by a manual examination The statistical significances of FIS were estimated using the allelic permutation method (10,000 permutations) implemented in GENETIX and their statistical significances were estimated by 5000 random permutations of individuals among populations A Bonferroni adjustment was applied to take into account multiple testing α = 0.05 was lowered to α = 0.00022 for 231 comparisons (22 × 21/2) Based on historical knowledge of the origins of G we excluded the hypothesis that some populations were native except for the South American populations (Bolivia Peru and Chile) and we draw for each target population (Canada Japan and Australia) two origins and main route hypotheses The first hypothesis is a direct South American origin the second hypothesis is an origin from Europe after the initial introduction into this continent 1,000,000 simulations datasets per scenario Possible scenarios were compared through the computation of the posterior probabilities of each scenario The relative posterior probabilities of the competing scenarios were assessed by a logistic regression estimate We also used the option ‘model checking’ with PCA in DIYABC to evaluate how well the best scenario and parameter posterior distributions combination fit the observed data The different Globodera rostochiensis populations were collected by the different co-authors who obtained the required authorizations They provided to INRAE the biological material following official endorsed Letter Of Authorization necessary for shipment of quarantine organisms between laboratories The INRAE laboratory is accredited to hold and perform experiments on this quarantine organism Authors declare that the cyst nematode species sampled in the present work is not concerned by both the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora The file (Esquibet_Gros.txt) containing the genotypic data (Genepop format) for the 22 Globodera rostochiensis populations is available at data.inrae.fr (https://doi.org/10.57745/3INH2O) Major emerging problems with minor Meloidogyne species Top 10 plant-parasitic nematodes in molecular plant pathology Inbreeding and population structure of the potato cyst nematode (Globodera pallida) in its native area (Peru) Population genetic structure of the sugar beet cyst nematode Heterodera schachtii: A gonochoristic and amphimictic species with highly inbred but weakly differentiated populations Human influence on the dispersal and genetic structure of French Globodera tabacum populations An assessment of potato cyst nematode (Globodera spp.) research from the Andean region of South America Distribution of species of potato cyst-nematodes in South America A cyst nematode ‘species factory’ called the Andes Heterodera schachtii found in a Long Island potato field The discovery of golden nematode in Newfoundland The golden potato cyst nematode Globodera rostochiensis pathotype Ro1 in the Saint-Amable regulated area in Quebec On the occurrence of the potato cyst-nematode Occurrence of the potato cyst nematode Globodera rostochiensis in Lebanon First record of the golden potato nematode Globodera rostochiensis in Yunnan and Sichuan provinces of China Area freedom from Globodera rostochiensis in Western Australia Identification of golden potato cyst nematode (Globodera rostochiensis) in Batu Potato cyst nematodes (Globodera species) in Africa First report of Globodera rostochiensis infesting potatoes in Kenya First report of potato cyst nematode (Globodera rostochiensis) infecting potato (Solanum tuberosum L.) in Rwanda infecting potato (Solanum tuberosum) in Uganda Genetic diversity of the golden potato cyst nematode Globodera rostochiensis and determination of the origin of populations in Quebec Mwangi, J. M. Resistance based integrated pest management strategy for Globodera rostochiensis and Globodera pallida in potato cropping systems. 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Populations 1.2.31. Population Genetic Software (Individuals or Populations Distances, Phylogenetic Trees) 2012. http://bioinformatics.org/~tryphon/populations/ (1999) Estimation of average heterozygosity and genetic distance from a small number of individuals logiciel sous Windows™ pour la génétique des populations (Laboratoire Génome Estimating F-statistics for the analysis of population structure Inference of population structure using multilocus genotype data Inference of population structure using multilocus genotype data: Linked loci and correlated allele frequencies The computer program structure for assigning individuals to populations: Easy to use but easier to misuse STRUCTURE HARVESTER: A website and program for visualizing STRUCTURE output and implementing the Evanno method Detecting the number of clusters of individuals using the software STRUCTURE: A simulation study CLUMPP: A cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure DIYABC v2.0: A software to make approximate Bayesian computation inferences about population history using single nucleotide polymorphism Detecting immigration by using multilocus genotypes Introduction history of Drosophila subobscura in the New World: A microsatellite based survey using ABC methods An evaluation of genetic distances for use with microsatellite loci Download references We acknowledge Gentyane INRAE platform for running the genotyping from the PCRs and providing the raw data for microsatellite genetic analyses Holley Center for Agriculture and Health and School of Integrative Plant Science Hokkaido Agricultural Research Centre (HARC) National Agriculture and Food Research Organization (NARO) ME performed the experiments according to a protocol elaborated jointly by JM and EG and approved by all authors JMM and SK described and provided the Kenyan populations XW described and provided the US population BM described and provided the population from Quebec WB and JH described and provided the Indonesian populations JW described and provided the Australian populations EG and JM provided the remaining populations from South America ME and JM analysed the data and prepared the figures All the authors wrote the text and have made substantial contributions to the interpretation of data All authors have approved the current version The authors declare no competing interests Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-024-64617-0 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science Words:  Owen Ralph The 24-seat installation follows the earlier supply of virtual reality surf simulators takes audiences on a 3D exploration to both the North and South Poles The accompanying 5 minute-long Arctic 4D film features previously unknown Arctic landscapes populated with rare species that are difficult to observe in their natural environment penguins and much more in this multi-sensory experience the custom-designed seats feature 3-DOF motion by D-BOX Technologies The programming by CL Corp provides precise and ultra realistic movements there is also a ‘cocktail’ of special effects integrated into the armrest of each seat Whether it’s a blast of air to give the sensation of speed or a breeze or smells that amplify the action on screen Based in Le Rheu, France, CL Corp boasts 20 years of experience providing motion bases and 3D/4D attractions. 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The installation of a scale and a parcel pass where the customer can drop off their parcel themselves allowed them to keep their job. An autistic postal worker, in the process of losing their job, took a training course in computer development with the help of Hipip in (i.e. a structure specialized in supporting autistic people with Asperger's syndrome). He regained his self-confidence and joined the IT department where everyone praises his remarkable performance. There is a lot of noise on the parcel platform in Le Rheu (Ille-et-Vilaine) and yet a deaf postal worker has found his place. Digital tablets have been configured and adapted to allow him to exchange with his colleagues and to work independently and safely. Now that he is comfortable in his position, he is considering becoming a team leader. In 2020, the overall promotion rate at La Poste was 10.33%. It will reach 8.34% for postal workers who are eligible for mandatory employment, (i.e. 945 disabled postal workers promoted). 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During the European Week for the Employment of People with Disabilities (SEPH) in November 2019, nearly 200 postal workers with "invisible disabilities" were honored throughout France in partnership with the Makanak&vous association. Their portraits were displayed on the facades of the group's headquarters and ten emblematic La Poste buildings, highlighting La Poste's commitment to non-discrimination in the eyes of all. A network of 130 disability advisors assists postal workers in all business lines and throughout France. And because digitalisation is accelerating, 5 referents are working to improve the accessibility of digital tools for visually impaired postal workers. Maintenance of letter carriers' electric bicycles, mailboxes and green spaces, repair of parcels or sorting of non-mechanizable parcels: La Poste has purchased 16.38 million euros worth of services from adapted and protected sectors in 2021. dedicated to the employment of people with disabilities in the 7th agreement (2019-2022) disabled people recruited in the past 4 years At BPE; the private bank of the La Poste Groupe, 1 out of 2 people hired is disabled. The number of beneficiaries of the employment obligation has tripled between 2019 and 2021, from 2% to 6% of the workforce. In 2020, BPE signed an agreement in favor of the employment of people with disabilities and family caregivers. Financial information Non-financial information Volume 6 - 2015 | https://doi.org/10.3389/fpls.2015.00772 All crop species are current or ancient polyploids structural and functional modifications result in differential gene content or regulation in the duplicated regions which can play a fundamental role in the diversification of genes underlying complex traits We have investigated this issue in Brassica napus with the aim of studying the structural and functional organization of duplicated regions involved in quantitative resistance to stem canker a disease caused by the fungal pathogen Leptosphaeria maculans Genome-wide association analysis on two oilseed rape panels confirmed that duplicated regions of ancestral blocks E and W were involved in resistance to stem canker The structural analysis of the duplicated genomic regions showed a higher gene density on the A genome than on the C genome and a better collinearity between homoeologous regions than paralogous regions The three ancestral sub-genomes were involved in the resistance to stem canker and the fractionation profile of the duplicated regions corresponded to what was expected from results on the B About 60% of the genes identified in these duplicated regions were single-copy genes while less than 5% were retained in all the duplicated copies of a given ancestral block Genes retained in several copies were mainly involved in response to stress Genes with resistance-associated markers were mainly retained in more than two copies These results suggested that some genes underlying quantitative resistance to stem canker might be duplicated genes Genes with a hydrolase activity that were retained in one copy or R-like genes might also account for resistance in some regions Further analyses need to be conducted to indicate to what extent duplicated genes contribute to the expression of the resistance phenotype Both expression bias and expression equivalence were observed at the transcriptomic level in these homoeologous regions: out of the 89 homoeologous gene pairs expressed in both regions 40% showed bias (higher transcript accumulation) toward the homoeologs on chromosome 10 To fully understand both the genetic control and the mechanisms underlying complex traits in polyploid plant species it is worthwhile to take into account the evolutionary processes through the dissection and comparison of the genomic regions underlying homoeologous duplicated QTL Comparative genomic analyses of homoeologous B napus regions involved in polygenic traits would then give the opportunity to study the impact of genome duplications on the structural and functional organization of such regions in a highly duplicated genome thaliana relatedness was used to explore gene ontology (GO) categories and the function of genes located in the duplicated regions According to the functions of the genes that were retained in all duplicated regions or were specific to some regions we discussed the putative functions associated to the stem canker quantitative resistance DNA was isolated from young leaves and DNA extracted using the DNeasy 96 Plant Kit (Qiagen DNA was quantified with the Quant-iT™ PicoGreen® Assay (Invitrogen using the Appliskan multiplate reader (Thermo Scientific Concentrations were adjusted to a minimum of 50 ng/μL and were submitted to a provider where the Infinium® assay was performed following the manufacturer's protocol (Illumina Inc. The automatic allele calling for each locus was accomplished using the Genome Studio software (Illumina Inc. The clusters were manually edited when necessary Technical replicates and signal intensities were controlled and only the most reliable calls were retained The proportion of false positive associations was controlled by a False Discovery Rate (FDR) test at 10% They included all the resistance-associated markers identified in all corresponding B For a visual overview of the synteny and the collinearity between the identified B. napus duplicated regions thus identified, we used the software MAUVE v2.3.1 (Darling et al., 2010) We took advantage of the close relatedness between B. napus and A. thaliana to identify the duplicated genes in the B. napus regions. The annotated B. napus genes (Chalhoub et al., 2014) were translated and aligned against the bank of A. thaliana proteins (https://www.arabidopsis.org/) napus genes showed the best matches to the same A thaliana protein then we considered that the two B napus genes were likely to be homoeologous (if one gene was on the A genome and the other on the C genome) or paralogous (if the two genes were on the same genome) We then looked at the distribution of the retained gene copies in the different duplicated regions in B thaliana genes can be present in one of the duplications in B genes can be present in one copy (single-copy) or in two or more copies (multi-copies) We deduced Gene Ontology (GO) categories from the A. thaliana best blast hits in order to functionally characterize and compare the duplicated B. napus regions between each other and with A. thaliana orthologous regions. The GO terms were obtained from the most recent release of predicted gene locus in the TAIR database (https://www.arabidopsis.org/) For GO term enrichment analysis, we used the online analysis tool “Singular Enrichment Analysis” (SEA) from AgriGO (http://bioinfo.cau.edu.cn/agriGO/analysis.php) A Fisher test with the Yekutielli multi-test correction method (significance threshold = 0.05) was used For comparison of GO terms of the genes present in 5–6 vs 1–2 duplicated regions of our studied five blocks we used a contingency Chi-2 test (α = 0.001) the previously identified flanking markers were mapped on our SNP map in order to deduce their putative position The results obtained confirmed that we have mainly assessed quantitative resistance in our panels all oilseed rape varieties in the experiment showed typical leaf lesions in the autumn indicating their lack of effective specific total resistance conferred by major genes that prevent leaf lesion development the varieties showed variability in stem canker resistance with DI ranging from 0.3 (resistant) to 8.4 (susceptible) Analysis of variance showed significant (P < 0.001) phenotypic variation among lines and replicates The proportion of total phenotypic variation which resulted from the genotypic variation within the oilseed rape collection was estimated at 0.94 Analysis of variance on the three control varieties did not show a significant genotype x replicate interaction (α = 0.05) The mean disease index of each variety (Table S1) was then used for GWA Association analyses were conducted on the two OSR panels with 13,973 informative SNP markers that were well distributed on our reference genetic map with an average density of 1 SNP every 0.15 centimorgans (cM) The average physical distance between two SNPs on the B napus reference sequence was estimated at 60 kb based on the physical position of the used SNP (Rousseau-Gueutin Linkage disequilibrium (LD) evaluated in the two panels extended up to 1.22 cM in the panel of 109 OSR and up to 1.37 cM in the panel of 170 OSR GWA analyses revealed 686 and 1019 markers significantly (p-value < 0.05) associated with resistance in the 109 and 170 OSR panels A set of 52 associated SNP markers was common to both panels After false discovery rate (FDR) correction no significant associated markers were detected in the 109 OSR panel and one associated marker located on linkage group (LG) A08 in block U was observed in the 170 OSR panel Out of the 686 and 1019 resistance-associated markers 505 (73%) and 467 (45%) were clearly anchored on one AK block (Table S3) The remaining markers were located in regions including several nested blocks or were putatively located on two different blocks We compared results obtained in the present two GWA analyses with those published by Fopa Fomeju et al. (2014), and we found 28 and 32 common regions containing resistance-associated markers between the three or two analyses, respectively (Table S3). Of these 60 regions, 27 (45%) corresponded to duplications of the AK blocks that were already identified by Fopa Fomeju et al. (2014) i.e. resistance-associated markers were identified on the six duplications of the block F In further analyses we focused on the AK blocks E as these were consistent between our different studies The A. thaliana gene intervals corresponding to the duplicated B. napus regions that carry resistance associated markers were delineated (Table 1) and their genomic sequence was extracted The alignment of the sequence of all the studied AK block intervals to the B napus sequence (Figure S1) revealed the expected number (six duplicated regions) and the expected location (according to the genetic map) of the duplications in the B The dotplots indicated that there was a good collinearity between A One small translocation appeared to be located on U block between chromosomes C3 and C8 napus regions indicated that there was globally a better collinearity between homoeologous regions than between paralogous regions (Figure S2) Interval definition of the five AK genomic blocks investigated in this study and U on the A genome and of block U on the C genome the regions with an intermediate number and the lowest number of conserved genes with A thaliana corresponded to the MF1 and MF2 sub-genomes For the duplicated regions of block W on the A genome and blocks E the differences between the number of conserved genes with A thaliana was not significant enough to distinguish between the MF1 and MF2 sub-genomes in these regions there were more tested SNPs in the regions located on the A than on the C genome There was no bias in the distribution of tested markers across the three sub-genomes The proportion of resistance-associated markers identified in the duplicated regions was similar on the A or C genomes except for a higher proportion observed on MF2 on A genome Structural organization of the duplicated regions of AK blocks E when gene copies were located in two duplicated regions one copy was on the A genome and the other was on the C genome (data not shown) a large majority of genes (85%) were maintained in one copy per duplicated region the proportion of genes in multiple copies is lower for genes that are located in one region compared with genes that are retained in several duplicated regions 7% of the genes retained in only one duplicate of block J were found in multiple copies within this duplicated region whereas 36% of the genes that were retained on the six duplicates of block J were in multiple copies on at least one of the six duplicated regions Distribution of genes in the studied duplicated regions in B Gene ontology terms associated to genes retained in 1–2 or 5–6 duplicated regions Gene ontology terms for biological process (A) and molecular activity (B) The three main molecular functions found were “hydrolase activity,” “nucleotide binding,” and “transferase activity.” These GO categories described genes located on five or six duplicated regions except for “hydrolase activity” which was preferentially identified for genes located in one duplicated region Gene ontology terms associated to SNP/genes associated to resistance to stem canker Out of the 425 NBS-LRR genes identified in the B. napus genome (Chalhoub et al., 2014) napus genes had a significant hit to NBS-LRR genes or resistance gene analogs (RGA) in A These were mostly distributed on duplicated regions corresponding to the blocks E Out of the 224 resistance-like genes (NBS-LRR and RGA) 21 were tested in the association study and four were significantly associated with resistance to stem canker (Table S2) Two were located on block E on LG A7 (LF sub-genome) and C2 and the two others on block U on LG C7 the Rlm3-Rlm4-Rlm7-Rlm9 cluster might be in the neighborhood of block E-LF sub-genome on chromosome A7 and Rlm1 was assigned to the studied region of block E-MF2 sub-genome on chromosome A7 The approximate mapping position of Rlm1 spread in a region of 1300 kb according to bridge SNP between linkage map and genomic sequence (Table S2) 22 resistance-associated markers have been tested and 12 were significantly associated to resistance to stem canker napus gene GSBRNA2G00135758001 which was located outside our investigated region on the block R on chromosome A10 and was not tested in association mapping In this study, we analyzed the structural and functional organization of duplicated regions involved in quantitative resistance of B. napus to stem canker. We took advantage of the recently released B. napus genome sequence (Chalhoub et al., 2014) for structural analyses and of the B thaliana relatedness for exploring GO categories and the putative function of genes located in the duplicated regions suggesting that fractionation might be ongoing in the B The proportions of resistance associated markers on the LF which suggests that all the subgenomes were involved in the control of the resistance trait This remains to be further studied since the number of associated markers identified does not provide information on the number of underlying causative genes nor on their contribution to the phenotype expression When considering gene distribution across the duplicated regions, we found that a large majority of genes (60%) were only present in one or two duplicated regions of the AK blocks and that these were mostly present in a single copy per region (on average, 85% of the genes). In general, genes present in two duplicated regions had one copy on the A genome and the other copy on the C genome. In a recent study, De Smet et al. (2013) estimated that 66% of the B This is consistent with our findings suggesting that these single-copy genes have not resulted from the allotetraploidization event but rather from the long-term evolution of diploid ancestors Twenty-two of the genes retained in five or six duplicated regions were associated to resistance and were related to response to stress and transferase activity we could not conclude on the involvement of duplicated genes in stem canker resistance due to the fact that only one of the duplicated copies was tested for 77% of the duplicated genes This has to be further analyzed in oilseed rape by investigating whether all duplicated copies are expressed and equally contribute to phenotype expression and if they have undergone sub-functionalization which appears to be a major force in maintaining genes in a duplicated state structural and functional sequence analyses CF carried out genetic mapping and BLAST analyses for the construction of the integrated map used in this study and the positioning of duplicated blocks on this map GL contributed to the structural and BLAST analyses RD and MM helped analyse the results and coordinated the project BF was a PhD student funded by INRA (Institut National de la Recherche Agronomique Département de Biologie et Amélioration des Plantes) and the Région Bretagne Cécile Baron and Laure Berton for providing the genotyping data obtained on the 170 OSR panel with the Brassica 60K SNP BeadChip Array in the RAPSODYN project We acknowledge the Genetic Resource Center (BrACySol We thank the team of the INRA Experimental Unit at Le Rheu for performing the disease evaluation trials The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2015.00772 Multiple flowering time QTLs within several Brassica species could be the result of duplicated copies of one ancestral gene Methods for sampling and assessment in relation to the spatial pattern of phoma stem canker (Leptosphaeria maculans) in oilseed rape The dispensable chromosome of Leptosphaeria maculans shelters an effector gene conferring avirulence towards Brassica rapa Genomic expression dominance in the natural allopolyploid Coffea arabica is massively affected by growth temperature Functional divergence of duplicated genes formed by polyploidy during Arabidopsis evolution Quantitative resistance increases the durability of qualitative resistance to Leptosphaeria maculans in Brassica napus A field method for evaluating the potential durability of new resistance sources: application to the Leptosphaeria maculans/Brassica napus pathosystem Identification of candidate genes of QTLs for seed weight in Brassica napus through comparative mapping among Arabidopsis and Brassica species Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome Detection of QTL for six yield-related traits in oilseed rape (Brassica napus) using DH and immortalized F-2 populations Deciphering the diploid ancestral genome of the mesohexaploid Brassica rapa Biased gene fractionation and dominant gene expression among the subgenomes of Brassica rapa Comparative analysis between homoeologous genome segments of Brassica napus and its progenitor species reveals extensive sequence-level divergence Homeologous gene expression in response to growing temperature in a recent allopolyploid (Coffea arabica L.) progressiveMauve: multiple genome alignment with gene gain Convergent gene loss following gene and genome duplications creates single-copy families in flowering plants A cluster of major specific resistance genes to Leptosphaeria maculans in Brassica napus Major gene and polygenic resistance to Leptosphaeria maculans in oilseed rape (Brassica napus) High-density SNP-based genetic map development and linkage disequilibroum assessment in Brassica napus L Evolutionary genetics of genome merger and doubling in plants doi: 10.1146/annurev.genet.42.110807.091524 Gene and genome duplications: the impact of dosage-sensitivity on the fate of nuclear genes World-wide importance of phoma stem canker (Leptosphaeria maculans and L biglobosa) on oilseed rape (Brassica napus) Homoeologous duplicated regions are involved in quantitative resistance of Brassica napus to stem canker The evolutionary position of subfunctionalization Bias in plant gene content following different sorts of duplication: tandem doi: 10.1146/annurev.arplant.043008.092122 Characterization of a tobacco epoxide hydrolase gene induced during the resistance response to TMV and utilization of single copy genes in 29 angiosperm genomes Genomic advances will herald new insights into the Brassica: Leptosphaeria maculans pathosystem Homoeologous loci control the accumulation of seed glucosinolates in oilseed rape (Brassica napus) Chromosomal and gene expression changes in Brassica Allopolyploids Association mapping of quantitative resistance for Leptosphaeria maculans in oilseed rape (Brassica napus L.) Assessment of a new strategy for selective phenotyping applied to complex traits in Brassica napus Connected populations for detecting quantitative resistance factors to Phoma stem canker in oilseed rape (Brassica napus L.) 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Differential formation of octadecadienoic acid and octadecatrienoic acid products in control and injured/infected potato tubers Meta-analysis of polyploid cotton QTL shows unequal contributions of subgenomes to a complex network of genes and gene clusters implicated in lint fiber development Screening and identification of resistance to Leptosphaeria maculans (stem canker) in Brassica napus accessions Capturing sequence variation among flowering-time regulatory gene homologs in the allopolyploid crop species Brassica napus Differentiation of the maize subgenomes by genome dominance and both ancient and ongoing gene loss The ABC's of comparative genomics in the Brassicaceae: building blocks of crucifer genomes Independent ancient polyploidy events in the sister families Brassicaceae and Cleomaceae Changes in twelve homoeologous genomic regions in soybean following three rounds of polyploidy Altered patterns of fractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Following tetraploidy in an Arabidopsis ancestor genes were removed preferentially from one homeolog leaving clusters enriched in dose-sensitive genes Genome analysis in Brassica with special reference to the experimental formation of B Dual control of avirulence in Leptosphaeria maculans towards a Brassica napus cultivar with ‘sylvestris-derived’ resistance suggests involvement of two resistance genes The evolution of Brassica napus FLOWERING LOCUST paralogues in the context of inverted chromosomal duplication blocks The genome of the mesopolyploid crop species Brassica rapa Genome and gene duplications and gene expression divergence: a view from plants Quantitative trait loci analyses for resistance to Sclerotinia sclerotiorum and flowering time in Brassica napus Epidemiology and management of Leptosphaeria maculans (phoma stem canker) on oilseed rape in Australia and gene regulatory consequences of genome dominance in polyploids (vol 111 CrossRef Full Text Loss/retention and evolution of NBS-encoding genes upon whole genome triplication of Brassica rapa Homoeolog expression bias and expression level dominance in allopolyploid cotton Identification of two novel genes for blackleg resistance in Brassica napus Identification and mapping of a third blackleg resistance locus in Brassica napus derived from B Identification and mapping of a novel blackleg resistance locus LepR4 in the progenies from Brassica napus x B Quantitative trait loci for resistance to Sclerotinia sclerotiorum and its association with a homeologous non-reciprocal transposition in Brassica napus L Comparative Analysis of FLC Homologues in Brassicaceae Provides Insight into Their Role in the Evolution of Oilseed Rape Manzanares-Dauleux MJ and Delourme R (2015) Comparative genomic analysis of duplicated homoeologous regions involved in the resistance of Brassica napus to stem canker Received: 15 June 2015; Accepted: 08 September 2015; Published: 25 September 2015 Copyright © 2015 Fopa Fomeju, Falentin, Lassalle, Manzanares-Dauleux and Delourme. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) provided the original author(s) or licensor are credited and that the original publication in this journal is cited *Correspondence: Régine Delourme, Institut National de la Recherche Agronomique, UMR IGEPP, BP35327, 35653 Le Rheu, France,cmVnaW5lLmRlbG91cm1lQHJlbm5lcy5pbnJhLmZy Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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. 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish. Volume 9 - 2021 | https://doi.org/10.3389/fevo.2021.618331 This article is part of the Research TopicSymbiotic Relationships as Shapers of BiodiversityView all 14 articles the influence of microbial symbionts on ecological and physiological traits of their hosts has been increasingly recognized most of these effects have been revealed under laboratory conditions which oversimplifies the complexity of the factors involved in the dynamics of symbiotic associations in nature which strongly differ in the prevalence of their facultative endosymbionts Some of the facultative endosymbionts of A pisum have been shown to confer protection against natural enemies among which Hamiltonella defensa is known to protect its host from parasitoid wasps we tested under natural conditions whether the endosymbiont communities of different A pisum biotypes had a protective effect on their hosts and whether endosymbiotic associations and parasitoid communities associated with the pea aphid complex were linked A space-time monitoring of symbiotic associations parasitoid pressure and parasitoid communities was carried out in three A pisum biotypes respectively specialized on Medicago sativa (alfalfa) (clover) throughout the whole cropping season parasitoid communities were stable over time and structured mainly by the A the parasitoid pressure strongly varied during the season and differed among the three biotypes This suggests a limited influence of parasitoid pressure on the dynamics of facultative endosymbionts at a seasonal scale we found a positive correlation between the α and β diversities of the endosymbiont and parasitoid communities indicating interactions between these two guilds we revealed a negative correlation between the prevalence of H defensa and Fukatsuia symbiotica in co-infection and the intensity of parasitoid pressure in the alfalfa biotype confirming in field conditions the protective effect of this symbiotic combination it was discovered that symbiont diversity could be driven by parasitoid diversity suggesting potential reciprocal influence of these two communities through their shared aphid hosts we examined in a subsample of our aphid collections whether variation in the strain of the protective endosymbiont H defensa was related to the intensity of parasitism pressures one sampling refers to an insects’ collection made at one date in one field of a given legume species alive pea aphid adults and nymphs were collected using beat sampling from plants separated by approximately 5 m to cover a larger spatial/genetic diversity at the field scale a dead aphid containing an immature parasitoid) were also collected by view in order to extract parasitoid specimens from field To estimate the diversity and structure of parasitoid communities, we considered parasitoid specimens emerging from pea aphid mummies collected from the fields or obtained in laboratory. Those parasitoid specimens were determined at species level (when possible) based on morphological criteria (Müller et al., 1999; Starý, 2006) The present study excluded all hyperparasitoids from the analyses (258 specimens were found) we determined 744 primary parasitoids from alfalfa which have confirmed the presence of seven facultative endosymbionts considered in our paper we are confident that we have surveyed the main fraction of the pea aphid symbionts we thus estimated the prevalence of these seven facultative heritable endosymbionts and their distribution among aphid individuals The endosymbiont infection status of each aphid individual (i.e. identity and number of endosymbionts) was then determined For each sample and each gene (accD and murE) sequences were used to build a phylogenetic tree using the Neighbor Joining method (Tamura-Nei distance) Bootstrap values were computed for each branch node (N = 1,000) Both the field ID and session number were fit as random factor to include data substructure For each combination of parasitism period and pea aphid biotype, the occurrence and co-occurrence of parasitoid species and facultative endosymbionts were visualized using the R package Mondrian (Siberchicot et al., 2016) For each combination biotype/session (i.e. we pooled the data obtained in the fields of a given legume crop during one sampling session) the α-diversity of parasitoid communities was estimated using the Shannon Index that accounts for both abundance and evenness of the species we calculated the α-diversity of the aphid endosymbiont infection statuses using the Shannon Index by considering the relative abundance of each infection status found in a given biotype/session combination we tested whether the parasitism period (i.e. three levels factor: “pre-parasitism,” “maximal parasitism,” and “post-parasitism”) or the pea aphid biotype (i.e. three levels factor: “alfalfa,” “clover,” and “pea” biotypes) affected the Shannon index of parasitoid communities or endosymbiont infection statuses of aphids to test whether α-diversities of both parasitoid communities and aphid endosymbiont infection statuses were linked a Pearson correlation coefficient between their Shannon indexes was calculated Model assumptions were verified by plotting residuals versus fitted values for each model To analyze the effect of parasitism pressure on the H defensa strain found before and after the parasitism peak in alfalfa fields using a χ2 test the rates of parasitism were the highest in clover fields (30.3%) and the lowest in alfalfa (15.9%) (GLMM: χ2 = 10.26 Temporal variation in parasitism pressure in alfalfa and pea crops estimated using two proxies: parasitism rate (black dots —) and the ratio of the number of parasitoids/pea aphid individuals (white dots The three shaded areas within each graph correspond to the three parasitism periods arbitrarily defined: “pre-parasitism,” “maximum parasitism,” and “post-parasitism” periods reaching 82% of parasitoids in post-parasitism period ervi as dominant species in the guild and leading to a decline in the Shannon index at this period Frequencies of the parasitoid species in the different pea aphid biotypes (“alfalfa,” “clover,” and “pea”) at different parasitism period (“pre-parasitism,” “maximal parasitism,” and “post-parasitism”) Shannon Index (H) of parasitoid communities and endosymbiont infection statuses and covariation between both Shannon Indices considering both the pea aphid biotypes and the parasitism periods The diversity and relative abundance of endosymbiont infection statuses of pea aphids in relation with aphid biotype (“alfalfa,” “clover,” and “pea”) and parasitism period (“pre-parasitism,” “maximal parasitism,” and “post-parasitism”) the colored area corresponds to the percentage of aphid individuals harboring the corresponding symbiont species When colored areas are present in several columns it indicates a co-infection with multiple symbiont species (Spi: Spiroplasma sp.; Reg: Regiella insecticola; Ham: Hamiltonella defensa; Rica: Rickettsiella viridis; Ser: Serratia symbiotica; Rick: Rickettsia sp.; Fuk: Fukatsuia symbiotica) When we analyzed the relationship between the Shannon Index (H) of parasitoid communities and the Shannon Index (H) of endosymbiont infection statuses of aphids, we found a significantly positive covariation (Pearson correlation, r = 0.499, p = 0.004) in α-diversities (Figure 3) When the Bray–Curtis distances were calculated between all pairwise combinations of parasitoid communities, we found that they varied significantly in composition between A. pisum biotypes (Figure 5) PERMANOVA detected a significant effect of aphid biotype on the parasitoid community assemblages (F = 3.84 p = 0.003) and this factor accounted for 21% of the variance in the data Pairwise comparisons between biotypes showed that the structure of parasitoid communities emerging from pea crops differed from the two other crops No temporal dynamics in β-diversity in parasitoid communities was found (PERMANOVA Non-metric MDS ordination plot comparing (A) endosymbiont infection statuses and (B) parasitoid communities from different pea aphid samples Each data point represents the (A) symbiont/(B) parasitoid community identified from aphid samples collected at one date for a given biotype The shape of the dots refers to the parasitism periods The Bray-Curtis dissimilarity index was used to rank distances calculated using the abundance community data Stress of the nMDS: (A) symbiont infection status = 0.083; (B) parasitoid community = 0.164 The Bray–Curtis dissimilarities between endosymbiont infection statuses of aphids presented highly contrasted values and showed that they differed strongly between biotypes while being stable during the season within each biotype (Figure 5) PERMANOVA confirmed this pattern since the biotype effect on infection statuses dissimilarity was highly significant (F = 79.08 This factor accounted for 83% of the variance in the data No temporal dynamics in β-diversity of endosymbiont infection statuses of aphids was found (PERMANOVA confirming their stability throughout the cropping season A positive correlation between the two dissimilarity matrices was found (Mantel test: zM = 0.186, p = 0.002, see Supplementary Figure 2) The dissimilarity between parasitoid communities was therefore correlated with the dissimilarity between endosymbiont infection statuses of aphids Results of statistical models (LMM: General Linear Mixed Models; GAMM: Generalized Additive Mixed Models) testing the effect of the most prevalent symbiotic associations on parasitism rate (for the dominant parasitoid species and all parasitoids) Relationship between Hamiltonella defensa/Fukatsuia symbiotica coinfection and parasitism rate (assessed for the dominant parasitoid species and for all parasitoids) in pea aphids from alfalfa fields Solid line represents the predicted values from the fitted generalized additive mixed models dominated aphid populations before and after parasitism peak A greater diversity of strains was observed after the peak of parasitism (0.11 before and 0.19 after) and a similar pattern was observed for the aphid clonal diversity (0.58 before and 0.64 after) defensa strains did not differ significantly before and after the parasitism peak in alfalfa fields (χ2 test: χ2 = 6.65 defensa strain diversity and haplotypes in the “pre-parasitism” and “post-parasitism” periods for pea aphids collected in alfalfa fields Since some microbial symbionts confer a protection against natural enemies to their hosts that can potentially alter food web interactions (Hafer and Vorburger, 2019; Mclean, 2019) our objectives were to test under natural conditions whether the parasitism rate of different A pisum biotypes depended on their endosymbiont communities and whether symbiont communities and parasitoid communities associated with pea aphids were related We found that facultative endosymbiont communities were highly structured by biotype and stable in time while the parasitoid communities showed moderate differences between pea aphid biotypes and some change in structure over time we revealed a correlation between diversities (i.e. α- and β-diversities) of endosymbiont infection statuses of aphids and parasitoid communities we found a negative correlation between the prevalence of H symbiotica in co-infection and the intensity of parasitoid pressure in the alfalfa biotype An essential step for linking facultative endosymbionts with parasitoid communities was to reliably assess the parasitism pressure exerted on aphid populations. Here, we used two proxies to reduce the bias of underestimating the parasitoid pressure by measuring only the mummification rate (Oliver et al., 2003) both parasitoid pressure proxies were highly positively correlated and showed the same temporal dynamics indicating that the actual parasitoid pressure exerted on field populations of the pea aphid in different ecological situations was reliably assessed the parasitism pressure over time presented a single peak of high parasitoids’ activity in early July This temporal dynamic was similar between alfalfa clover and pea biotypes but varied quantitatively: the pressure exerted by parasitoids was the highest in clover intermediate in pea and the lowest in alfalfa crops One explanation for these quantitative differences could be that clover is typically grown in a more complex agricultural mosaic than the other crops An alternative could be that clover is more attractive to parasitoids Our study and the previous ones thus confirm the effectiveness of symbiont-mediated protection in natural environments exploited by complex and diverse parasitoid communities More field works are needed to assess the influence of environmental factors on symbiont composition of the various pea aphid biotypes Two earlier field studies showed that protective symbionts could influence the third trophic level by shaping the structure of parasitoid community attacking aphid populations (Hrcek et al., 2016; Rothacher et al., 2016). In addition, an experimental evolution study showed that parasitoid diversity could maintain diversity in protective symbionts (Hafer-Hahmann and Vorburger, 2020) Our study is in line with this body of work linking symbiont and parasitoid diversities we found that both α- and β-diversities of symbionts and parasitoids were correlated suggesting some interactions between these two communities through their aphid hosts or other environmental factors (i.e. this endosymbiont was not present in the pea biotype in our survey and it is not known whether S More work is needed to test whether the most prevalent symbiotic combination in each biotype confers an optimal protection to the corresponding parasitoid communities We also showed that the three pea aphid biotypes despite their distinct endosymbiont composition were exposed to similar range of parasitism pressures suggesting that other protective alternatives than hosting H may be used by the various biotypes of the pea aphid complex we detected a link between communities of parasitoids and symbionts suggesting interactions through shared resources or other environmental filters The study of other communities of natural enemies of aphids (e.g. pathogens) could reveal more such links and allow to better measure the importance of symbionts in food webs The datasets generated and analyzed in this article are available in Zenodo at https://doi.org/10.5281/zenodo.4548282 and YO conceived and designed the field works and experiments and managed the field samplings FM and ML performed both molecular studies and symbiont detection YO and J-CS contributed to the supervision of this study All authors contributed critically to the drafts and gave final approval for publication We are very grateful to all people who helped in aphid sampling to Anne-Lise Boixel and Antoine Briand for their strong investment in field surveys and laboratory works during summer 2014 and to Bernard Chaubet for his help in parasitoid species 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Yannick Outreman, eWFubmljay5vdXRyZW1hbkBhZ3JvY2FtcHVzLW91ZXN0LmZy †These authors have contributed equally to this work ‡ORCID: Jean-Christophe Simon, orcid.org/0000-0003-0620-5835; Yannick Outreman, orcid.org/0000-0002-7337-3049 Volume 6 - 2015 | https://doi.org/10.3389/fpls.2015.00539 This article is part of the Research TopicAdvances in plant microbe interactionsView all 30 articles Camalexin has been reported to play defensive functions against several pathogens in Arabidopsis we investigated the possible role of camalexin accumulation in two Arabidopsis genotypes with different levels of basal resistance to the compatible eH strain of the clubroot agent Plasmodiophora brassicae Camalexin biosynthesis was induced in infected roots of both Col-0 (susceptible) and Bur-0 (partially resistant) accessions during the secondary phase of infection the level of accumulation was four-to-seven times higher in Bur-0 than Col-0 This was associated with the enhanced transcription of a set of camalexin biosynthetic P450 genes in Bur-0: CYP71A13 brassicae growth in Bur-0 compared to Col-0 thus suggesting a relationship between the levels of camalexin biosynthesis and the different levels of resistance Clubroot-triggered biosynthesis of camalexin may also participate in basal defense in Col-0 as gall symptoms and pathogen development were enhanced in the pad3 mutant (Col-0 genetic background) which is defective in camalexin biosynthesis Clubroot and camalexin responses were then studied in Heterogeneous Inbred Families (HIF) lines derived from a cross between Bur-0 and Col-0 The Bur/Col allelic substitution in the region of the previously identified clubroot resistance QTL PbAt5.2 (Chromosome 5) was associated with both the enhanced clubroot-triggered induction of camalexin biosynthesis and the reduced P our results suggest that high levels of clubroot-triggered camalexin biosynthesis play a role in the quantitative control of partial resistance of Arabidopsis to clubroot In a preliminary screen to identify defense response patterns triggered by clubroot infection we also observed that one of the most prominent features of the Bur-0 response to clubroot is high-levels of camalexin (data not published) Figure 1. Camalexin biosynthesis pathway according to Millet et al. (2010) and Geu-Flores et al. (2011) Camalexin is derived from tryptophan which is firstly converted to indole 3 acetaldoxime (IAOx) by the cytochrome P450 enzymes CYP79B2 and CYP79B3 IAOx is then converted to indole 3 acetonitrile (IAN) by the CYP71A12 and CYP71A13 enzymes IAN is converted to the Cys(IAN) conjugate after intermediate steps (represented in dashed arrows) The final two steps in camalexin biosynthesis are catalyzed by the P450 enzyme CYP71B15/PAD3 Clubroot-induced camalexin accumulation was previously reported in Col-0 and several other Arabidopsis accessions (Siemens et al., 2008) The absence of enhanced clubroot susceptibility in the pad3 mutant led the authors to conclude that camalexin was not likely to play a role in clubroot resistance our preliminary assays indicated that camalexin levels accumulate at high levels in the partially resistant accession Bur-0 compared to the susceptible accession Col-0 we carried out an in depth investigation of the role of camalexin in the defense response toward P We first evaluated the time-course of camalexin accumulation and camalexin biosynthesis gene expression during clubroot infection in Bur-0 and Col-0 We also followed pathogen growth dynamics using a combination of histological and PCR-based pathogen quantification the time-course of camalexin accumulation and post-invasive partial resistance establishment could be compared The role of clubroot-triggered camalexin biosynthesis in Col-0 was reassessed by phenotyping the pad3 mutant challenged with the eH isolate we tested possible genetic links between two major partial resistance QTL from Bur-0 and the intensity of the clubroot-induced camalexin response camalexin accumulation and camalexin-biosynthesis gene transcription was evaluated in pairs of appropriate near isogenic Heterogeneous Inbred Lines (HIF) developed from the Bur-0 × Col-0 cross The inoculum used in all the clubroot tests was the “selected” eH isolate (Fähling et al., 2003) which belongs to the pathotype P1 according to Somé et al. (1996). The host differential set described by Somé et al. (1996) was included in each test as a control Heterogeneous Inbred Families (HIF) pairs were obtained from the Versailles Arabidopsis Stock Centre (publiclines.versailles.inra.fr). The HIFs were derived from RIL lines described in Simon et al. (2008) The HIF pair 499 shows polymorphism at the QTL PbAt5.2 harboring either the Col-0 susceptibility or the Bur-0 resistance allele This HIF pair harbors the Bur-0 resistance allele at the QTL PbAt1 The HIF pair 508 shows polymorphism at the QTL PbAt1 This HIF pair harbors the Bur-0 resistance allele at the QTL PbAt5.2 (Supplementary Figures S2 Erich Glawischnig (Technische Universität München) kindly provided the seeds of the camalexin deficient homozygous phytoalexin deficient 3 (pad3) T-DNA mutant (SALK_026585) 3 cm of roots was collected from all plants frozen in liquid nitrogen and stored at −80°C for molecular and biochemical analysis The primer sets used were as follows: Pb F 5′-AAACAACGAGTC AGCTTGAATGC-3′; Pb R 5′-AGGACTTGGCT GCGGATCAC-3′; F-Box F 5′-TTTCGGCTGAGA GGTTCGAGT-3′; F-Box R 5′-GATTCCAAGACGT AAAGCAGATCAA -3′ Quantitative PCR reactions were carried out with 50 cycles of denaturation at 95°C for 15 s and annealing/extension at 61°C for 30 s Amplification specificity was assessed by both melt curve analyses and agarose gel electrophoresis Four biological replicates were analyzed for each time point The results were expressed as the ratio between the DNA quantities of P brassicae and the corresponding plant genotype DNA multiplied by 100 Infected and non-infected roots collected at 17 dpi were fixed in a glutaraldehyde (2%) and paraformaldehyde (1%) phosphate buffer (0.1 M pH 7.2) then washed with phosphate buffer (0.1 M pH 7.2) and distilled water dehydrated in different ethanol:water solutions (10 and 100%) and finally embedded in resin with the Technovit 7100 kit (Heraeus Kulzer) 4 μm thick histological sections were cut with a microtome (Microm Microtech) and stained in cotton blue (1%) and safranin (1%) to differentiate the pathogen plasmodia and the root plant cells respectively during microscopic investigations The impact of the infection on xylem vessel upkeep was visualized with an epifluorescence Nikon Eclipse E200 microscope (BP 365 nm LP400 nm) after staining the sections with aniline blue (0.5%) dissolved in lactophenol Amplification reactions were carried out with 50 cycles of denaturation at 95°C for 15 s annealing/extension at 60°C for 30 and 72°C for 30 s CP values were obtained for each gene studied and converted to arbitrary units The final results were expressed as the ratio between the gene of interest and the housekeeping gene in arbitrary units Two technical and three biological replicates were analyzed The accumulation of camalexin in infected and non-infected roots of each genotype was determined at 10 camalexin was extracted from approximately 200 mg of freshly ground roots in 1.5 mL tubes After addition of 1 mL of a methanol:water:formic acid (80:19:1) (v:v:v) mixture solvent tubes were ultrasonicated and agitated at room temperature for 30 min The tubes were then centrifuged at 1200 g for 10 min and the supernatants were removed into new 1.5 mL tubes The pellets were re-extracted with 1 ml of the extraction solvent and the supernatants were pooled with those from the first extraction and dried in a speed vacuum centrifuge Dried residues were then resuspended in 100 μl of acidified methanol and 5 μl were injected and analyzed on an Acquity UPLC system (Waters) coupled to a Quattro Premier XE equipped with an electrospray ionization (ESI) source Chromatographic separation was performed on an Acquity HSS C18 T3 1.8 μm (2.1 × 150 mm) column using a gradient of two mobile phases corresponding to an A solution (0.1% of formic acid and water) and B solution (0.1% of formic acid and methanol) The elution gradient started with 99% of A and 1% of B then 20 min later 100% of B and returned to the initial conditions 25 min after the start of the elution This separation step was at 40°C with a flow rate of 0.35 ml min−1 and the retention time of the camalexin was determined at 12.77 min The eluted camalexin was ionized in negative mode at the ESI source of the mass spectrometer and fragmented at 40 V Data were acquired in Multi Reaction Monitoring (MRM) mode with Masslynx software and results were expressed by reporting MS peak areas of the corresponding camalexin concentration in ng mL−1 determined using a camalexin standard (kindly provided by Pr P Statistical analyses were performed with R software by using Wald tests applied on Linear Mixed Models (function “lme,” package “nlme”) the kinetic time point of sampling and the inoculation as fixed factors and biological replicates as random factors pairwise comparisons of Least Squares Means were computed (function “lsmeans,” package “lsmeans”) The alpha level was set at a standard level of 5% the camalexin content showed a weak increase in infected roots of both plant genotypes with no significant differences between Col-0 and Bur-0 at this time point (P = 0.060) the camalexin content in infected roots increased in both genotypes and was seven times higher in the partial resistant Bur-0 genotype than in the susceptible Col-0 the camalexin content was again enhanced in the infected roots of both genotypes and reached more than four times higher levels in Bur-0 than in Col-0 Camalexin content in infected (continuous lines) and non-infected (dashed lines) roots of the partially resistant accession Bur-0 and the susceptible accession Col-0 at 10 Camalexin was quantified in root methanol extracts using UPLC-MS/MS and is expressed as ng g−1 of fresh weight Error bars represent standard error (three biological replicates 12–54 plants analyzed per biological replicate) Asterisks indicate statistically significant differences according to the Wald tests applied on a linear mixed model (P < 0.05) and (C) PAD3 in infected (black bars) and non-infected roots (white bars) of the partially resistant accession Bur-0 and the susceptible accession Col-0 at 10 Expression levels were normalized using the reference gene PP2A Error bars represent standard error (four biological replicates both symptom severity and pathogen content in infected roots were enhanced in pad3 compared to the wild type Col-0 This suggests that the camalexin response does contribute to a late and weak basal control of clubroot symptoms and pathogen development in Col-0 (A) Clubroot symptoms and (B) quantification of Plasmodiophora brassicae DNA in infected roots of the clubroot susceptible WT Col-0 and pad3 (A) Clubroot symptoms were evaluated using the GA/LA disease index calculated by image analysis at 21 dpi GA/LA is the ratio between gall area (GA in cm2) and an estimation of the rosette extent (LA in cm2) Error bars represent standard error (Four biological replicates (B) Pathogen DNA quantification (Pb) by qPCR expressed as a ratio relative to the expression level of the plant Fbox gene quantification of Plasmodiophora brassicae DNA in infected roots of the partially resistant accession Bur-0 and the susceptible accession Col-0 (A) Clubroot symptoms were evaluated using the GA/LA disease index calculated by image analysis at 17 and 21 dpi GA/LA was calculated from gall area (GA in cm2) divided by an estimation of the rosette extent (LA in cm2) Error bars represent standard error (Four biological replicate 18 plants analyzed per biological replicate) Asterisks indicate statistically significant differences according to the (P < 0.05) (B) Illustration of clubroot symptoms (C) Pathogen DNA quantification (Pb) by qPCR expressed as a ratio relative to the expression level of the plant Fbox gene at 10 12–54 plants per biological replicate) (A) Safranin and blue cotton and (B) aniline blue stained radial sections of roots infected by Plasmodiophora brassicae in the partially resistant accession Bur-0 and the susceptible accession Col-0 then fixed and immobilized in resin as described in Materials and Methods part Histological sections were cut with a microtome and stained in cotton blue and safranin to visualize pathogen plasmodia (colored in blue in A) and vascular vessels colored by aniline blue in (B) respectively White arrows indicate plasmodia structures in infected cells the image shown is representative of the observations performed on at least six independent root samples We previously showed that QTL PbAt1 and PbAt5.2 are the two genetic regions which mainly contribute to the quantitative partial resistance in Bur-0 (Jubault et al., 2008) The objective was then to establish whether the presence of the Bur-0 allele in the PbAt1 and PbAt5.2 regions is associated with the high levels of camalexin accumulation in Bur-0 We used two pairs of Heterogeneous Inbred Families (HIF) lines derived from the Bur-0 × Col-0 RIL lines 499 and 508 (cf Materials and Methods) The HIF paired lines 499-Col and 499-Bur have an identical homozygous genetic background issued from the recombination between the Col-0 and Bur-0 genomes (Supplementary Figure S2) except in the genomic region between markers c5_14766 and c5_21319 which comprise the confidence interval of QTL PbAt5.2 and 499-Bur carry either the Col-0 or Bur-0 alleles in the homozygous state Comparison of the phenotypic behaviors of 499-Col and 499-Bur near isogenic lines thus allowed the effect of the Bur-0/Col-0 allelic variation at QTL PbAt5.2 to be tested have the same homozygous recombinant genetic background (different from the genetic background of the 499 line) derived from both the Col-0 and Bur-0 genomes and differ only from each other in the QTL PbAt1 region between markers c1_00593 and c1_08385 (Supplementary Figure S3) Comparison of the phenotypic behaviors of 508-Col and 508-Bur allowed the phenotypic consequences of the Bur-0/Col-0 allelic variation at the QTL PbAt1 to be evaluated (Supplementary Figure S3) suggesting that different genetic factors may control gall development and pathogen growth Clubroot symptoms and accumulation of DNA from Plasmodiophora brassicae in infected roots of the HIFs 499 and 508 499-Bur and 499-Col harbor the Bur and Col alleles 508-Bur and 508-Col harbor the Bur and Col alleles (A,B) Clubroot symptoms evaluated using the GA/LA disease index from image analysis as described in the Materials and Methods Section Error bars represent standard error (Four independent biological replicates (C,D) Pathogen DNA quantification (Pb) by qPCR after normalization with the Fbox gene from Arabidopsis (A,B) Camalexin content in infected (continuous lines) and non-infected (dashed lines) roots of the HIFs 499 (A) and 508 (B) at 10 499-Bur and 499-Col harbor the Bur-0 and Col-0 alleles 508-Bur and 508-Col harbor the Bur-0 and Col-0 alleles and is expressed as ng g−1 of the fresh weight Error bars represents standard error (Three biological replicates Asterisks represent statistically significant differences according to the Wald tests applied on a linear mixed model (P < 0.05) in the genetic background of the 499 HIF lines allelic variation at QTL PbAt5.2 was linked to both clubroot-induced biosynthesis of camalexin and transcriptional regulation of CYP71A13 and PAD3 PAD3 in infected (black bars) and non-infected roots (white bars) of the HIF 499 at 10 499-Bur and 499-Col harbors the Bur-0 and Col-0 allele Error bars represent standard error (Four independent replicates A preliminary screen for contrasted biochemical defense responses to clubroot between the Bur-0 and Col-0 accessions highlighted the defense-related compound camalexin as a promising marker associated with partial resistance The initial aim of this study was to clarify the extent to which the induction of camalexin contributes to the different degrees of basal/partial resistance to clubroot in Col-0 and Bur-0 and could explain the differences in the results that genetic resistance factors often inhibit plasmodia development during the secondary rather than the primary infection the setup of partial resistance correlated with the camalexin response enhanced induction of camalexin biosynthesis in Bur-0 appears to contribute to its partial post-invasive resistance Several reports associated induction of camalexin biosynthesis with the transcriptional induction of a set of genes encoding key enzymes in the pathway. For example, Millet et al. (2010) showed that PAD3 and CYP71A12 expression increased in response to Flg22 treatment in Arabidopsis and CYP71A12 were significantly more induced during the secondary phase of infection in Bur-0 compared to Col-0 enhanced camalexin biosynthesis in Bur-0 appears to be controlled at the transcriptional level through the induction of several camalexin biosynthetic enzymes The induction of CYP71A13 in infected Bur-0 roots was interesting as it occurred as early as 10 dpi and was sustained all along the secondary phase of infection This suggests that the IAN biosynthetic step plays a prominent role in the regulation of camalexin biosynthesis during clubroot infection we can speculate that the translation of the PAD3 transcript or PAD3 protein stability is higher in Bur-0 thus explaining why low levels of PAD3 transcription do not impair the biosynthetic flux toward camalexin The introgression of a pad3 mutation into the Bur-0 genetic background could be of great interest to solve this tricky question Our data clearly demonstrated that the Bur/Col allelic substitution in the PbAt5.2 region (chromosome 5) drove the level of clubroot-induced camalexin biosynthesis (including the induction of key genes involved in the camalexin biosynthetic pathway) and contributed to partial inhibition of P Together with other features discussed above (enhanced symptoms observed in the pad3 mutant and paralleled kinetics of partial resistance and camalexin biosynthesis in Bur-0) these results give additional support to a model where camalexin levels are related to the quantitative control of clubroot infection Our findings provide an additional example of the role of camalexin in post-invasive pathogen inhibition in the context of partial quantitative resistance in the root to a compatible isolate of an obligatory biotrophic pathogen The confidence interval of PbAt5.2 is bordered by the genes At5G46260 and At5G47690 The interval includes several plant defense-related genes but no single gene (such as PAD3 or CYP71A13) that would be directly linked with the camalexin biosynthesis pathway Gene regulation in the HIF pair 499-Bur/499-Col suggests instead that the allelic variation in the PbAt5.2 region is associated with a signaling process potentially controlling the expression of genes involved in all steps of the camalexin biosynthetic pathway Substantial additional studies are now needed to identify the nature of the causal nucleotide variation(s) underlying QTL PbAt5.2 and to clarify which molecular mechanisms are driving the modulation of clubroot-triggered camalexin biosynthesis MM contributed to initial hypotheses and to the design of the whole project and MJ contributed to the design and the conduct of clubroot assays and samplings NM developed the analytical method for the quantification of camalexin and NM contributed to all metabolite extractions and biochemical analyses SL and JL performed PCR and RT-qPCR analyses All co-authors contributed to the interpretation of data This work was supported by the CETIOM (The Technical Center for Oilseed Crops and Industrial Hemp) SL is a PhD student funded by a CJS grant of the National Institute for Agronomic Research (INRA) AR is supported by a Marie Curie FP7 fellowship Erich Glawischnig for providing us the camalexin standard for UPLC quantification and seeds of pad3 We express our thanks to Pascal Glory for technical support The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2015.00539 Exploiting natural genetic diversity and mutant resources of Arabidopsis thaliana to study the A thaliana-Plasmodiophora brassicae interaction Use of Arabidopsis thaliana defense-related mutants to dissect the plant response to pathogens Characterisation of an Arabidopsis-Leptosphaeria maculans pathosystem: resistance partially requires camalexin biosynthesis and is independent of salicylic acid Böttcher The multifunctional enzyme 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in Arabidopsis thaliana Quantitative trait loci mapping in five new large recombinant inbred line populations of Arabidopsis thaliana genotyped with consensus single-nucleotide polymorphism markers Variation for virulence on Brassica napus L amongst Plasmodiophora brassicae collections from France and derived single-spore isolates Transgressive segregation reveals two Arabidopsis TIR-NB-LRR resistance genes effective against Leptosphaeria maculans Glutathione-indole-3-acetonitrile is required for camalexin biosynthesis in Arabidopsis thaliana Deficiency in phytoalexin production causes enhanced susceptibility of Arabidopsis thaliana to the fungus Alternaria brassicicola Histochemical and genetic analysis of host and non-host interactions of Arabidopsis with three Botrytis species: an important role for cell death control a gene required for camalexin biosynthesis encodes a putative cytochrome P450 monooxygenase Manzanares-Dauleux MJ and Gravot A (2015) Camalexin contributes to the partial resistance of Arabidopsis thaliana to the biotrophic soilborne protist Plasmodiophora brassicae Received: 11 May 2015; Accepted: 03 July 2015; Published: 21 July 2015 Copyright © 2015 Lemarié, Robert-Seilaniantz, Lariagon, Lemoine, Marnet, Levrel, Jubault, Manzanares-Dauleux and Gravot. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Antoine Gravot, UMR 1349 IGEPP, Université de Rennes 1, Domaine de la Motte au Vicomte, BP 35327, 35653 Le Rheu, France,YW50b2luZS5ncmF2b3RAdW5pdi1yZW5uZXMxLmZy Volume 6 - 2015 | https://doi.org/10.3389/fpls.2015.00221 Rapid and uniform seed germination is a crucial prerequisite for crop establishment and high yield levels in crop production A disclosure of genetic factors contributing to adequate seed vigor would help to further increase yield potential and stability Here we carried out a genome-wide association study in order to define genomic regions influencing seed germination and early seedling growth in oilseed rape (Brassica napus L.) A population of 248 genetically diverse winter-type B napus accessions was genotyped with the Brassica 60k SNP Illumina genotyping array Automated high-throughput in vitro phenotyping provided extensive data for multiple traits related to germination and early vigor absolute germination rate and radicle elongation The data obtained indicate that seed germination and radicle growth are strongly environmentally dependent but could nevertheless be substantially improved by genomic-based breeding Conditions during seed production and storage were shown to have a profound effect on seed vigor and a variable manifestation of seed dormancy appears to contribute to differences in germination performance in B Several promising positional and functional candidate genes could be identified within the genomic regions associated with germination speed napus orthologs of the Arabidopsis thaliana genes SNOWY COTYLEDON 1 (SCO1) ARABIDOPSIS TWO-COMPONENT RESPONSE REGULATOR (ARR4) and ARGINYL-t-RNA PROTEIN TRANSFERASE 1 (ATE1) which have been shown previously to play a role in seed germination and seedling growth in A For breeding of vigorous plant cultivars with fast and uniform field emergence, it is crucial to understand the genetic factors that contribute to an adequate germination performance and seedling growth. As seed germination is a very complex trait controlled at transcriptional, translational and metabolic level (Rajjou et al., 2012) it is difficult to identify the contributing genetic factors by conventional genetic or physiological analyses A preferential method for identification of genomic regions associated with complex quantitative traits is genome-wide association mapping in which marker-trait associations are calculated across a broad set of diverse germplasm in order to define chromosome regions harboring promising genes In such a case statistical methods have to be applied which correct for population stratification In this study we used genome-wide association studies for the investigation of quantitative trait loci (QTL) linked to germination performance and early vigor in winter oilseed rape (Brassica napus L.). An automated high throughput phenotyping platform (Demilly et al., 2014) was used to assay diverse traits related to the three different phases of seed germination and post-germination radicle growth in a large set of diverse winter rapeseed lines genotyped with genome-wide single-nucleotide polymorphism (SNP) markers Genome-wide association analyses using these data sets enabled identification of highly promising candidate genes and markers for breeding towards improved germination in rapeseed Phenotyping of seed lots produced in different environments helped to evaluate the effects of germination-independent factors napus inbred lines were used in this study 20 winter fodder (WF) and 10 exotic lines (Table S1) Seeds of all inbred lines were produced by controlled self-pollination in two distinct environments during the growing seasons of 2010/2011 (SL 2011) in Le Rheu France and 2011/2012 (SL 2012) in Asendorf The genotype panel comprises lines derived from modern oilseed rape cultivars with low seed erucic acid and glucosinolate content old rapeseed varieties with high seed erucic acid and high glucosinolate content (++ quality) napus derived from interspecific hybridizations between its two diploid progenitors The following parameters were determined: Volume increase within first 8 h (VI; in %) imbibition speed during first 4 h after initiation of imbibition (IS; in mm3/h) total germination rate within 72 h after initiation of imbibition (GR72; in %) time to reach 50% of germination (T50; in h) and germination rate within 36 h after initiation of imbibition (GR36; in %) thousand seed weight (TSW; in g) was measured before germination monitoring Correlations were calculated applying the Pearson's product-moment correlation SNPs were blasted using the following criteria: minimum overlap of 50 bp length SNPs which failed across the entire genotype collection As heterozygous SNPs cannot be distinguished from multi-locus hemi-SNPs or false calls heterozygous calls were treated as missing values For visualization of population structure and calculation of genome-wide associations SNP markers with more than 20% missing calls across the panel were excluded all individuals which had more than 20% missing calls across the genotype data were excluded In order to incorporate rare alleles with potential effects on germination performance only markers with a minor allele frequency ≤ 0.025 were excluded from analysis 218 individuals and 22,169 SNP markers remained for further analyses Optimal number of clusters was estimated by calculating the within-cluster sum of squares (WSS) Linkage disequilibrium (LD) was calculated for each chromosome individually quoting coefficients of determination (r2) for all locus pairs localized on the same chromosome Inter-chromosomal genome-wide LD decay was calculated for trait-associated markers that exhibited unexpected patterns of local LD Locally paired scatterplot smoothing in R was employed for graphical representation of LD curves with a span of 0.1 For characterization of genomic kinship, genome-wide allele identity by descent was computed for the whole diversity panel as well as separately for the four identified subpopulation clusters, using the R package GenABEL (Aulchenko et al., 2007). Additionally, to determine the proposition of genetic differentiation explained by differentiation among the subclusters, overall FST values were calculated using the software Genepop version 4.2.2 (Rousset, 2008) Narrow-sense heritability for each trait was calculated separately for each seed lot, again using the R package GenABEL. Genome-wide associations were calculated with GenABEL. Adjustment for stratification was performed by mixed-model approximation combined with PC adjustment (Price et al., 2010; Svishcheva et al., 2012) For PC adjustment the first 4 components were taken into account Marker haplotypes were defined for each associated region and genotypes were bulked in regard to their associated-marker haplotypes For T50 and ES boxplots were generated showing the phenotypic distribution of 4 and 6 classes representing the cumulative phenotypes from haplotypes with favorable effects on the trait performance Significant differences were calculated with Student's t-test Phenotypic variation for different traits associated with seed germination performance in two seeds lots of a Brassica napus diversity panel (n=248) produced in Le Rheu standard deviation (SD) and correlation between seed lots (r) Correlation coefficients (r) for different trait pairs associated with seed germination performance measured in two seed lots (SL2011 and SL2012) from an oilseed rape diversity panel (n = 248) The largest cluster “blue” comprised 79 genotypes and showed the narrowest distribution whereas the smallest cluster “green” comprised only 29 genotypes but showed the widest distribution of IBD values Genetic relatedness of 218 genetically diverse winter-rapeseed lines shown in a principal component analysis regarding the first 2 components Different clusters were represented by different colors Winter oilseed rape lines were shown as “O,” winter fodder lines as “F” and others as “X.” Top right: Within-cluster sum of squares depending on the number of clusters applied for k-means clustering Genetic relationship between all individual pairs within the total diversity set (n = 218) and within the single clusters from k-means clustering Histograms show the frequency of individual pairs depending on their kinship coefficients IBD (identity by descent) which score allelic identity by descent For all chromosomes except C1, C3, C4, and C8, LD decayed to r2 = 0.1 at a distance between marker pairs ranging from 480 kbp (A1) to 1283 kbp (A9) (Figure 3) r2 did not drop to 0.1 until the distance between marker pairs reached 2014 kbp and 1939 kbp strongly conserved LD was observed for marker distances between 4500 and 6000 kbp Particularly strong patterns of LD were observed on chromosomes C1 and C4 with r2-values above 0.1 for up to 6651 kbp and 4048 kbp The very slow average LD decay on chromosome C4 was mainly determined by the presence of a large conserved LD block localized between 15,429 and 20,449 kbp whereas conserved LD between 17,787 and 26,635 kbp caused the extremely slow LD decay on chromosome C1 Overall chromosome-wide decay of linkage disequilibrium (LD) shown as smoothed r2-values for all marker pairs on each chromosome depending on the distance between marker pairs correction for stratification was performed by mixed-model approach including principal component covariates Subdivision into clusters caused an increase of IBD values within all clusters except for cluster “green,” which mainly comprises fodder rapes and exotic accessions Intervals of linkage disequilibrium (LD) containing significant marker-trait associations for different traits related to germination performance in seeds of an oilseed rape diversity panel produced in Le Rheu Chromosomal position in base pairs [LD interval (bp)] logarithmic scaled P-value (−log10(p)) The chromosomal regions delineated by haplotype blocks in strong LD (r2 > 0.4) with trait-associated markers harbored a total of 681 genes (Table S2). Several genes ascribed to seed germination, seed dormancy and seed and embryo development were disclosed, associated with T50, GR72, ES, FG, and TSW (Table 4). Further investigation showed that none of the candidate genes listed in Table 4 was known to be involved in a mutual pathway Identical chromosome regions were associated with T50 and GR72 hence the same candidate genes were assumed on chromosomes A9 and C6 is localized within the non-coding region of the candidate gene BnaA06g34100D Very strong local LD values (r2 > 0.8) were found between markers Bn-A04-p4074166 whereas LD with the other surrounding markers was very low (0 < r2 < 0.06) these markers showed strong extra-chromosomal LD to markers Bn-A03-p5072729 and Bn-A03-p16990947 on chromosome A03 both of which also showed associations with GR72 Although this might be due to co-selection of two epistatically interacting gene loci this may also suggest a spurious allocation of markers Bn-A04-p4074166 Bn-A04-p4217227 and Bn-A04-p10196289 to the wrong chromosome hence they were excluded from further analysis List of candidate genes ascribed to seed germination dormancy and seed development: Chromosome (Chr) Name of the candidate gene (Candidate gene) and absolute chromosomal position in base pairs [Position (bp)] thaliana gene implicated in germination and early development ARGINYL-t-RNA PROTEIN TRANSFERASE 1 (ATE1) Manhattan plot describing marker-trait associations for the trait ES within the chromosomal region around candidate BnaA09g48160D (Bna.ARR4) on chromosome A9 and correlations between surrounding markers Red dots represent associations calculated for SL2011 Black dots represent associations for SL2012 Manhattan plot describing marker-trait associations for the trait T50 within the chromosomal region around candidate BnaA09g12770D (Bna.SCO1) on chromosome A9 and correlations between the associated marker and both flanking markers Manhattan plot describing marker-trait associations for the trait ES for the chromosomal region around candidate BnaA10g24850D (Bna.ATE1) on chromosome A10 and correlations between markers of the linkage group harboring BnaA10g24850D Pigment concentrations were measured in the cotyledons of 5d old seedlings from the two genotype bulks, featuring consistently low and high ES values, respectively. Mean concentrations of chlorophyll a and b were found to be significantly lower in genotypes with poor performance in ES (Figure 7) (A) Phenotypic variation for cotyledon chlorophyll pigmentation in B napus genotypes showing high (ES+) and low (ES−) elongation radicle rates (B) Box plots representing chlorophyll a and chlorophyll b concentrations of 10 genotypes each showing high and low elongation radicle rates Significant differences were calculated with student's t-test at p < 0.05* and p < 0.01** For T50 an increase in the number of undesirable haplotypes was associated with a decrease in the number of individuals per bulk (Table S3) While the main fraction of lines (n=133) exhibited the favorable haplotype at all three loci a combination of all three unfavorable alleles was observed in only one line with most individuals (n = 105) exhibiting 2 favorable alleles (Table S3) Loci-specific alleles of single SNP markers and different haplotypes from LD-blocks harboring various SNP markers associated with T50 and ES Favorable alleles (+) were marked in green alleles with negative effect on trait performance (−) were shown in red Corresponding marker names were listed below (A) Boxplot representing the phenotypic distribution for T50 (Time to reach 50% of germination) in two different seed lots produced in 2011 (SL2011) and 2012 (SL2012) Each whisker represents a bulk of genotypes exhibiting no favorable haplotype (0) two favorable haplotypes (2) and three favorable haplotypes (3) regarding the three associated regions on chromosome A9 Population means were indicated as dashed lines (B) Boxplot representing the phenotypic distribution for ES (Elongation speed of the radicle) in two different seed lots two favorable haplotypes (2) three favorable haplotypes (3) four favorable haplotypes (4) and five favorable haplotypes (5) regarding the five disclosed associated regions on chromosome A9 Seed germination has a decisive influence on homogeneous field emergence and successful seedling establishment and is therefore a basic target for the development of vigorous crop plants with stable yield performance. According to Finch-Savage et al. (2010) germination speed is a major key element of vigorous seeds along with a rapid initial downward and upward seedling growth According to our observations TSW should be regarded as a seed yield parameter in oilseed rape rather than a contributing factor for germination performance As both seed lots were tested for germination under constant laboratory conditions the observed differences in performance between the two seed lots must reflect genotype by environment interactions during seed ripening in the field and/or during the subsequent seed storage One possible mechanism could be epigenetic imprinting during seed development due to stress conditions in the maternal environment there is still a lack of knowledge about individual genes involved in these complex traits Both germination and dormancy underlie polygenic control that can only be elucidated by quantitative genetics approaches so that selection for T50 may also lead to improvement of GR72 Strong LD conservation can complicate candidate gene identification from association peaks hence it is important to consider local LD when defining QTL confidence intervals for candidate gene selection The validity of the trait associations we detected appears to be confirmed by the identification of comparatively small regions of strong LD that harbor highly interesting genes involved in processes closely related to seed germination and seedling development Allelic differences in Bn.ATE1 within LD haplotypes associated with ES are hence also strong positional and functional candidates for control of radicle growth during and after germination Genotypic classification for T50 and ES revealed that both traits can potentially be improved by pyramiding of favorable alleles (Figures 9A,B) For both traits the absence among the diversity panel of individuals with three (T50) or five (ES) unfavorable haplotypes indicates that strong natural and artificial selection has occurred in cultivated forms of B napus against individuals with very poor germination and vigor effects of loci associated with these traits might be underestimated because specific undesirable allele combinations are absent thus restricting the extent of phenotypic variability within the diversity set For ES we also observed a lack of favorable haplotype combinations indicating that early seedling growth could be substantially improved beyond the phenotypic diversity in the present study Large-scale automated phenotyping revealed a broad phenotypic variability in germination performance across a B narrow-sense heritability revealed that germination performance is influenced by both genetic as well as environmental factors As the latter were constant during in vitro phenotyping the observed differences in germination performance between two different seed lots could be attributed to the conditions predominant during seed production and storage No relationship of germination traits to seed weight could be detected the results indicate that genetic determinants for the manifestation of seed dormancy may be a decisive factor influencing inheritance of germination performance in B In a genome-wide association analysis several promising genes were found within regions showing LD with QTL for seed germination and vigor Combination of positive alleles for the loci we identified should facilitate a decisive increase in germination performance and early seedling growth in oilseed rape improving prospects for breeding of these complex As such the study provides a basis for the establishment of genomic selection tools for improved seed germination and seed vigor in rapeseed SH and RS designed the study and interpreted the results and GL developed the diversity panel and generated seed lots for the phenotypic analysis and FB performed field phenotyping and analyzed the field phenotype data SD and MW performed the automated germination phenotyping SH and RS wrote the manuscript and all authors corrected and approved the final version The work described in this manuscript was performed within the framework of the transnational PLANT-KBBE cooperation project CONVIGOUR with funding from BMBF (Germany) and ANR (France) We thank Deutsche Saatveredelung AG (Lippstadt France) for generation of seed materials and Sarah Schiessl and Kai Voss-Fels for helpful discussions and biostatistics support The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fpls.2015.00221/abstract thaliana gene two-component response regulator ARR4 (AT1G10470.1); 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Rod J. Snowdon, Department of Plant Breeding, Justus Liebig University, IFZ Research Centre for Biosystems, Land Use and Nutrition, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germanycm9kLnNub3dkb25AYWdyYXIudW5pLWdpZXNzZW4uZGU= Metrics details has a complex reproductive mode in which some lineages reproduce by continuous parthenogenesis whereas others reproduce sexually once a year The climate is thought to act directly on the reproductive mode because sexual eggs are the only form that can resist frost in cold regions Sexual reproduction necessitates an obligatory host alternation that may result in long-distance dispersal we examined the genetic variability at seven microsatellite loci of populations of M We provide clear genetic evidence that the breeding system affects genotypic variability as cyclically parthenogenetic aphids are far more variable than their obligately parthenogenetic counterparts A temporal decrease in genetic variability and a temporal genetic differentiation effect suggest the existence of selective factors that play an important role in shaping the genetic structure of M differences in the population structure between reproductive modes suggest that the migration associated with the change of host during sexual reproduction lowers the level of population differentiation the coexistence of both reproductive modes results from the loss of sexuality in some lineages An understanding of the genetical consequences of both reproductive modes in these organisms may help to explain the maintenance of sex The continuous black arrows indicate the migration of winged aphids The dashed black arrows indicate several parthenogenetic generations The grey arrow corresponds to the development of the eggs spring–summer) and sample characteristics (aerial and sexual) are indicated for both reproductive modes: ‘ssas’ corresponds to spring–summer aerial samples ‘aas’ to autumn aerial samples and ‘ss’ to sexual samples The different life cycle forms of M. persicae are detailed in Figure 1 cyclically parthenogenetic aphids give birth to gynoparae (precursor forms of sexual females) and winged males that both fly to peach trees where gynoparae give birth to sexual females Mating occurs on these trees and sexual females lay eggs that hatch by the beginning of spring most peach trees grow in commercial orchards in southern regions but some of them are also found further north males and precursors of sexual females may have to occasionally fly long distances because of host alternation associated with sexual reproduction aphids migrate from primary to secondary hosts both obligately and cyclically parthenogenetic aphids produce winged morphs that spread among secondary hosts obligately parthenogenetic females can also participate in the autumn flight to disperse among secondary hosts three main flight activities take place per year may correspond to long-distance migration because of the necessity to change host for sexual reproduction c) linkage disequilibrium should be more frequent in obligately parthenogenetic populations than in cyclically parthenogenetic ones (d) drift and/or selection may strongly affect neutral genetic variability during the parthenogenetic phase of both life cycles due to the absence of recombination and the rapid rate of increase during clonal reproduction and (e) in the specific case of M a larger differentiation is expected between strictly asexual populations than between cyclically parthenogenetic populations because of the migration associated with the change of hosts we analysed the effects of the reproductive mode on the genetic variability of M persicae and tested the hypotheses described above persicae individuals collected in France in suction (aerial) traps and on primary hosts was performed to answer the following questions: Can we infer the reproductive modes from microsatellite data and can we validate hypotheses (a)–(c) What is the extent of genetic differentiation and migration among populations of M persicae in France and do they depend on the reproductive mode (hypothesis (e)) Does this genetic structure imply drift or selection during the parthenogenetic phase of the life cycle (hypothesis (d)) Two sets of aphids were sampled by different methods from the air and from peach trees Geographical locations of the 10 sampling sites. The open circles correspond to aerial samples. persicae (males and females pooled) caught at 12.2 m in 2000 The black bars show the number of aphids analysed in the genetic study and the arrows delimit the two periods considered Peach orchard samples: In France, peach trees generally grow in southern regions. Five samples of aphids were collected in November 2000 from peach orchards located in the Côte d'Azur and Provence in southern France (Table 1 and Figure 2) wingless sexual females and winged females (thought to be precursors of sexual females) were sampled These samples will be referred to as sexual samples hereafter All aphids were stored at −80°C until DNA extraction the three types of samples should a priori include different proportions of aphids with different reproductive modes: (1) in late spring and summer both cyclically and obligately parthenogenetic aphids disperse among secondary hosts; therefore the spring–summer aerial samples should correspond to a mixture of both reproductive modes and (2) autumn aerial samples should a priori correspond to cyclically parthenogenetic aphids in cold regions and to obligately parthenogenetic aphids in mild regions The aerial samples collected in Valence and Le Rheu should be mainly constituted of obligately parthenogenetic aphids because of their geographical locations (3) aphids caught on the primary host are considered to be cyclically parthenogenetic aphids Allele sizes were estimated by use of a sequencing size ladder (sequence of pGEM - 3Zf(+) vector PROMEGA) Linkage disequilibrium between loci within each population and departure from Hardy–Weinberg equilibrium at each locus were tested using the pseudoexact test procedures in GENEPOP 3.3 Individual multilocus heterozygosity was calculated on one individual without missing data representative of each genotype per sample considered (one copy per genotype) it is important to use common statistical methods The Isolde program from the GENEPOP package was used to perform Mantel tests of independence between matrices (genetic versus geographical or temporal distance matrices) whereas the autumn flight was clearly observable in all five traps although its quantitative importance was variable between traps This variation could not be attributed to the geographic location of the traps The number of alleles at the microsatellite loci ranged from four at locus S.24 to 18 at locus R5.180 except locus S.24 in the Loos-en-Gohelle samples The genotypes of locus S.17b could not be obtained for the two samples from Colmar Analysis of genotypic disequilibrium between each pair of loci in each sample did not reveal any significant linkage in sexual samples (Table 2) significant associations were frequently found in aerial samples The number of loci showing significant linkage disequilibrium increased between the two periods in Valence and Le Rheu and decreased in Colmar and Loos the autumn flight sample from Colmar presented the lowest number of loci in linkage disequilibrium Although Hardy–Weinberg (H–W) equilibrium was not expected in groups of individuals that had been reproducing parthenogenetically for several generations before sampling, we tested for this equilibrium and measured the corresponding deviation in each sample (Table 2) Three sexual samples did not deviate from H–W equilibrium and two samples presented a significant heterozygote deficiency H–W equilibrium was rejected in all aerial samples and generally corresponded to heterozygote deficiency No heterogeneity in observed heterozygosities (Ho) was found whatever the grouping of the samples defined as the proportions of heterozygous loci per individual representative of each genotype (ie using the ‘one copy per genotype’ method) were not significantly different between samples (Fisher's exact test between both periods in aerial samples (P=0.75) or between each period and the sexual samples (P>0.05 for both comparisons) The mean number of alleles per locus and per sample was significantly higher in aerial samples collected in spring–summer (nall=4.58±0.49) than in autumn (nall=3.49±0.38; Wilcoxon's signed rank test on period means P=0.016) and in sexual samples (nall=3.34±0.28; N=7 Aerial samples contained 92% of the number of alleles found in the sexual samples and 88% of the alleles found in autumn aerial samples were common to spring-summer aerial samples 30% of the total number of alleles found in spring–summer samples were private alleles an impoverishment through time was detectable at the allelic level Table 2 shows the number of different genotypes per sample when considering the 237 individuals without missing data (including the samples from Colmar that were characterised at six loci) 163 multilocus genotypes were distinguished 63 different genotypes were found among 64 individuals Le Rheu was the location where repeated genotypes were the most frequent with only eight genotypes present in a single copy in the 50 aphids without missing data the number of repeated genotypes was lowest at Colmar with 37 different genotypes among 42 individuals without missing data The only genotype that was common to aerial and sexual samples was shared by an individual from the Colmar autumn sample and by two females from the Roquebrune sexual sample The proportion of different genotypes (ratio ng/nind) was larger in sexual samples (0.98) than in spring–summer samples (0.75) and was larger in spring–summer than in autumn aerial samples (0.54) (Fisher's exact test The four most frequent multilocus genotypes (G1 The frequency of G1 and G4 increased significantly from 6.4 to 19% and from 0 to 6.3% The frequencies of G2 and G3 did not vary significantly with time we observed maximal genotypic variability in sexual samples the number of genotypes being almost equal to the number of individuals with no linkage disequilibrium linkage disequilibrium and departure from H–W equilibrium were frequent in most aerial samples The Colmar autumn sample showed intermediate characteristics between the sexual and the aerial samples These samples were grouped for subsequent tests On a regional scale (distances of about 150–200 km) we found a low but significant genetic differentiation between Côte d'Azur and Avignon orch-ards including sexual samples and aerial samples revealed significant genetic differentiation The estimated Fst between the Colmar autumn and sexual samples were rather low (4.6±2.6%) when compared to that between other aerial samples and sexual samples (10.7±2.7%) All aerial samples were significantly differentiated with moderate and high Fst estimates in spring and autumn For each suction trap, we compared the two periods corresponding to the two main flight activities: spring–summer and autumn (Table 3) Genotypic/genic differentiation was significant in Valence and Le Rheu with Fst estimates of 3 and 8% We performed an ‘isolation by time’ test in each suction trap that is we tested the null hypothesis of independence between genetic distances and time between individual samplings Our sampling scheme allowed us to test this hypothesis because the aphids were caught every day for several months and because temporal coordinates (day of capture) were assigned to each aphid The results of the ‘isolation by time’ test were significant in Valence and Le Rheu (â(r)=0.45+0.0026r all aerial samples were differentiated from each other and the level of differentiation increased between summer and autumn Sexual samples were moderately differentiated or undifferentiated Sexual and most autumn aerial samples (except that of Colmar) were strongly differentiated Differentiation was observed in Le Rheu and Valence between autumn and summer an isolation by time effect was observed in these two sites we analysed the population structure of three groups of aphids: cyclically parthenogenetic aphids collected on peach trees (also referred to as sexual samples) and a mixture of obligately and cyclically parthenogenetic aphids collected aerially in spring–summer and autumn with more obligately parthenogenetic aphids in milder regions (ie in Valence and Le Rheu) We expected to find (1) a greater genotypic diversity (2) less linkage disequilibrium between loci and (3) a lower heterozygosity in cyclically than in obligately parthenogenetic populations The two first expectations proved to be true in which a high genotypic diversity was found despite numerous parthenogenetic generations and showed that heterozygosity was globally higher in obligately than in cyclically parthenogenetic aphids These authors explained these results by either an ancient loss of sexuality or a hybrid origin of asexual lineages in this species The general tendency for heterozygote deficiency found in our study may be explained by the sampling design as all of the aphids analysed were migrating Those caught in aerial traps were obviously migrating and sexual individuals captured on peach trees were at the end point of the autumn migration to the primary host these samples of aphids corresponded to pools of migrants originating from diverse populations If those original populations were genetically differentiated that is an heterozygote deficiency due to the fact that several subpopulations were pooled A major result of this study is that the reproductive system that is cyclical or obligate parthenogenesis is correlated to microsatellite variability in M Samples caught on primary hosts (Prunus persica) can be used as a reference for sexual samples in genetic studies aerial samples from Valence and Le Rheu might mostly correspond to obligately parthenogenetic aphids whereas most aphids caught in Colmar in autumn might generally be cyclically parthenogenetic aphids The autumn aerial sample collected at Colmar was closer to the sexual samples in terms of genotypic diversity and linkage disequilibrium low Fst estimates were found between the Colmar autumn and sexual samples and the only common genotype between sexual and aerial samples was found in the autumn flight of Colmar while males were very rare in Valence and Le Rheu These results strongly suggest that autumn aerial samples are mostly constituted of obligately parthenogenetic aphids except in Colmar where the aphids display biological and genetic characteristics of cyclically parthenogenetic individuals Genetic differentiation between sexual samples was low and not significant over small geographic distances (<60 km) and low but significant on a larger geographical scale (150–200 km) Putatively obligately parthenogenetic aphids from aerial samples were significantly differentiated on the national scale in both periods We observed a temporal genetic differentiation between samples collected in spring–summer and autumn in Valence and Le Rheu A pattern of ‘isolation by time’ at a fine temporal scale was also found in these samples This means that the temporal distribution of genotypes and/or genes is nonrandom and that two M persicae individuals collected at different periods are more likely to be genetically different than two aphids collected in a short temporal window Temporal variability in the genetic structure of M persicae populations also indicates an impoverishment through time of genetic variability: the number of alleles and pairs of loci in linkage equilibrium decreased from spring–summer to autumn in Valence and Le Rheu whereas some multilocus genotypes increased in frequency between sample differentiation increased sharply between summer and autumn suggesting that the genetic compositions of the samples varied in time and that these variations were different between populations during the parthenogenetic phase and also interpreted this phenomenon as clonal selection rather than drift The results of this microsatellite analysis show that the reproductive mode polymorphism in M persicae is detectable at the population level the major difference between cyclically and obligately parthenogenetic populations concerns genotypic variability with cyclically parthenogenetic aphids being the most variable persicae in France may be genetically structured by at least three factors: (1) limited migration that is not sufficient to homogenise the genetic structures of populations on a national scale in obligately parthenogenetic aphids (2) the geographic distribution of reproductive modes and probably (3) the effect of strong selection other than climate that causes geographic and temporal genetic differentiation As asexual reproduction occurs most of the year in M strong selective forces together with the absence of genetic recombination may have major effects on the ‘neutral’ population genetic structure: departure from H–W equilibrium and temporal differentiation can all occur as a result of these forces a combined analysis of microsatellites and insecticide resistance genes would be of great interest to quantify the relative importance of selection and drift on ‘neutral’ genetic variability The inheritance of life-cycle differences in Myzus persicae (Sulz.) (Hem. Life-cycle variation of Myzus persicae (Sulz.) (Hom. Aphididae) in different parts of the world Aphids on the World's Crops: An Identification Guide Geographic and microgeographic genetic differentiation in two aphid species over southern England using the multilocus (GATA)4 probe Coexistence in space and time of sexual and asexual populations of the cereal aphid Sitobion avenae Genetic architecture of sexual and asexual populations of the aphid Rhopalosiphum padi based on allozyme and microsatellite markers The evolution of insecticide resistance in the peach–potato aphid Devonshire AL (eds) Insecticide Resistance: From Mechanisms to Management Analysis of clonal diversity of the peach–potato aphid UK and evidence for the existence of a predominant clone The ups and down of insecticide resistance in peach–potato aphids (Myzus persicae) in the UK Temporal and spatial dynamics of insecticide resistance in Myzus persicae (Hemiptera: Aphididae) parthenogenesis and genetic structure of rotifers: microsatellite analysis of contemporary and resting egg bank populations Evidence for predominant clones in a cyclically parthenogenetic organism provided by a combined demographic and genetic analyses Evolutionary and genetic aspect of aphid biology: a review un réseau de surveillance des populations de pucerons: base de données associée et domaines d'application Biological and molecular characterisation of the autumn migrants of the bird cherry-oat aphid Tracking the evolution of insecticide resistance in the mosquito Culex pipiens Migration and genetic structure of the grain aphid (Sitobion avenae) in Britain related to climate and clonal fluctuation as revealed using microsatellites Estimating levels of gene flow between natural populations of cereal aphids (Homoptera: Aphididae) The relative importance of short- and long-range movement in flying aphids Slaves of the environment: the movement of herbivorous insects in relation to their ecology and genotype Evidence for evolution of Bdelloid rotifers without sexual reproduction or genetic exchange Assessing the nucleotide diversity of three aphid species by RAPD Genetic Structure and Local Adaptation in Natural Insect Populations: Effects of Ecology Long term stability in the frequency of insecticide resistance in the peach–potato aphid In: BCPC (ed) Brighton Crop Protection Conference – Pests and Diseases Evolution in a putatively ancient asexual aphid lineage: recombination and rapid karyotype change a population genetics software for exact tests and ecumenicism An exact test for population differentiation obligate parthenogens and intermediates in a fluctuating environment Effect of climate on the proportion of males in the autumn flight of the aphid Rhopalosiphum padi L Models of sexual and asexual coexistence in aphids based on constraints Reproductive mode and population genetic structure of the cereal aphid Sitobion avenae studied using phenotypic and microsatellite markers Isolation and characterization of microsatellite loci in the aphid species linkage group identification and determination of frequency in the peach–potato aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae) Genetic structure of an aphid studied using microsatellites: cyclic parthenogenesis differentiated lineages and host specialization Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae) Migration and the spatial dynamics of an aphid Host-based genetic differentiation in the aphid Aphis gossypii Glover Mortality during dispersal and the cost of host-specificity in parasites: how many aphids find hosts Microsatellite variation in cyclically parthenogenetic populations of Myzus persicae in south-eastern Australia low clonal diversity and genetic affinities of parthenogenetic Sitobion aphids in New Zealand Download references We thank M Hullé for permission to use Agraphid samples and the trap operators for their precious work X Fauvergue and D Forcioli for their advice and comments during this work Paul Sunnucks and Alex Wilson for sharing unpublished microsatellites of aphids Part of this work was financed by the Department ‘Santé des Plantes et Environnement’ of the Institut National de la Recherche Agronomique Equipe ‘Ecotoxicologie et Résistance aux Insecticides’ Equipe ‘Biologie et Génétique des Populations d'Insectes’ UMR BIO3P Download citation DOI: https://doi.org/10.1038/sj.hdy.6800292 Journal of Oceanology and Limnology (2021) Metrics details Although loss of sex is widespread among metazoans the genetic mechanisms underlying the transition to asexuality are poorly understood Aphids are good models to address this issue because they frequently show reproductive-mode variation at the species level involving cyclical parthenogens (CP) that reproduce sexually once a year and obligate parthenogens (OP) that reproduce asexually all year round we explore the genetic basis of OP in the cereal aphid Sitobion avenae by crossing several genotypes with contrasting reproductive modes and then characterising the reproductive phenotypes of F1 and F2 offspring The analysis of phenotypic variation in F1 and F2 progenies suggests that at least two autosomal loci control OP in S the transition to asexuality seems to depend on a single recessive locus because the offspring from self-crossed cyclical parthenogenetic genotypes contain either 0 or 25% OP as we observed OP in the F1 progenies from crosses between CP and OP and some CP in the offspring from outcrossed OP a dominant ‘suppressor’ gene may also be involved being inactive when in a recessive homozygous state in CP; this is the most parsimonious explanation for these results avenae appears to be an efficient genetic system to generate new OP genotypes continually It also allows asexuality-inducing alleles to be protected locally during harsh winters when extreme frost kills most OP and then to spread very quickly after winter recessive and (2) that the mixed strategy (intermediate phenotype producing sexual and asexual forms in similar quantities) could be a modification of cyclical parthenogenesis because of an epistatic recessive suppressor the phenotypes of the F2 progenies did not conform well to the second hypothesis we investigated the genetic basis of OP in S avenae by crossing several genotypes with distinct reproductive modes characterising the reproductive phenotypes of their F1 and F2 offspring and testing genetic models for the inheritance of OP in S Seven S. avenae clones were used for the crossing experiments (Table 1) When induced for sexual morph production under the right stimuli (see below) CP14 and CP5 produce only males and sexual females and are therefore classified as CP genotypes OP21 and OP26 exposed to the same conditions mainly produce parthenogenetic females (>90%) and are therefore classified as OP genotypes Some clones have been used in previous experiments and have not shown any change with time either in their reproductive phenotype (since 1990) or in their genotype as assessed with five microsatellite loci (since 1999) Egg hatching rates were most often around 60% the number of parthenogenetic females newly born from eggs (fundatrices) were counted every 2 or 3 days Each fundatrix was carefully removed with a fine brush Crossing design used to generate F2 offspring F2 were produced (a) by selfing F1 CP clones or (b) by allowing sexual morphs of different CP clones from the same F1 progeny to mate freely (: male; : sexual female; : parthenogenetic female) DNA was then resuspended in 20 μl of water DNA extractions were checked and roughly quantified on a 1% agarose gel electrophoresis and dilutions were made correspondingly in order to reach a DNA concentration of about 10 ng μl−1 A 1.5% agarose gel electrophoresis was used to check for good amplification and the concentration of the PCR products These were diluted with water before electrophoresis One microlitre of diluted PCR products was added to 5 μl of high-dye formamide containing 1% of 500 LIZ DNA ladder (Applied Biosystems USA) and electrophoresis was performed in the capillary sequencer ABI 3130 Xl (Applied Biosystems) Allele calls were automatically assigned by GeneMapper (version 3.7 by Life Technologies Corporation) and visually checked male production was not taken into consideration in further analyses A grand total of 1335 aphid clones were analysed for their reproductive phenotype The strong differences between clones producing mostly sexual females and clones producing almost exclusively parthenogenetic females allowed characterising CP and OP clones The numbers of parthenogenetic females produced by OP clones were not precisely assessed but ranged from 40 to 60 per seedling batch Production of parthenogenetic females by CP clones appeared to be negligible (0–5 per batch) A two-way ANOVA implemented in S-Plus 6.2 (Insightful Corporation USA) was performed to assess the effects of reproductive mode (CP vs OP) and of the cross (CPRoum × OP1 CPR2 × OP1 and CP14 × OP1) on the numbers of sexual females produced by F1 progenies of the three crosses between CP and OP clones Conformity to expected proportions of clones with different reproductive modes in the F1 progenies was further assessed by χ2 tests Bimodal frequency curve of abundance classes of sexual females in the progenies of three crosses between cyclical and obligate parthenogenetic clones of the aphid S compared with the monomodal frequency curves of abundance classes of sexual females in the progeny of the OP (white bars) and CP (hatched bars) parents For the six autosomal loci, allele frequencies conformed to Mendelian proportions in the four F1 families genotyped (Table 4) Mendelian proportions were also observed for the two X-linked loci in the offspring of both selfings indicating no transmission bias involving the sexual chromosome in males of CPRoum and CP14 F1 progenies from crosses involving OP1 males were not in Mendelian proportions for either of the two X-linked loci presumably because of the lack of transmission of one of the two X chromosomes in OP1 Results from the OP clone outcrosses, however, are not compatible with a monogenic model, and more generally with the previous hypothesis that OP clones should be aa (Table 5) a minority of CP clones were observed in F1 when only OP clones should be produced if parents were aa results from crosses between CP and OP clones showed segregation in F1 whereas no OP clones should be produced under the monogenic hypothesis Let us consider a model involving two independent genes (A) and (S), a dominant suppressor of (A), inactive in the recessive state. In this case, genotypes of CP clones would be AAss (CP-A) or Aass (CP-B), and OP clones, AASs (Table 5) This hypothesis predicts the production of (1) 75% OP clones in OP × OP crosses (or 66% if we assume that SS F1 clones are unviable) which differs from observations (the proportion was closer to 33%) It also conforms to the 100% sexual clones observed in the F2 from CP-A × OP crosses (in this case some OP should be produced (3% were observed) A three-gene model, involving a recessive restorer (b), inhibiting the effects of (S), gives a better prediction of the observed percentage of CP clones produced in CP-A × OP crosses (37.5%), but is not compatible with the homogenous F2 observed for such crosses (Table 5) but the numbers of sexual females and males produced were much lower than for the two above species crossing cyclical and obligate parthenogens gave F1 progenies whose production of sexual females was clearly bimodal and could not be used to study the genetic determinism of the male function reproductive-mode variation displayed by the S avenae genotypes used in this study fell into two well-differentiated categories: (1) cyclical parthenogens (CP) which produce large quantities of sexual females in response to short-day conditions which produce few sexual females and a large quantity of asexual females when exposed to the same conditions Phenotypic variation may be greater under a larger range of genotypes and/or environmental conditions as our aim was to explore the genetic basis of OP avenae genotypes with contrasting reproductive phenotypes characterised in the same controlled environmental conditions Our results on S. avenae cannot be fully compared with those of Blackman (1972) because we did not observe ‘intermediate’ clones in our F1 and F2 progenies we confirmed Blackman’s results on the dominance of CP vs OP: we found that selfing one of our CP genotypes gave 25% F1 OP offspring and that crosses between this CP genotype and OP genotypes also gave heterogeneous progeny Blackman's genetic model fails to account for the production of heterogeneous progeny in most of our S avenae CP genotypes crossed with OP genotypes Further research is needed to compare the proportions of both aphid reproductive modes in progenies of CP × OP crosses obtained in different conditions of photoperiod and temperature the involvement of a third gene restoring sexuality is an alternative explanation for some of the proportions observed in CP × OP F1 but it implies having heterogeneous F2 in such crosses Even if none of the tested genetic models fully explain the outcomes of all the crosses we performed on CP and OP S it is likely that OP in this aphid is controlled by a limited set of loci Further genetic analyses on a wider range of phenotypes are nevertheless needed to explore all the mechanisms underlying reproductive-mode variation in the species This article does not report new empirical data or software Variation in the photoperiodic response within natural populations of Myzus persicae (Sulz.) Life-cycle in the cereal aphid Sitobion avenae F.: polymorphism and comparison of life history traits associated with sexuality Seasonal and annual genotypic variation and the effect of climate on population genetic structure of the cereal aphid Sitobion avenae in Northern France Delayed setting of the photoperiodic response in recombinant clones of the aphid species Sitobion avenae Multiple routes to asexuality in an aphid species Proc Royal Soc London Ser B 268: 2291–2299 The developmental genetics of the temperature-sensitive lethal allele of the suppressor of Forked The spread of a transposon insertion in Rec8 is associated with obligate asexuality in Daphnia Strong biases in the transmission of sex chromosomes in the aphid Rhopalosiphum padi Two recessive genes controlling thermophotoperiod-sensitive male sterility in wheat Gene flow beween sexual and facultative asexual lineages of an aphid species and the maintenance of reproductive mode variation Production of sexual morphs by the monoecious cereal aphid Sitobion avenae Life-cycle variation in the aphid Sitobion avenae: costs and benefits of male production The origin and genetic basis of obligate parthogenesis in Daphnia pulex A single locus determines thelytokous parthenogenesis of laying honeybee workers (Apis mellifera capensis) Transcriptomic and proteomic analyses of seasonal photoperiodism in the pea aphid Seasonal photoperiodism regulates the expression of cuticular and signalling protein genes in the pea aphid The role of photoperiod and temperature in the determination of parthenogenetic and sexual forms in the aphid Megoura viciae Buckton I The influence of those factors on apterous virginoparae and their progeny II The operation of the ‘interval timer’ in young clones Molecular strategies to use nuclear male sterility in plant hybrid breeding general-purpose genotypes and geographic parthenogenesis Localization of the genetic determinants of meiosis suppression in Daphnia pulex Transcription regulation of sex-biaised genes during ontogeny in the Malaria vector Anopheles gambiae Fine tuning of Notch signalling by differential co-repressor recruitement during eye development of Drosophila A genetic suppressor of two dominant temperature-sensitive lethal proteasome mutants of Drosophila melanogaster is itself a mutated proteasome subunit gene Evolutionary history of contagious asexuality in Daphnia pulex Single-locus recessive inheritance of asexual reproduction in a parasitoid wasp Phenotypic effects of an allele causing obligate parthenogenesis in a rotifer Local variability in the life cycle of the bird cherry-oat aphid Rhopalosiphum padi (Homoptera: Aphididae) in western France Phylogenetic relationships between parthenogens and their sexual relatives: the possible routes to parthenogenesis in animals Evolutionary and functional insights into reproductive strategies of aphids Genomics of environmentally induced phenotypes in two extremely plastic arthropods Loss of sexual reproduction and dwarfing in a small metazoan Numerous transposed sequences of mitochondrial cytochrome oxidase I–II in aphids of the genus Sitobion (Hemiptera: Aphididae) Microsatellite and chromosome evolution of parthenogenetic Sitobion aphids in Australia Explaining the coexistence of asexuals with their sexual progenitors: no evidence for general-purpose genotypes in obligateparthenogens of the peach-potato aphid Cross-species amplification of microsatellite loci in aphids: assessment and application Random loss of X chromosome at male determination in an aphid detected using an X-linked polymorphic microsatellite marker The genetic outcomes of sex and recombination in long-term functionally parthenogenetic lineages of Australian Sitobion aphids Download references We are especially grateful to Christoph Vorburger (Institute of Integrative Biology Switzerland) for constructive comments on a previous version of the manuscript for their helpful suggestions for improving this paper Environnement et Protection des Plantes (IGEPP) Domaine de la Motte – 35653 The authors declare no conflict of interest Download citation Didier Azam has been director of the INRAE U3E Experimental Unit for Aquatic Ecology and Ecotoxicology in Rennes for over 24 years His enthusiasm and determination led to the development of an ambitious project for experimental facilities aimed at furthering knowledge of aquatic environments going from technician to research engineer and earning him the 2022 INRAE Innovation Award for research This film enthusiast never imagined his name in lights nor did he see himself as a research engineer understanding ecosystems and what happens if you change one element of them: pollution And this has been Didier’s leitmotiv during his 40-year career at INRAE: developing experimental facilities we started to imagine the first installations to better understand these aquatic ecosystems Combining work and passion was an obvious choice After earning a vocational diploma in pisciculture He then spent several years in vocational education an activity that left him some time to explore parallel paths: an internship on an oceanographic boat to inventory Gadidae larvae (cod to work on the development of frog and crayfish farms two rivers that would always flow through his life I was more interested in the experimental part we started to imagine the first installations to better understand these aquatic ecosystems” Didier never would have guessed that he would become director of the experimental unit that only existed in his imagination at the time.. What I wanted was to develop these tools for experimentation and observation adding wooden boards and plastic film; it was all very low-tech the first aquaculture hall was built with ponds and greenhouses to study the ecological aspects of the environment and ponds were built in which the ecosystems were reproduced and the parameters controlled But it was in 1998 that the U3E experimental unit was really created with six staff and none other than Didier Azam at its head "I was one of the few people to be a unit head while being a technician!” He then proposed grouping together the experimental facilities in Rennes and Le Rheu as well as the Scorff and Oir monitoring sites Didier has no shortage of ideas for developing his experimental unit and he knows how to defend them vigorously He opens the facilities to other scientists and institutions "This has enabled us to participate in ambitious research projects and to obtain funding to maintain our facilities and experiments” Consolidate the team to make sure these projects go on And I went from being a technician to a research engineer What I wanted was to develop these tools for experimentation and observation and to be recognised” U3E joins the European Aquacosm infrastructure (with about thirty partners in 15 countries) the LIFE (Living In Freshwaters and Estuaries) infrastructure is recognised by INRAE the biological resource repository COLISA (ichthyological samples) is recognised by the GIS IBISA and joins the National RARe infrastructure 2017 a large part of the Unit's activities are included in the DCF: European network for data collection in support of scientific advice for the European Common Fisheries Policy development and innovation centre between the OFB and INRAE is attached to the U3E 2011 The U3E is attached to the National Infrastructure in Biology and Health "AnaEE-France” 2008 U3E becomes part of the Environmental Research Observatory on Diadromous Fish in Coastal Rivers (ORE DiaPFC) We look at the overall state of the system through the prism of these fish Didier's determination and perseverance have helped the U3E unit conduct experiments for ambitious scientific projects The unit's teams carry out long-term observations in three rivers: the Oir The aim is to monitor migratory fish (trout eels) in order to better understand how they function and determine whether these rivers are still adequate homes for them "We look at the overall state of the system through the prism of these fish since 2017 the unit has been working to support European policy makers by providing information to European authorities about the best management methods for these species (quotas it follows whatever research projects are happening at the time The ponds help researchers understand the influence of various parameters on ecosystems: pollution technicians observe the impact of climate change and decreasing biodiversity on ecosystems or how ecosystems evolve if the species are changed or the supply of nutrients via wastewater modified the researchers are currently studying the primrose willow an invasive plant which clogs canals and upsets ecosystems Judging by the harmonious atmosphere in the labs and facilities Didier's leadership is based on mutual trust This faithful captain at the helm of a ship of 40 crew members finds the same trust more broadly throughout the Institute: "I was able to express and defend my ideas his enthusiasm remains intact: "The experimental units are very dear to me they are one of the distinguishing features of INRAE Scientists from other institutes work with us to use these infrastructures Centre technique du génie rural des eaux et forêts INRA and IRSTEA merged in 2020 to become INRAE "Normally these are things that only happen to other people but it's also difficult because I don't like being in the spotlight but I got recognition through the success of the internal competitions that I won and also through the confidence that I was given to carry out my projects the INRAE Award is a bit like the cherry on the cake” The time has come for Didier to pass the baton with whom he built this successful unit 40 years ago Experimental Unit for Aquatic Ecology and Ecotoxicology Siège : 147 rue de l'Université 75338 Paris Cedex 07 - tél PRESS RELEASE - Farmers in the main production areas of northern France and the Paris Basin will have a new bread winter wheat variety available for sowing in autumn 2025 this variety not only features very high yields and excellent protein levels but is also one of the first wheat varieties to be considered agroecological being resistant to diseases such as septoria leaf blotch eyespot and leaf rust and efficient in terms of nitrogen fertiliser use the result of 10 years of research and experimentation by INRAE teams now registered in the Official Catalogue of Species and Varieties of Cultivated Crops in France is one of Agri Obtentions' most important launches in recent years with an estimated potential growing area of 30,000 hectares beginning with the 2026 harvest Winter bread wheat is the main crop grown in France mainly for the production of bread flour both for the domestic market and for export the start of 2025 is marked by the inclusion of a new multi-resistant winter wheat variety in the Official Catalogue of Species and Varieties of Cultivated Crops in France[1] designed for low-input farming practices (reduced use of fungicides and nitrogen fertilisers) After 10 years of field evaluations supervised by scientists and technicians from INRAE's experimental units (Estrées-Mons (80) GEOPOLIS has achieved remarkable results in terms of both agronomic and agroecological efficiency during the two years of official trials for registration under contrasting climatic conditions (hot and dry in 2023 where some thirty new winter bread wheat varieties are released every year GEOPOLIS stands out for its characteristics as assessed by the Technical Committee for Plant Breeding (CTPS)[2] it currently has the best score for resistance to septoria leaf blotch underlining the effectiveness of the INRAE-Agri Obtentions breeding programme combined with appropriate crop management practices will enable farmers to reduce their costs while complying with the environmental objectives set out in the Ecophyto II+ Plan The development of a new variety is the result of a breeding scheme that lasts an average of 10 years involving scientific experts and cutting-edge selection tools The various stages involved in obtaining GEOPOLIS were: Agri Obtentions has already begun seed multiplication on 600 hectares marking an important step in making this innovation accessible to as many farmers as possible [1] Catalogue managed by GEVES, the French Variety and Seed Study and Control Group which conducts trials and studies for the description analysis and control of seeds and has a role of expert and reference at national and international level for all cultivated species each new variety is registered by decision of the Minister for Agriculture published in the Official Journal of the French Republic The Ministry relies on the advice of an advisory committee Diversity & Ecophysiology of Cereals Unit Crop Innovation & Environment Facility – Picardy The fungus Magnaporthe oryzae ravages rice and wheat crops and poses a formidable threat to human food on a global scale together with the Hunan Hybrid Rice Research Centre (HHRRC) and the International Rice Research Institute (IRRI) have discovered a gene in rice for resistance to this pathogen challenging the findings of a study that remained the reference for 25 years these results pave the way to new solutions to improve disease resistance in crops that are compatible with the development of sustainable agriculture PRESS RELEASE - Re-thinking the agriculture of tomorrow means developing sustainable and efficient production techniques This practice has been in full swing for more than a decade but is still too unpredictable in terms of performance CNRS and Institut Agro Montpellier carried out a vast genetic analysis They showed that one particular region of durum wheat DNA is pivotal: if there is diversity to be found there the performance of cultivar mixtures is compromised These novel and counter-intuitive findings were published in New Phytologist on 26 January 2022 Metrics details The aim of this work was to evaluate random amplified polymorphic DNA (RAPD) as a source of markers for race identification and variability analysis of D dipsaci were tested and one population of both D Sufficient levels of variation were detected to allow a clear distinction between the two D This result was in agreement with the characterization based on morphological differences among races and on the type of faba bean disease symptoms faba was low and no strong correlation could be found between the geographical origin and molecular or morphological characterization RAPDs are a powerful tool for identification of D The objectives of the present work were to study the intraspecific variability in a set of D symptomatology) and molecular (RAPD-PCR) methods in order to infer relationships between the normal and giant races Ditylenchus destructor was extracted from bulbous iris collected from fields Means were classified according to the Newman–Keuls test at P≤0.05 DNA was resuspended in TE buffer [0.01 M Tris (pH 8.0) 0.001 M EDTA (ethylene diamine tetraacetic acid) ] to a final concentration of 5 ng μL−1 and stored at −20°C Nineteen random decamer oligonucleotides of the Operon kit G (Operon Technologies and used according to the manufacturer's directions RAPD reactions were carried out in a 12.5 μL final volume containing 10 ng of genomic DNA; 2.5 μmol of primer; 4.4 nmol MgCl2; dATP dGTP and dTTP (Boehringer) each at 100 μM final concentration; 0.5 U Taq polymerase (Appligene) and 1×Taq incubation buffer Each reaction was overlaid with a droplet of mineral oil to prevent evaporation Amplifications were performed in a 480 Perkin Elmer thermal cycler 2 min; and (iii) a final incubation of 72°C Amplification products were stored at 4°C before use and separated by electrophoresis in 1.5% agarose gels in TBE buffer (89 mM Tris pH 8.0) at a constant current of 200 mA for about 2 h Products were visualized by ethidium bromide staining (0.5 μg mL−1) and examined under ultraviolet (UV) light Marker VI (Boehringer) was used as the molecular weight marker Similarity (S) was calculated according to the equation: Among the 13 stem nematode populations tested, eight populations were scored as giant (BOU, CHE, DAR, SAD, COR, LR, LKH and ARI) and five as normal (TIA, AIL, DCH, BEJ and AP) by the pathologies induced (Table 2) The comparison of female length means showed five significantly different groups c and d included eight populations with mean lengths ranging from 1649 to 1987 μm (average=1832 μm) These populations were those previously classified as giant according to plant symptoms Lengths of females from the two last groups (e and f) ranged from 1289 to 1410 μm (average=1325 μm) corresponded to the five populations which caused necrotic lesions specific to a normal race Analysis of male size showed the same grouping of populations the significantly larger nematodes with an average length of 1704 μm and the normal D Very similar results were obtained comparing J4 lengths differences between J4 length were smaller than those observed for males and females (average length=1048 μm for the normal race and 1180 μm for the giant race) UPGMA dendrogram showing estimated average genetic distances among different Ditylenchus species based on polymorphism generated by RAPD This dendrogram was built using 124 polymorphic bands produced with 13 random primers Average linkage cluster analysis was performed on the (1−S) values Bootstrap values (%) based on 1000 resamplings are given on appropriate clusters This suggests that RAPD–PCR should be considered as a powerful method for the accurate differentiation between giant and normal races within D dipsaci populations pathogenic to faba bean as no bands were common to both groups of races the tree topology clearly defined four groups when all D dipsaci populations were computed with the outgroup species A principal coordinate analysis (not shown) calculated from the similarity matrix confirmed the separation of these four groups the divergence between the two groups of D dipsaci races seemed to be as great as between the three species Such a clear separation between populations from the giant and the normal races provides new support for the sibling species hypothesis Only the four giant Moroccan populations are clustered together More extensive sampling of species and populations is required to confirm the informative value of the specific and subspecific clusters detected in this study and more investigations are needed to select the best experimental conditions (primer combinations but the RAPD approach appears more than promising for diagnostic purposes within the Ditylenchus genus (Nematoda: Tylenchida) parasite de Vicia faba L Morphometric relationships in the genus Ditylenchus Vicia faba lines resistant to the giant race of stem nematode (Ditylenchus dipsaci (Kühn) Fil.) Estimation de la résistance à la race géante de Ditylenchus dipsaci par les symptômes chez la féverole (Vicia faba L.) Contribution à l'étude de la biologie de Ditylenchus myceliophagus Goodey Tylenchus devastatrix (Kühn) et la maladie vermiculaire des fèves en Algérie Confidence limits on phylogenies: an approach using the bootstrap Inter and intraspecific variation between populations of Globodera rostochiensis and G pallida revealed by Random Amplified Polymorphic DNA An assessment of serological procedures for the differentiation of the biological races of Ditylenchus dipsaci Applications of random amplified polymorphic DNA (RAPD) in molecular ecology Molecular diversity amongst Radopholus similis populations from Sri Lanka detected by RAPD analysis Genomic diversity between Radopholus similis populations from around the world detected by RAPD-PCR analysis Disc-electrophoretic patterns of enzymes and soluble proteins of Ditylenchus dipsaci and D The relevance of races in Ditylenchus dipsaci (Kühn) Filipjev Analysis of Heterodera avenae populations by the random amplified polymorphic DNA technique Mathematical model for studying genetic variation in terms of restriction endonucleases The use of DNA probes to identify Ditylenchus dipsaci (eds) Plant Parasitic Nematodes in Subtropical and Tropical Agriculture The use of the subspecies and the superspecies categories in nematode taxonomy Cytogenetics and morphology in relation to evolution and speciation of plant-parasitic nematodes Separation of three species of Ditylenchus and some host races of D dipsaci by restriction fragment length polymorphism Download references Panchaud-Mattei for supplying original populations of D myceliophagus and the anonymous reviewers for their comments on the manuscript and additional statistical analysis This research is part of the GRAM programme (Groupe de Recherches Agronomiques Mediterranéen) concerning food legumes supported by the INRA Direction des Relations Internationales (M Institut National de la Recherche Agronomique Download citation DOI: https://doi.org/10.1046/j.1365-2540.1998.00367.x Vous utilisez un ancien navigateur. Ce site risque de ne pas s'afficher correctement. Mettez à jour votre navigateur pour améliorer votre expérience Maison fissurée à cause de la retractation et du gonflement de l'argile (Pas-de-Calais Vulnerability of the Rennes Métropole territory to clay shrinkage and swelling: hazard map drawn up for the Ille-et-Vilaine département The shrinkage and swelling of clays causes considerable damage every year in France; it can be compensated as a natural disaster It is therefore necessary to characterise it precisely two-thirds of the département’s surface is exposed to this phenomenon medium (4% of the area) or high (less than 1% of the area) levels BRGM teams will carry out a field campaign in twenty communes in the Rennes metropolitan area in order to produce a precise map of the nature of the clays and the shrink-swell phenomenon In sectors of the Rennes metropolitan area which swell in the presence of water and shrink in the event of drought When buildings are built on these formations the alteration of these clays can cause damage to the structure: cracks While there is already a departmental map of this geological risk (available on the Géorisques website) it is not accurate enough to describe the phenomenon at the communal and plot level through the Ille-et-Vilaine Departmental authority for inland and marine areas Rennes Métropole and BRGM have joined forces to carry out a study to determine plot by plot those sectors vulnerable to a high and medium risk This study is co-financed up to 60% by the State In order to obtain accurate data on the nature of the ground a team of geophysicists and geologists from BRGM will be visiting certain sectors of the communes of Rennes Métropole during the second half of May and the first half of June The following municipalities are concerned: Le Rheu The data collected will be used to produce a precise map of the shrinkage-swelling of clay The new hazard maps will be available in a public report from BRGM Information leaflets on the phenomenon of clay shrinkage and swelling recommendations and construction measures will also be made available to the population by Rennes Métropole Subscribe to our newsletter and receive our news every month [2020 International Year of Plant Health] The potato is the number one non-grain food crop in the world which farmers have been managing with an arsenal of pesticides for over 70 years the "potato team” has dedicated itself heart and soul to developing alternative strategies This research has resulted in tangible benefits for human health and the environment The team’s work has earned its members the 2019 INRA Science with an Impact Award Members of the "potato team” can be found in Rheu and Ploudaniel the two sites of the Institute for Genetics which is part of the INRAE centre of Brittany-Normandy and the work atmosphere is warm and cheery in both places Receiving a collective award suits the team perfectly The 2019 INRA Science with an Impact Award recognises the team's major contribution to reducing pesticide use even before the issue became a major societal concern "It wasn't always easy to be ahead of the curve,” comments Jean-Eric Chauvin it is important to play the long game when improving a plant's genetic resistance to pathogens and nematodes—can adapt to and circumvent resistance mechanisms the potato team has shown that French populations of blight have adapted to the resistance strategies of the Bintje the most commonly cultivated potato variety in France It must utilise combinations of resistance mechanisms and brainstorm about the conditions under which such mechanisms would be the most durable and effective the team has put into place extensive research programmes that focus on three major tasks The first task is to monitor plant health and safety explains: "Any potato carrying a virus is eliminated because planting an infected tuber means that the virus will be spread to the next round of crops." To monitor pathogen population dynamics the potato team has drawn on its innovative research to develop kits and analytical tools Cultivated varieties are enhanced using a traditional approach: individuals with the most promising traits are crossed Some of the experimental research involved is conducted in the field molecular and cell biology techniques are used to describe key genes and examine their functions Researchers then confirm the presence of these genes in varieties displaying traits of interest a unique virus resistance mechanism was first discovered in a pepper species by scientists at INRAE Avignon; the mechanism's utility was then explored in the potato the team developed nematode-resistant breeding lines These lines all carry a gene whose effect is to change the sex ratio of the roundworms The team also identified a second gene that operates in tandem with the first gene to prevent nematodes from entering potato plants “Many other research groups in Europe are envious of what we have been able to do," remarks Kerlan "We are now trying to see if these mechanisms can help stop outbreaks of virulent nematodes such as those seen in Germany in 2014." To study how nematode populations adapt in the face of plant resistance nematologists carried out experiments in secure facilities where non-virulent nematodes were allowed to interact with resistant plants over several generations the researchers identified and characterised the nematodes that had managed to overcome plant resistance Such work is essential in the quest to discover more effective and longer lasting resistance mechanisms in plants The third task is to ensure the long-term effectiveness of the chosen resistance mechanisms we thought that breeding resistant varieties would spell the end of chemical pesticides plant resistance isn't enough," explains nematologist Sylvain Fournet For pathologist and epidemiologist Didier Andrivon the team's work on plant architecture was a major step forward: more air flow through the plant cover layer reduces humidity levels and thus the risk of leaf infections “By changing plant architecture while also applying other methods moderate levels of disease can be controlled,” comments Claudine Pasco "Farmers must utilise a suite of solutions: they need to select an appropriate variety with a suitable form of resistance such as those that our team is currently developing as was underscored in the recent joint scientific report 'Can organic farming give up copper as a crop protection product?’” If we want farmers to adopt these solutions we must provide support as they change their practices To promote advances in the technical regulations associated with variety registration in the French catalogue Andrivon and other experts helped define the "environmental value” of plant varieties a metric that expresses a variety's level of resistance to both blight and nematodes “This new score has significantly influenced the perspectives of plant breeders who now pay much more attention to this criterion,” he asserts which puts us far ahead of other countries." *Members of ACVNPT (Association for the Creators of New Potato Varieties) The potato is the fourth most important crop in the world Bolstering potato farming in Africa and Asia could help promote food security it is crucial to control the plant’s plethora of pathogens without resorting to chemical pesticides likes to remind people of the potato's historical significance: thanks to Antoine-Augustin Parmentier the plant spread throughout Europe in the late 18th century helping bring an end to famine on the continent The potato is much loved the world over and features prominently in both holiday feasts and everyday meals many other species also have a penchant for the potato a microscopic soil roundworm whose root cysts can last 10 years There are also numerous bacterial species that cause problems for potato storage and that negatively affect potato appearance the potato's number-one enemy remains the water mold responsible for potato blight potato crops are treated 12–22 times per year with fungicides When this pathogen arrived in Europe in 1845 it took out susceptible varieties within the space of just two years One of the consequences was Ireland's Great Famine which resulted in mass emigration to the United States France’s potato plants are in an excellent state of health all thanks to the 1.5 million immunochemical assays performed annually to eliminate individuals that are infected with viral or bacterial pathogens These testing procedures arose from the innovative research carried out by the potato team in the 1980s The team subsequently used genetics to create resistant potatoes a winning approach for more sustainably controlling potato blight and cyst nematodes The team’s detailed understanding of the mechanisms underlying pathogen virulence and evolution is directly responsible for its research successes The potato team is composed of geneticists while others are experts affiliated with FN3PT (the French National Federation of Potato Plant Producers) a group that has provided support for INRAE's research over the last 70 years this collaborative research effort was formalised via the creation of Innoplant ** Unité de Génétique et d'amélioration des fruits et légumes The "4 parts per 1000" initiative proposes to increase organic matter contents and encourage carbon sequestration in soils through the application of appropriate farming and forestry practices The Joint Programming Initiative on Agriculture Food Security and Climate Change (FACCE-JPI) has published its new brochure and a flyer on soil management in the context of climate change mitigation The brochure presents the achievements and future actions of FACCE-JPI and includes several key interviews The flyer highlights some of the research projects that are part of the 'FACCE-JPI Multi-Partner Call on Agricultural Greenhouse Gas Research' Both documents were presented at the International Conference on Agricultural GHG Emissions and Food Security organised by GRA and FACCE-JPI in Berlin (10-13 September 2018) The stability and security of dams are crucial research engineer Claudio Carvajal must visit the site and provide immediate support Retrained Grade One-winning racehorse Saphir Du Rheu has made a phenomenal start to his eventing career jumping clear across country at novice level on his third ever horse trials retired from racing in March 2018 and enjoyed a holiday before starting his new hunting team chasing and eventing career with Charlotte Alexander before storming clear around Nigel Taylor’s British Eventing course at Aston-le-Walls (8 May) in the fastest time of the day They ended the day 13th in the 35-strong section Charlotte told H&H she is “thrilled” with Saphy “Getting any horse to novice I think is a particular achievement but to get a horse that has only been out of training for less than a year to novice on his third run — only his third ever dressage test “A special thank you should go to Brian Hutton at Talland who has been helping me with my dressage and showjumping along with Owen Moore who is also trying to ‘knock off the rough edges’ with my riding “There is still a long way to go and it’s not easy when you have an elderly team chase rider added she will step Saphy back down to BE100 level for his next run He will then move back up to novice at West Wilts the following weekend and is being aimed for Barbury in July “He is much better at novice level in all phases as the dressage gives him more to think about; the showjumping just needs more practice although he is green at the combinations — a little wobbly between the skinny combinations — he has plenty of scope and did find it easy.” previously a Highflyer Bloodstock purchase was gifted to Charlotte on his retirement by the Stewart family who owned him during his racing years In the year since he has been off the track, the popular grey has led The Boring Gorings around open team chase courses, hunted with the Beaufort, and helped Ballycasey — who retired from racing this week — school over Cheltenham’s cross-country fences He also returned to the Cheltenham Festival in March to take part in the Retraining of Racehorses parade and escort Champion Hurdle victor Espoir D’Allen to the winners’ enclosure Remote control: Plouzané may be 'the most remote team in France' but they are enjoying some success Along with its quaint fishing villages and magnificent beaches Brittany boasts four clubs in the French first division – Rennes despite the fact Bretons have a reputation for being a tough race Basques have hot blood but Bretons have hard heads along with a fierce independence – a similarity they share with their Celtic cousins in Cornwall But while rugby has a long and proud tradition among the Cornish in Brittany the sport never established the same foothold even though it was in the far north of the country where rugby was first played in France in the early 1870s In the decades that followed rugby migrated south leaving Brittany to pursue its obsession with the round ball rugby has started to lay down roots in Brittany Nantes hosted three pool matches during the 2007 World Cup and the semi-finals of last season’s Top 14 were also held in the city Harlequins thrashed Racing 32-8 in a Heineken Cup pool match a stunning performance that silenced the 35,000 Bretons who had come to the Stade de la Beaujoire in Nantes who have to travel three hours to play their local derbies Two hundred miles north-west of Nantes is Plouzané a seaside town with a famous old lighthouse and a rugby team that is a beacon of hope for rugby in Brittany The coach of the first XV is Steven Sparks a name that may stir memories for aficionados of English rugby The 39-year-old Sparks began his career propping the scrum of his hometown club before moving to France at the end of the 1990s Spells followed with La Rochelle and Castres (in what was then the Top 16) before he returned to England in 2003 playing in a front row that included Raphael Ibanez and Cobus Visagie before joining Worcester for a couple of years the pull of France proved too much and Sparks returned to finish his career with Morlaàs a Federale One side in the foothills of the Pyrenees Calling time on his career a few years ago Sparks moved to Brittany with his young family (his wife is French) and for a while dabbled in the property business But he missed his rugby and when he heard Plouzané were looking for a full-time coach in 2011 he successfully applied “I’ve got all my coaching badges,” he explains “It took two years in total and was quite difficult Plouzané have enjoyed two promotions and now reside in Federale Three But success brings its own challenges as with every promotion the club is forced to literally broaden its horizons As Midi Olympique explained in a recent feature on Plouzané, the club is the “most isolated” in the French league system, a fact for which Sparks can vouch. “Our local derbies are against Le Rheu and Auray,” he tells Rugby World “They’re both in Brittany but they take about three hours to reach.” which is a round trip of 550 miles – in English terms that’s like going from London to Newcastle and back “For the really long trips we leave on the Saturday play on the Sunday and then drive straight back,” explains Sparks “Often we don’t arrive back until the early hours of Monday morning and a lot of the boys then have to be at work a few hours later.” Such demands naturally affect the demographic of the squad students but the average age is probably around 23 and they’re players who don’t have young families,” says Sparks Then there’s the financial cost of each away game a sum Sparks estimates to be around €3,000 every time they hit the road “We have a lot of small sponsors who help us out and the players also contribute to the costs.” But the dedication, allied to Plouzane’s on-field success, is reaping its rewards The junior section is thriving with more than 160 youngsters playing each week and 80% of the first XV squad have come up through the system Sparks is evidently relishing the challenge of coaching France’s most remote club and for their part Plouzané are delighted to have a coach of his calibre “As much as he is English,” says club president Guillaume Renault so who better to front a new look than England’s man… Sie haben erfolgreich Ihre Einwilligung in die Nutzung von Transfermarkt mit Tracking und Cookies widerrufen Sie können sich jetzt zwischen dem Contentpass-Abo und der Nutzung mit personalisierter Werbung \"our\") of the service provided by this web site (\"Service\") are not responsible for any user-generated content and accounts Content submitted express the views of their author only This Service is only available to users who are at least {age} years old you represent that you are this age or older or otherwise make available to the Service (\"Content\") may be reviewed by staff members All Content you submit or upload may be sent to third-party verification services (including Do not submit any Content that you consider to be 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designated a Privacy Officer who is accountable for Chart’s compliance with this Privacy Policy and PIPEDA If at any time you have any questions or complaints about this Privacy Policy or your personal information and how it is being used collected or disclosed (including if you wish to request to update or correct any personally identifiable information you have provided) or you wish to withdraw your consent to “Use of Personal Information” Chart Communications Inc.5255 Yonge Street Ryerson University's Department of Architectural Science is kicking off its 2015-2016 lecture series this Thursday with a lecture from Nasrine Seraji of Paris-based firm Atelier Seraji Architectes et Associés (ASAA) to be held at 6:30 PM in The Pit (ARC 202) Endlesshaus by Atelier Seraji Architectes et Associés Seraji has participated in lectures and exhibitions worldwide She has taken on several educational roles throughout her career teaching at Columbia University in New York between 1993 and 2001 at the Architectural Association in London as Diploma Unit Master and Princeton University as Visiting Professor She then served as Professor and Chair of the Department of Architecture at Cornell University from 2001 to 2005 before assuming the role of Dean of the École Nationale Supérieure d’Architecture Paris-Malaquais the following year Seraji served as Professor of Ecology as well as Head of the Institute for Art and Architecture at the Academy of Fine Arts in Vienna In January 2014, she was appointed centennial visiting professor of design at Hong Kong University Seraji has overseen a number of acclaimed projects including the award-winning Temporary American Centre in Paris a student housing development in Paris and an extension to the School of Architecture in Lille were nominees for the Mies Van der Rohe Prize Seraji has received numerous awards for her work including the medal for Chevalier des Arts et des Letters from the Minister of Culture in France in 2006 for her role as an architect contributing to excellence in art and humanities she was awarded both the medal of Chevalier dans l’Ordre National du Mérite by Presidential decree and the Medaille d’Argent by the French Academy of Architecture for her contribution to academic endeavours in architecture Some of her most recent awards include a July 2011 Chevalier de l’Ordre National de la Légion d’Honneur and last year's prize of Mention de l’Équerre d’Argent.  Current projects from Nasrine Seraji and Atelier Seraji Architectes et Associés includes a new complex for the Paris Transportation Authority comprised of 212 housing units and a bus depot competitions for student housing and affordable housing and large scale planning projects in the French cities of le Rheu