Highly-multiplexed SNP genotyping for genetic mapping and germplasm diversity studies in pea

Background: Single Nucleotide Polymorphisms (SNPs) can be used as genetic markers for applications such as genetic diversity studies or genetic mapping. New technologies now allow genotyping hundreds to thousands of SNPs in a single reaction. In order to evaluate the potential of these technologies in pea, we selected a custom 384-SNP set using SNPs discovered in Pisum through the resequencing of gene fragments in different genotypes and by compiling genomic sequence data present in databases. We then designed an Illumina GoldenGate assay to genotype both a Pisum germplasm collection and a genetic mapping population with the SNP set. Results: We obtained clear allelic data for more than 92% of the SNPs (356 out of 384). Interestingly, the technique was successful for all the genotypes present in the germplasm collection, including those from species or subspecies different from the P. sativum ssp sativum used to generate sequences. By genotyping the mapping population with the SNP set, we obtained a genetic map and map positions for 37 new gene markers. Conclusion: Our results show that the Illumina GoldenGate assay can be used successfully for high-throughput SNP genotyping of diverse germplasm in pea. This genotyping approach will simplify genotyping procedures for association mapping or diversity studies purposes and open new perspectives in legume genomics

An EST-SSR Linkage Map of Raphanus sativus and Comparative Genomics of the Brassicaceae†

Raphanus sativus (2n = 2x = 18) is a widely cultivated member of the family Brassicaceae, for which genomic resources are available only to a limited extent in comparison to many other members of the family. To promote more genetic and genomic studies and to enhance breeding programmes of R. sativus, we have prepared genetic resources such as complementary DNA libraries, expressed sequences tags (ESTs), simple sequence repeat (SSR) markers and a genetic linkage map. A total of 26 606 ESTs have been collected from seedlings, roots, leaves, and flowers, and clustered into 10 381 unigenes. Similarities were observed between the expression patterns of transcripts from R. sativus and those from representative members of the genera Arabidopsis and Brassica, indicating their functional relatedness. The EST sequence data were used to design 3800 SSR markers and consequently 630 polymorphic SSR loci and 213 reported marker loci have been mapped onto nine linkage groups, covering 1129.2 cM with an average distance of 1.3 cM between loci. Comparison of the mapped EST-SSR marker positions in R. sativus with the genome sequence of A. thaliana indicated that the Brassicaceae members have evolved from a common ancestor. It appears that genomic fragments corresponding to those of A. thaliana have been doubled and tripled in R. sativus. The genetic map developed here is expected to provide a standard map for the genetics, genomics, and molecular breeding of R. sativus as well as of related species. The resources are available at http://marker.kazusa.or.jp/Daikon

A SNP-based consensus genetic map for synteny-based trait targeting in faba bean (Vicia faba L.)

Faba bean (Vicia faba L.) is a globally important nitrogen-fixing legume, which is widely grown in a diverse range of environments. In this work, we mine and validate a set of 845 SNPs from the aligned transcriptomes of two contrasting inbred lines. Each V. faba SNP is assigned by BLAST analysis to a single Medicago orthologue. This set of syntenically anchored polymorphisms were then validated as individual KASP assays, classified according to their informativeness and performance on a panel of 37 inbred lines, and the best performing 757 markers used to genotype six mapping populations. The six resulting linkage maps were merged into a single consensus map on which 687 SNPs were placed on six linkage groups, each presumed to correspond to one of the six V. faba chromosomes. This sequence-based consensus map was used to explore synteny with the most closely related crop species, lentil and the most closely related fully sequenced genome, Medicago. Large tracts of uninterrupted colinearity were found between faba bean and Medicago, making it relatively straightforward to predict gene content and order in mapped genetic interval. As a demonstration of this, we mapped a flower colour gene to a 2-cM interval of Vf chromosome 2 which was highly colinear with Mt3. The obvious candidate gene from 78 gene models in the collinear Medicago chromosome segment was the previously characterized MtWD40-1 gene controlling anthocyanin production in Medicago and resequencing of the Vf orthologue showed a putative causative deletion of the entire 50 end of the gene.Peer reviewe

SNP Discovery and Linkage Map Construction in Cultivated Tomato

Few intraspecific genetic linkage maps have been reported for cultivated tomato, mainly because genetic diversity within Solanum lycopersicum is much less than that between tomato species. Single nucleotide polymorphisms (SNPs), the most abundant source of genomic variation, are the most promising source of polymorphisms for the construction of linkage maps for closely related intraspecific lines. In this study, we developed SNP markers based on expressed sequence tags for the construction of intraspecific linkage maps in tomato. Out of the 5607 SNP positions detected through in silico analysis, 1536 were selected for high-throughput genotyping of two mapping populations derived from crosses between ‘Micro-Tom’ and either ‘Ailsa Craig’ or ‘M82’. A total of 1137 markers, including 793 out of the 1338 successfully genotyped SNPs, along with 344 simple sequence repeat and intronic polymorphism markers, were mapped onto two linkage maps, which covered 1467.8 and 1422.7 cM, respectively. The SNP markers developed were then screened against cultivated tomato lines in order to estimate the transferability of these SNPs to other breeding materials. The molecular markers and linkage maps represent a milestone in the genomics and genetics, and are the first step toward molecular breeding of cultivated tomato. Information on the DNA markers, linkage maps, and SNP genotypes for these tomato lines is available at http://www.kazusa.or.jp/tomato/

SNiPlay: a web-based tool for detection, management and analysis of SNPs. Application to grapevine diversity projects

<p>Abstract</p> <p>Background</p> <p>High-throughput re-sequencing, new genotyping technologies and the availability of reference genomes allow the extensive characterization of Single Nucleotide Polymorphisms (SNPs) and insertion/deletion events (indels) in many plant species. The rapidly increasing amount of re-sequencing and genotyping data generated by large-scale genetic diversity projects requires the development of integrated bioinformatics tools able to efficiently manage, analyze, and combine these genetic data with genome structure and external data.</p> <p>Results</p> <p>In this context, we developed SNiPlay, a flexible, user-friendly and integrative web-based tool dedicated to polymorphism discovery and analysis. It integrates:</p> <p>1) a pipeline, freely accessible through the internet, combining existing softwares with new tools to detect SNPs and to compute different types of statistical indices and graphical layouts for SNP data. From standard sequence alignments, genotyping data or Sanger sequencing traces given as input, SNiPlay detects SNPs and indels events and outputs submission files for the design of Illumina's SNP chips. Subsequently, it sends sequences and genotyping data into a series of modules in charge of various processes: physical mapping to a reference genome, annotation (genomic position, intron/exon location, synonymous/non-synonymous substitutions), SNP frequency determination in user-defined groups, haplotype reconstruction and network, linkage disequilibrium evaluation, and diversity analysis (Pi, Watterson's Theta, Tajima's D).</p> <p>Furthermore, the pipeline allows the use of external data (such as phenotype, geographic origin, taxa, stratification) to define groups and compare statistical indices.</p> <p>2) a database storing polymorphisms, genotyping data and grapevine sequences released by public and private projects. It allows the user to retrieve SNPs using various filters (such as genomic position, missing data, polymorphism type, allele frequency), to compare SNP patterns between populations, and to export genotyping data or sequences in various formats.</p> <p>Conclusions</p> <p>Our experiments on grapevine genetic projects showed that SNiPlay allows geneticists to rapidly obtain advanced results in several key research areas of plant genetic diversity. Both the management and treatment of large amounts of SNP data are rendered considerably easier for end-users through automation and integration. Current developments are taking into account new advances in high-throughput technologies.</p> <p>SNiPlay is available at: <url>http://sniplay.cirad.fr/</url>.</p

Development and implementation of a highly-multiplexed SNP array for genetic mapping in maritime pine and comparative mapping with loblolly pine

<p>Abstract</p> <p>Background</p> <p>Single nucleotide polymorphisms (SNPs) are the most abundant source of genetic variation among individuals of a species. New genotyping technologies allow examining hundreds to thousands of SNPs in a single reaction for a wide range of applications such as genetic diversity analysis, linkage mapping, fine QTL mapping, association studies, marker-assisted or genome-wide selection. In this paper, we evaluated the potential of highly-multiplexed SNP genotyping for genetic mapping in maritime pine (<it>Pinus pinaster </it>Ait.), the main conifer used for commercial plantation in southwestern Europe.</p> <p>Results</p> <p>We designed a custom GoldenGate assay for 1,536 SNPs detected through the resequencing of gene fragments (707 <it>in vitro </it>SNPs/Indels) and from Sanger-derived Expressed Sequenced Tags assembled into a unigene set (829 <it>in silico </it>SNPs/Indels). Offspring from three-generation outbred (G2) and inbred (F2) pedigrees were genotyped. The success rate of the assay was 63.6% and 74.8% for <it>in silico </it>and <it>in vitro </it>SNPs, respectively. A genotyping error rate of 0.4% was further estimated from segregating data of SNPs belonging to the same gene. Overall, 394 SNPs were available for mapping. A total of 287 SNPs were integrated with previously mapped markers in the G2 parental maps, while 179 SNPs were localized on the map generated from the analysis of the F2 progeny. Based on 98 markers segregating in both pedigrees, we were able to generate a consensus map comprising 357 SNPs from 292 different loci. Finally, the analysis of sequence homology between mapped markers and their orthologs in a <it>Pinus taeda </it>linkage map, made it possible to align the 12 linkage groups of both species.</p> <p>Conclusions</p> <p>Our results show that the GoldenGate assay can be used successfully for high-throughput SNP genotyping in maritime pine, a conifer species that has a genome seven times the size of the human genome. This SNP-array will be extended thanks to recent sequencing effort using new generation sequencing technologies and will include SNPs from comparative orthologous sequences that were identified in the present study, providing a wider collection of anchor points for comparative genomics among the conifers.</p

Genomics-assisted breeding in four major pulse crops of developing countries: present status and prospects

The global population is continuously increasing and is expected to reach nine billion by 2050. This huge population pressure will lead to severe shortage of food, natural resources and arable land. Such an alarming situation is most likely to arise in developing countries due to increase in the proportion of people suffering from protein and micronutrient malnutrition. Pulses being a primary and affordable source of proteins and minerals play a key role in alleviating the protein calorie malnutrition, micronutrient deficiencies and other undernourishment-related issues. Additionally, pulses are a vital source of livelihood generation for millions of resource-poor farmers practising agriculture in the semi-arid and sub-tropical regions. Limited success achieved through conventional breeding so far in most of the pulse crops will not be enough to feed the ever increasing population. In this context, genomics-assisted breeding (GAB) holds promise in enhancing the genetic gains. Though pulses have long been considered as orphan crops, recent advances in the area of pulse genomics are noteworthy, e.g. discovery of genome-wide genetic markers, high-throughput genotyping and sequencing platforms, high-density genetic linkage/QTL maps and, more importantly, the availability of whole-genome sequence. With genome sequence in hand, there is a great scope to apply genome-wide methods for trait mapping using association studies and to choose desirable genotypes via genomic selection. It is anticipated that GAB will speed up the progress of genetic improvement of pulses, leading to the rapid development of cultivars with higher yield, enhanced stress tolerance and wider adaptability

Féverole : des outils génétiques d'aide à la sélection

National audienceLa féverole est une légumineuse, protéagineuse, partiellement allogame. Pour soutenir cette culture, les partenaires de la filière (organismes de recherche publique, instituts techniques, interprofession et sélectionneurs privés) se fixent les objectifs suivants : (1) Améliorer la qualité nutritionnelle de la graine Des variétés FEVITA®, sans tanin et à faible teneur en vicine-convicine, sont disponibles. Cette composition améliorée de la graine assure une valorisation optimale des nutriments par les animaux monogastriques et une réduction du favisme, chez l’Homme. Des outils de diagnostic pour la teneur en vicine-convicine sont disponibles : la méthode quantitative ‘HPLC’ ou la méthode rapide qualitative ‘spectrométrie de masse’ ; les marqueurs moléculaires. (2) Stabiliser les rendements a) Améliorer la tolérance aux bruches La bruche affecte la qualité visuelle et sanitaire des graines, ce qui impacte le débouché vers le marché de l’alimentation humaine (≤ 3% de graines bruchées). Et les matières actives sont inefficaces ! • Pour étudier le déterminisme génétique de la tolérance aux bruches, 2 géniteurs sont utilisés pour construire des populations de lignées recombinantes. - Les dégâts sur les graines sont évalués par tomographie. Cette méthode haut-débit de phénotypage permet de quantifier le volume des galeries réalisées par les bruches, - Un génotypage et une détection de QTL sont réalisés, - Des études transcriptomiques et protéomiques sont réalisées dans les conditions de présence versus quasi-absence de bruches, - Les Composés Organiques Volatiles sont analysés et comparés à deux stades de développement des plantes. • Pour aborder la génétique d’association sur génome entier (GWAS), le phénotypage de la tolérance aux bruches et un séquençage d’exomes sont réalisés sur une Collection de Référence de 248 accessions. b) Améliorer la résistance au froid Les politiques actuelles encouragent la culture des protéagineux, mais la féverole est confrontée à la rigueur climatique de l’hiver dans les régions du centre et du Nord-Est de la France. • Pour étudier le déterminisme génétique de la résistance au froid et détecter des QTL : - Un géniteur (-20°C) est utilisé pour construire des populations de lignées recombinantes. Les RILs sont phénotypées (échelle de notation visuelle des dégâts de froid) et génotypées. - La Collection de Référence est utilisée pour la GWAS. • Pour fournir des géniteurs de résistance au froid, un programme de sélection phénotypique de populations et de lignées est en cours, via un travail d’évaluation multilocal

La bruche, état des lieux en France : biologie de l’insecte, solutions actuelles limitées, pistes de recherche

La vente des graines de féveroles, en alimentation humaine est le plus rémunérateur mais exige un bon niveau de qualité visuelle avec un taux maximum de 3% de graines bruchées. Les dégâts de bruches sont des taches et des cavités circulaires sur les graines matures. Rappel du cycle de vie et des différents stades de développements de l’insecte, de sa répartition géographique en France. La forte pression bruche et les difficultés de maîtrise du ravageur présentent un frein à la culture de féverole en France. La bruche de la féverole est présente sur tout le territoire, tandis que la bruche du pois étant, à l’heure actuelle surtout présente dans le sud de la France, mais présence sur le territoire s’agrandit, et remonte vers le nord, année après année. La lutte chimique en culture et lors du stockage est la seule solution actuelle contre la bruche. Les matières actives disponibles pour les producteurs de pois et de féveroles sont limitées, peu efficaces et la loi abeille engendre des limites réglementaires à l’utilisation des insecticides en culture. Pour lors, il n’y a pas de variétés commercialisées de féveroles ni de pois tolérantes aux bruches. Vers des recherches de solutions à moyen terme: La sélection et la recherche se tournent vers de nouvelles pistes pour lutter contre les bruches. L’étude de nouveaux génotypes de féverole tolérants aux bruches, l’intégration de ces génotypes d’intérêt dans des programmes de création variétale, la recherche de gènes impliqués dans les mécanismes de tolérance aux bruches, utilisation de la sélection assistée par marqueurs pour compléter la sélection variétale classique. Enfin une dernière piste prometteuse est la mise au point d’un attractif de synthèse pour piéger les bruches de la féverole. En effet les bruches sont attirées par l’odeur émise par les plantes à la floraison, les composés organiques volatiles pourraient donc devenir de vrais outils de lutte contre ces ravageurs (voir présentation Ené Leppik INRA)

DNA Analysis of Herbarium Specimens of the Grass Weed <em>Alopecurus myosuroides</em> Reveals Herbicide Resistance Pre-Dated Herbicides

International audienceAcetyl-CoA carboxylase (ACCase) alleles carrying one point mutation that confers resistance to herbicides have been identified in arable grass weed populations where resistance has evolved under the selective pressure of herbicides. In an effort to determine whether herbicide resistance evolves from newly arisen mutations or from standing genetic variation in weed populations, we used herbarium specimens of the grass weed Alopecurus myosuroides to seek mutant ACCase alleles carrying an isoleucine-to-leucine substitution at codon 1781 that endows herbicide resistance. These specimens had been collected between 1788 and 1975, i.e., prior to the commercial release of herbicides inhibiting ACCase. Among the 734 specimens investigated, 685 yielded DNA suitable for PCR. Genotyping the ACCase locus using the derived Cleaved Amplified Polymorphic Sequence (dCAPS) technique identified one heterozygous mutant specimen that had been collected in 1888. Occurrence of a mutant codon encoding a leucine residue at codon 1781 at the heterozygous state was confirmed in this specimen by sequencing, clearly demonstrating that resistance to herbicides can pre-date herbicides in weeds. We conclude that point mutations endowing resistance to herbicides without having associated deleterious pleiotropic effects can be present in weed populations as part of their standing genetic variation, in frequencies higher than the mutation frequency, thereby facilitating their subsequent selection by herbicide applications