This paper aims at providing reliable and cost effective genotyping conditions, level of polymorphism in a range of genotypes and map position of newly developed microsatellite markers in order to promote broad application of these markers as a common set for genetic studies in pea. Optimal PCR conditions were determined for 340 microsatellite markers based on amplification in eight genotypes. Levels of polymorphism were determined for 309 of these markers. Compared to data obtained for other species, levels of polymorphism detected in a panel of eight genotypes were high with a mean number of 3.8 alleles per polymorphic locus and an average PIC value of 0.62, indicating that pea represents a rather polymorphic autogamous species. One of our main objectives was to locate a maximum number of microsatellite markers on the pea genetic map. Data obtained from three different crosses were used to build a composite genetic map of 1,430 cM (Haldane) comprising 239 microsatellite markers. These include 216 anonymous SSRs developed from enriched genomic libraries and 13 SSRs located in genes. The markers are quite evenly distributed throughout the seven linkage groups of the map, with 85% of intervals between the adjacent SSR markers being smaller than 10 cM. There was a good conservation of marker order and linkage group assignment across the three populations. In conclusion, we hope this report will promote wide application of these markers and will allow information obtained by different laboratories worldwide in diverse fields of pea genetics, such as QTL mapping studies and genetic resource surveys, to be easily aligned.

SUMMARYSingle nucleotide polymorphism (SNP) arrays represent important genotyping tools for innovative strategies in both basic research and applied breeding. Pea is an important food, feed and sustainable crop with a large (about 4.45 Gbp) but not yet available genome sequence. In the present study, 12 pea recombinant inbred line populations were genotyped using the newly developed GenoPea 13.2K SNP Array. Individual and consensus genetic maps were built providing insights into the structure and organization of the pea genome. Largely collinear genetic maps of 3918-8503 SNPs were obtained from all mapping populations, and only two of these exhibited putative chromosomal rearrangement signatures. Similar distortion patterns in different populations were noted. A total of 12 802 transcript-derived SNP markers placed on a 15 079-marker high-density, high-resolution consensus map allowed the identification of ohnologue-rich regions within the pea genome and the localization of local duplicates. Dense syntenic networks with sequenced legume genomes were further established, paving the way for the identification of the molecular bases of important agronomic traits segregating in the mapping populations. The information gained on the structure and organization of the genome from this research will undoubtedly contribute to the understanding of the evolution of the pea genome and to its assembly. The GenoPea 13.2K SNP Array and individual and consensus genetic maps are valuable genomic tools for plant scientists to strengthen pea as a model for genetics and physiology and enhance breeding.

BackgroundPea has a complex genome of 4.3 Gb for which only limited genomic resources are available to date. Although SNP markers are now highly valuable for research and modern breeding, only a few are described and used in pea for genetic diversity and linkage analysis.ResultsWe developed a large resource by cDNA sequencing of 8 genotypes representative of modern breeding material using the Roche 454 technology, combining both long reads (400 bp) and high coverage (3.8 million reads, reaching a total of 1,369 megabases). Sequencing data were assembled and generated a 68 K unigene set, from which 41 K were annotated from their best blast hit against the model species Medicago truncatula. Annotated contigs showed an even distribution along M. truncatula pseudochromosomes, suggesting a good representation of the pea genome. 10 K pea contigs were found to be polymorphic among the genetic material surveyed, corresponding to 35 K SNPs.We validated a subset of 1538 SNPs through the GoldenGate assay, proving their ability to structure a diversity panel of breeding germplasm. Among them, 1340 were genetically mapped and used to build a new consensus map comprising a total of 2070 markers. Based on blast analysis, we could establish 1252 bridges between our pea consensus map and the pseudochromosomes of M. truncatula, which provides new insight on synteny between the two species.ConclusionsOur approach created significant new resources in pea, i.e. the most comprehensive genetic map to date tightly linked to the model species M. truncatula and a large SNP resource for both academic research and breeding.

Partial resistances, often controlled by quantitative trait loci (QTL), are considered to be more durable than monogenic resistances. Therefore, a precursor to developing efficient breeding programs for polygenic resistance to pathogens should be a greater understanding of genetic diversity and stability of resistance QTL in plants. In this study, we deciphered the diversity and stability of resistance QTL to Aphanomyces euteiches in pea towards pathogen variability, environments and scoring criteria, from two new sources of partial resistance (PI 180693 and 552), effective in French and USA infested fields. Two mapping populations of 178 recombinant inbred lines each, derived from crosses between 552 or PI 180693 (partially resistant) and Baccara (susceptible), were used to identify QTL for Aphanomyces root rot resistance in controlled and in multiple French and USA field conditions using several resistance criteria. We identified a total of 135 additive-effect QTL corresponding to 23 genomic regions and 13 significant epistatic interactions associated with partial resistance to A. euteiches in pea. Among the 23 additive-effect genomic regions identified, five were consistently detected, and showed highly stable effects towards A. euteiches strains, environments, resistance criteria, condition tests and RIL populations studied. These results confirm the complexity of inheritance of partial resistance to A. euteiches in pea and provide good bases for the choice of consistent QTL to use in marker-assisted selection schemes to increase current levels of resistance to A. euteiches in pea breeding programs.

A pathosystem between Aphanomyces euteiches, the causal agent of pea root rot disease, and the model legume Medicago truncatula was developed to gain insights into mechanisms involved in resistance to this oomycete. The F83005.5 French accession and the A17-Jemalong reference line, susceptible and partially resistant, respectively, to A. euteiches, were selected for further cytological and genetic analyses. Microscopy analyses of thin root sections revealed that a major difference between the two inoculated lines occurred in the root stele, which remained pathogen free in A17. Striking features were observed in A17 roots only, including i) frequent pericycle cell divisions, ii) lignin deposition around the pericycle, and iii) accumulation of soluble phenolic compounds. Genetic analysis of resistance was performed on an F7 population of 139 recombinant inbred lines and identified a major quantitative trait locus (QTL) near the top of chromosome 3. A second study, with near-isogenic line responses to A. euteiches confirmed the role of this QTL in expression of resistance. Fine-mapping allowed the identification of a 135-kb sequenced genomic DNA region rich in proteasome-related genes. Most of these genes were shown to be induced only in inoculated A17. Novel mechanisms possibly involved in the observed partial resistance are proposed.

Aphanomyces root rot, caused by Aphanomyces euteiches Drechs, is the most-important disease of pea ( Pisum sativum L.) worldwide. No efficient chemicals are available to control the pathogen. To facilitate breeding for Aphanomyces root rot resistance and to better understand the inheritance of partial resistance, our goal was to identify QTLs associated with field partial resistance. A population of 127 RILs from the cross Puget (susceptible) x 90-2079 (partially resistant) was used. The lines were assessed for resistance to A. euteiches under field conditions at two locations in the United States (Pullman, Wash. and LeSueur, Minn.) in 1996 and 1998 for three criteria based on symptom intensity and disease effects on the whole plant. The RILs were genotyped using automated AFLPs, RAPDs, SSRs, ISSRs, STSs, isozymes and morphological markers. The resulting genetic map consisted of 324 linked markers distributed over 13 linkage groups covering 1,094 cM (Kosambi). Twenty seven markers were anchored to other published pea genetic maps. A total of seven genomic regions were associated with Aphanomyces root rot resistance. The first one, located on LG IVb and named Aph1, was considered as "major" since it was highly consistent over the years, locations and resistance criteria studied, and it explained up to 47% of the variation in the 1998 Minnesota trial. Two other year-specific QTLs, namely Aph2 and Aph3, were revealed from different scoring criteria on LG V and Ia, respectively. Aph2 and Aph3 mapped near the r (wrinkled/round seeds) and af (normal/afila leaves) genes, and accounted for up to 32% and 11% of the variation, respectively. Four other "minor" QTLs, identified on LG Ib, VII and B, were specific to one environment and one resistance criterion. The resistance alleles of Aph3 and the two "minor" QTLs on LG Ib were derived from the susceptible parent. Flanking markers for the major Aphanomyces resistance QTL, Aph1, have been identified for use in marker-assisted selection to improve breeding efficiency.

BackgroundDevelopment of durable plant genetic resistance to pathogens through strategies of QTL pyramiding and diversification requires in depth knowledge of polygenic resistance within the available germplasm. Polygenic partial resistance to Aphanomyces root rot, caused by Aphanomyces euteiches, one of the most damaging pathogens of pea worldwide, was previously dissected in individual mapping populations. However, there are no data available regarding the diversity of the resistance QTL across a broader collection of pea germplasm. In this study, we performed a meta-analysis of Aphanomyces root rot resistance QTL in the four main sources of resistance in pea and compared their genomic localization with genes/QTL controlling morphological or phenological traits and with putative candidate genes.ResultsMeta-analysis, conducted using 244 individual QTL reported previously in three mapping populations (Puget x 90–2079, Baccara x PI180693 and Baccara x 552) and in a fourth mapping population in this study (DSP x 90–2131), resulted in the identification of 27 meta-QTL for resistance to A. euteiches. Confidence intervals of meta-QTL were, on average, reduced four-fold compared to mean confidence intervals of individual QTL. Eleven consistent meta-QTL, which highlight seven highly consistent genomic regions, were identified. Few meta-QTL specificities were observed among mapping populations, suggesting that sources of resistance are not independent. Seven resistance meta-QTL, including six of the highly consistent genomic regions, co-localized with six of the meta-QTL identified in this study for earliness and plant height and with three morphological genes (Af, A, R). Alleles contributing to the resistance were often associated with undesirable alleles for dry pea breeding. Candidate genes underlying six main meta-QTL regions were identified using colinearity between the pea and Medicago truncatula genomes.ConclusionsQTL meta-analysis provided an overview of the moderately low diversity of loci controlling partial resistance to A. euteiches in four main sources of resistance in pea. Seven highly consistent genomic regions with potential use in marker-assisted-selection were identified. Confidence intervals at several main QTL regions were reduced and co-segregation among resistance and morphological/phenological alleles was identified. Further work will be required to identify the best combinations of QTL for durably increasing partial resistance to A. euteiches.

SummaryThe use of quantitative disease resistance (QDR) is a promising strategy for promoting durable resistance to plant pathogens, but genes involved in QDR are largely unknown. To identify genetic components and accelerate improvement of QDR in legumes to the root pathogen Aphanomyces euteiches, we took advantage of both the recently generated massive genomic data for Medicago truncatula and natural variation of this model legume.A high-density (%5.1 million single nucleotide polymorphisms (SNPs)) genome-wide association study (GWAS) was performed with both in vitro and glasshouse phenotyping data collected for 179 lines.GWAS identified several candidate genes and pinpointed two independent major loci on the top of chromosome 3 that were detected in both phenotyping methods. Candidate SNPs in the most significant locus (r 2 A = 23%) were in the promoter and coding regions of an F-box protein coding gene. Subsequent qRT-PCR and bioinformatic analyses performed on 20 lines demonstrated that resistance is associated with mutations directly affecting the interaction domain of the F-box protein rather than gene expression.These results refine the position of previously identified QTL to specific candidate genes, suggest potential molecular mechanisms, and identify new loci explaining QDR against A. euteiches.