Fine mapping of Ae-Ps4.5, a major locus for resistance to pathotype III of Aphanomyces euteiches in pea.

KEY MESSAGE: QTL mapping and recombinant screening confirmed the major effect of QTL Ae-Ps4.5 on pea resistance to pathotype III of Aphanomyces euteiches and fine-mapped the QTL to a 3.06-Mb interval. Aphanomyces root rot, caused by Aphanomyces euteiches, is the most important disease of pea (Pisum sativum L.) worldwide. The development of pea-resistant varieties is a major challenge to control the disease. Previous linkage studies identified seven main resistance quantitative trait loci (QTL), including the QTL Ae-Ps4.5 associated with partial resistance in US nurseries infested by the pea pathotype III of A. euteiches. This study aimed to confirm the major effect of Ae-Ps4.5 on A. euteiches pathotype III, refine its interval, and identify candidate genes underlying the QTL. QTL mapping on an updated genetic map from the Puget × 90-2079 pea recombinant inbred line population identified Ae-Ps4.5 in a 0.8-cM confidence interval with a high effect (R2 = 89%) for resistance to the Ae109 reference strain of A. euteiches (pathotype III) under controlled conditions. However, the QTL mapping did not detect Ae-Ps4.5 for resistance to the RB84 reference strain of A. euteiches (pathotype I). Screening 224-pea BC5F2 plant progeny derived from three near-isogenic lines (NILs) carrying the 90-2079 allele at Ae-Ps4.5 in the Puget genetic background with 26 SNP markers identified 15 NILs showing recombination in the QTL interval. Phenotyping of the recombinant lines for resistance to the Ae109 strain of A. euteiches reduced the QTL to a physical interval of 3.06 Mb, containing 50 putative annotated genes on the Caméor pea genome V1a among which three candidate genes highlighted. This study provides closely linked SNP markers and putative candidate genes to accelerate pea breeding for resistant varieties to Aphanomyces root rot.

A major-effect genetic locus, ApRVII, controlling resistance against both adapted and non-adapted aphid biotypes in pea.

KEY MESSAGE: A genome-wide association study for pea resistance against a pea-adapted biotype and a non-adapted biotype of the aphid, Acyrthosiphon pisum, identified a genomic region conferring resistance to both biotypes. In a context of reduced insecticide use, the development of cultivars resistant to insect pests is crucial for an integrated pest management. Pea (Pisum sativum) is a crop of major importance among cultivated legumes, for the supply of dietary proteins and nitrogen in low-input cropping systems. However, yields of the pea crop have become unstable due to plant parasites. The pea aphid (Acyrthosiphon pisum) is an insect pest species forming a complex of biotypes, each one adapted to feed on one or a few related legume species. This study aimed to identify resistance to A. pisum and the underlying genetic determinism by examining a collection of 240 pea genotypes. The collection was screened against a pea-adapted biotype and a non-adapted biotype of A. pisum to characterize their resistant phenotype. Partial resistance was observed in some pea genotypes exposed to the pea-adapted biotype. Many pea genotypes were completely resistant to non-adapted biotype, but some exhibited partial susceptibility. A genome-wide association study, using pea exome-capture sequencing data, enabled the identification of the major-effect quantitative trait locus ApRVII on the chromosome 7. ApRVII includes linkage disequilibrium blocks significantly associated with resistance to one or both of the two aphid biotypes studied. Finally, we identified candidate genes underlying ApRVII that are potentially involved in plant-aphid interactions and marker haplotypes linked with aphid resistance. This study sets the ground for the functional characterization of molecular pathways involved in pea defence to the aphids but also is a step forward for breeding aphid-resistant cultivars.

Aggressiveness of Diverse French Aphanomyces euteiches Isolates on Pea Near Isogenic Lines Differing in Resistance Quantitative Trait Loci.

Aphanomyces root rot is a major disease in many pea growing regions worldwide. Development of resistant varieties is necessary to manage the disease. Near isogenic lines (NILs) carrying resistance alleles at main quantitative trait loci (QTLs) were developed by marker-assisted backcrossing. This study aimed to evaluate the aggressiveness of diverse French isolates of Aphanomyces euteiches on NILs carrying different resistance QTLs. Forty-three A. euteiches isolates from different French pea growing regions were tested for aggressiveness on eight NILs carrying single or combinations of resistance QTLs and two susceptible or resistant control lines, in controlled conditions. Three clusters of isolates, unrelated to geographical origin, were identified, including 37, 56, and 7% of isolates with high, moderate, and low average levels of aggressiveness, respectively. Three groups of pea lines were also identified. The first group consisted of a pea resistant control line, moderately to highly resistant to all of the isolates. The second group included five NILs carrying a major-effect resistance allele at QTL Ae-Ps7.6, with a medium to broad range of effects on the isolates. The third group consisted of three NILs carrying minor-effect resistance alleles, with a narrow range of effects on the isolates. The results suggest that highly aggressive isolates occur naturally, which may be selected by future partially resistant pea varieties carrying QTLs and increase the risk of erosion of QTL effect. QTL pyramiding strategies for a higher level and a broader range of effect of quantitative resistance on A. euteiches populations will be required for breeding for durable pea resistant varieties.

Confirmation of Fusarium root rot resistance QTL Fsp-Ps 2.1 of pea under controlled conditions.

BACKGROUND: Dry pea production has increased substantially in North America over the last few decades. With this expansion, significant yield losses have been attributed to an escalation in Fusarium root rots in pea fields. Among the most significant rot rotting pathogenic fungal species, Fusarium solani fsp. pisi (Fsp) is one of the main causal agents of root rot of pea. High levels of partial resistance to Fsp has been identified in plant genetic resources. Genetic resistance offers one of the best solutions to control this root rotting fungus. A recombinant inbred population segregating for high levels of partial resistance, previously single nucleotide polymorphism (SNP) genotyped using genotyping-by-sequencing, was phenotyped for disease reaction in replicated and repeated greenhouse trials. Composite interval mapping was deployed to identify resistance-associated quantitative trait loci (QTL). RESULTS: Three QTL were identified using three disease reaction criteria: root disease severity, ratios of diseased vs. healthy shoot heights and dry plant weights under controlled conditions using pure cultures of Fusarium solani fsp. pisi. One QTL Fsp-Ps 2.1 explains 44.4-53.4% of the variance with a narrow confidence interval of 1.2 cM. The second and third QTL Fsp-Ps3.2 and Fsp-Ps3.3 are closely linked and explain only 3.6-4.6% of the variance. All of the alleles are contributed by the resistant parent PI 180693. CONCLUSION: With the confirmation of Fsp-Ps 2.1 now in two RIL populations, SNPs associated with this region make a good target for marker-assisted selection in pea breeding programs to obtain high levels of partial resistance to Fusarium root rot caused by Fusarium solani fsp. pisi.

A local score approach improves GWAS resolution and detects minor QTL: application to Medicago truncatula quantitative disease resistance to multiple Aphanomyces euteiches isolates.

Quantitative trait loci (QTL) with small effects, which are pervasive in quantitative phenotypic variation, are difficult to detect in genome-wide association studies (GWAS). To improve their detection, we propose to use a local score approach that accounts for the surrounding signal due to linkage disequilibrium, by accumulating association signals from contiguous single markers. Simulations revealed that, in a GWAS context with high marker density, the local score approach outperforms single SNP p-value-based tests for detecting minor QTL (heritability of 5-10%) and is competitive with regard to alternative methods, which also aggregate p-values. Using more than five million SNPs, this approach was applied to identify loci involved in Quantitative Disease Resistance (QDR) to different isolates of the plant root rot pathogen Aphanomyces euteiches, from a GWAS performed on a collection of 174 accessions of the model legume Medicago truncatula. We refined the position of a previously reported major locus, underlying MYB/NB-ARC/tyrosine kinase candidate genes conferring resistance to two closely related A. euteiches isolates belonging to pea pathotype I. We also discovered a diversity of minor resistance QTL, not detected using p-value-based tests, some of which being putatively shared in response to pea (pathotype I and III) and/or alfalfa (race 1 and 2) isolates. Candidate genes underlying these QTL suggest pathogen effector recognition and plant proteasome as key functions associated with M. truncatula resistance to A. euteiches. GWAS on any organism can benefit from the local score approach to uncover many weak-effect QTL.

Development of two major resources for pea genomics: the GenoPea 13.2K SNP Array and a high-density, high-resolution consensus genetic map.

Single 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.

SNP discovery and genetic mapping using genotyping by sequencing of whole genome genomic DNA from a pea RIL population.

BACKGROUND: Progress in genetics and breeding in pea still suffers from the limited availability of molecular resources. SNP markers that can be identified through affordable sequencing processes, without the need for prior genome reduction or a reference genome to assemble sequencing data would allow the discovery and genetic mapping of thousands of molecular markers. Such an approach could significantly speed up genetic studies and marker assisted breeding for non-model species. RESULTS: A total of 419,024 SNPs were discovered using HiSeq whole genome sequencing of four pea lines, followed by direct identification of SNP markers without assembly using the discoSnp tool. Subsequent filtering led to the identification of 131,850 highly designable SNPs, polymorphic between at least two of the four pea lines. A subset of 64,754 SNPs was called and genotyped by short read sequencing on a subpopulation of 48 RILs from the cross 'Baccara' x 'PI180693'. This data was used to construct a WGGBS-derived pea genetic map comprising 64,263 markers. This map is collinear with previous pea consensus maps and therefore with the Medicago truncatula genome. Sequencing of four additional pea lines showed that 33 % to 64 % of the mapped SNPs, depending on the pairs of lines considered, are polymorphic and can therefore be useful in other crosses. The subsequent genotyping of a subset of 1000 SNPs, chosen for their mapping positions using a KASP™ assay, showed that almost all generated SNPs are highly designable and that most (95 %) deliver highly qualitative genotyping results. Using rather low sequencing coverages in SNP discovery and in SNP inferring did not hinder the identification of hundreds of thousands of high quality SNPs. CONCLUSIONS: The development and optimization of appropriate tools in SNP discovery and genetic mapping have allowed us to make available a massive new genomic resource in pea. It will be useful for both fine mapping within chosen QTL confidence intervals and marker assisted breeding for important traits in pea improvement.

Genome-wide association mapping of partial resistance to Aphanomyces euteiches in pea.

BACKGROUND: Genome-wide association (GWA) mapping has recently emerged as a valuable approach for refining the genetic basis of polygenic resistance to plant diseases, which are increasingly used in integrated strategies for durable crop protection. Aphanomyces euteiches is a soil-borne pathogen of pea and other legumes worldwide, which causes yield-damaging root rot. Linkage mapping studies reported quantitative trait loci (QTL) controlling resistance to A. euteiches in pea. However the confidence intervals (CIs) of these QTL remained large and were often linked to undesirable alleles, which limited their application in breeding. The aim of this study was to use a GWA approach to validate and refine CIs of the previously reported Aphanomyces resistance QTL, as well as identify new resistance loci. METHODS: A pea-Aphanomyces collection of 175 pea lines, enriched in germplasm derived from previously studied resistant sources, was evaluated for resistance to A. euteiches in field infested nurseries in nine environments and with two strains in climatic chambers. The collection was genotyped using 13,204 SNPs from the recently developed GenoPea Infinium® BeadChip. RESULTS: GWA analysis detected a total of 52 QTL of small size-intervals associated with resistance to A. euteiches, using the recently developed Multi-Locus Mixed Model. The analysis validated six of the seven previously reported main Aphanomyces resistance QTL and detected novel resistance loci. It also provided marker haplotypes at 14 consistent QTL regions associated with increased resistance and highlighted accumulation of favourable haplotypes in the most resistant lines. Previous linkages between resistance alleles and undesired late-flowering alleles for dry pea breeding were mostly confirmed, but the linkage between loci controlling resistance and coloured flowers was broken due to the high resolution of the analysis. A high proportion of the putative candidate genes underlying resistance loci encoded stress-related proteins and others suggested that the QTL are involved in diverse functions. CONCLUSION: This study provides valuable markers, marker haplotypes and germplasm lines to increase levels of partial resistance to A. euteiches in pea breeding.

Bean pod mottle virus: a new powerful tool for functional genomics studies in Pisum sativum.

Pea (Pisum sativum L.) is an important legume worldwide. The importance of pea in arable rotations and nutritional value for both human and animal consumption have fostered sustained production and different studies to improve agronomic traits of interest. Moreover, complete sequencing of the pea genome is currently underway and will lead to the identification of a large number of genes potentially associated with important agronomic traits. Because stable genetic transformation is laborious for pea, virus-induced gene silencing (VIGS) appears as a powerful alternative technology for determining the function of unknown genes. In this work, we present a rapid and efficient viral inoculation method using DNA infectious plasmids of Bean pod mottle virus (BPMV)-derived VIGS vector. Six pea genotypes with important genes controlling biotic and/or abiotic stresses were found susceptible to BPMV carrying a GFP reporter gene and showed fluorescence in both shoots and roots. In a second step, we investigated 37 additional pea genotypes and found that 30 were susceptible to BPMV and only 7 were resistant. The capacity of BPMV to induce silencing of endogenes was investigated in the most susceptible genotype using two visual reporter genes: PsPDS and PsKORRIGAN1 (PsKOR1) encoding PHYTOENE DESATURASE and a 1,4-ß-D-glucanase, respectively. The features of the 'one-step' BPMV-derived VIGS vector include (i) the ease of rub-inoculation, without any need for biolistic or agro-inoculation procedures, (ii) simple cost-effective procedure and (iii) noninterference of viral symptoms with silencing. These features make BPMV the most adapted VIGS vector in pea to make low- to high-throughput VIGS studies.

Transcriptome sequencing for high throughput SNP development and genetic mapping in Pea.

BACKGROUND: Pea 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. RESULTS: We 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. CONCLUSIONS: Our 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.

High-density genome-wide association mapping implicates an F-box encoding gene in Medicago truncatula resistance to Aphanomyces euteiches.

• The 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 (σ(A)²= 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.

Pathotype characterization of Aphanomyces euteiches isolates collected from pea breeding nurseries.

Introduction: Aphanomyces euteiches Drechsler is an oomycete pathogen that affects legume crops, causing root rot, a severe disease of peas (Pisum sativum L.) worldwide. While significant research progress has been made in breeding pea-resistant varieties, there is still a need for a deeper understanding of the diversity of pathogen populations present in breeding nurseries located in various legume-growing regions around the world. Methods: We analysed the diversity of 51 pea-infecting isolates of A. euteiches, which were recovered from four American (Athena, OR; Le Sueur, MN; Mount Vernon, WA; Pullman, WA) and three French (Riec-sur-Belon, Templeux-le-Guérard, Dijon) resistance screening nurseries. Our study focused on evaluating their aggressiveness on two sets of differential hosts, comprising six pea lines and five Medicago truncatula accessions. Results: The isolates clustered into three groups based on their aggressiveness on the whole pea set, confirming the presence of pathotypes I and III. Pathotype I was exclusive to French isolates and American isolates from Athena and Pullman, while all isolates from Le Sueur belonged to pathotype III. Isolates from both pathotypes were found in Mount Vernon. The M. truncatula set clustered the isolates into three groups based on their aggressiveness on different genotypes within the set, revealing the presence of five pathotypes. All the isolates from the French nurseries shared the same Fr pathotype, showing higher aggressiveness on one particular genotype. In contrast, nearly all-American isolates were assigned to four other pathotypes (Us1, Us2, Us3, Us4), differing in their higher aggressiveness on two to five genotypes. Most of American isolates exhibited higher aggressiveness than French isolates within the M. truncatula set, but showed lower aggressiveness than French isolates within the P. sativum set. Discussion: These results provide valuable insights into A. euteiches pathotypes, against which the QTL and sources of resistance identified in these nurseries displayed effectiveness. They also suggest a greater adaptation of American isolates to alfalfa, a more widely cultivated host in the United States.

QTL meta-analysis provides a comprehensive view of loci controlling partial resistance to Aphanomyces euteiches in four sources of resistance in pea.

BACKGROUND: Development 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. RESULTS: Meta-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. CONCLUSIONS: QTL 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.

Natural diversity in the model legume Medicago truncatula allows identifying distinct genetic mechanisms conferring partial resistance to Verticillium wilt.

Verticillium wilt is a major threat to alfalfa (Medicago sativa) and many other crops. The model legume Medicago truncatula was used as a host for studying resistance and susceptibility to Verticillium albo-atrum. In addition to presenting well-established genetic resources, this wild plant species enables to investigate biodiversity of the response to the pathogen and putative crosstalk between disease and symbiosis. Symptom scoring after root inoculation and modelling of disease curves allowed assessing susceptibility levels in recombinant lines of three crosses between susceptible and resistant lines, in a core collection of 32 lines, and in mutants affected in symbiosis with rhizobia. A GFP-expressing V. albo-atrum strain was used to study colonization of susceptible plants. Symptoms and colonization pattern in infected M. truncatula plants were typical of Verticillium wilt. Three distinct major quantitative trait loci were identified using a multicross, multisite design, suggesting that simple genetic mechanisms appear to control Verticillium wilt resistance in M. truncatula lines A17 and DZA45.5. The disease functional parameters varied largely in lines of the core collection. This biodiversity with regard to disease response encourages the development of association genetics and ecological approaches. Several mutants of the resistant line, impaired in different steps of rhizobial symbiosis, were affected in their response to V. albo-atrum, which suggests that mechanisms involved in the establishment of symbiosis or disease might have some common regulatory control points.

Genetic variability and QTL mapping of freezing tolerance and related traits in Medicago truncatula.

Freezing is a major environmental limitation to crop productivity for a number of species including legumes. We investigated the genetic determinism of freezing tolerance in the model legume Medicago truncatula Gaertn (M. truncatula). After having observed a large variation for freezing tolerance among 15 M. truncatula accessions, the progeny of a F6 recombinant inbred line population, derived from a cross between two accessions, was acclimated to low above-freezing temperatures and assessed for: (a) number of leaves (NOL), leaf area (LA), chlorophyll content index (CCI), shoot and root dry weights (SDW and RDW) at the end of the acclimation period and (b) visual freezing damage (FD) during the freezing treatment and 2 weeks after regrowth and foliar electrolyte leakage (EL) 2 weeks after regrowth. Consistent QTL positions with additive effects for FD were found on LG1, LG4 and LG6, the latter being the most explanatory (R (2) ≈ 40 %). QTL for NOL, QTL for EL, NOL and RDW, and QTL for EL and CCI colocalized with FD QTL on LG1, LG4 and LG6, respectively. Favorable alleles for these additive effects were brought by the same parent suggesting that this accession contributes to superior freezing tolerance by affecting plants' capacity to maintain growth at low above-freezing temperatures. No epistatic effects were found between FD QTL, but for each of the studied traits, 3-6 epistatic effects were detected between loci not detected directly as QTL. These results open the way to the assessment of syntenic relationships between QTL for frost tolerance in M. truncatula and cultivated legume species.

Five Regions of the Pea Genome Co-Control Partial Resistance to D. pinodes, Tolerance to Frost, and Some Architectural or Phenological Traits.

Evidence for reciprocal links between plant responses to biotic or abiotic stresses and architectural and developmental traits has been raised using approaches based on epidemiology, physiology, or genetics. Winter pea has been selected for years for many agronomic traits contributing to yield, taking into account architectural or phenological traits such as height or flowering date. It remains nevertheless particularly susceptible to biotic and abiotic stresses, among which Didymella pinodes and frost are leading examples. The purpose of this study was to identify and resize QTL localizations that control partial resistance to D. pinodes, tolerance to frost, and architectural or phenological traits on pea dense genetic maps, considering how QTL colocalizations may impact future winter pea breeding. QTL analysis revealed five metaQTLs distributed over three linkage groups contributing to both D. pinodes disease severity and frost tolerance. At these loci, the haplotypes of alleles increasing both partial resistance to D. pinodes and frost tolerance also delayed the flowering date, increased the number of branches, and/or decreased the stipule length. These results question both the underlying mechanisms of the joint control of biotic stress resistance, abiotic stress tolerance, and plant architecture and phenology and the methods of marker-assisted selection optimizing stress control and productivity in winter pea breeding.

Advanced backcross QTL analysis and comparative mapping with RIL QTL studies and GWAS provide an overview of QTL and marker haplotype diversity for resistance to Aphanomyces root rot in pea (Pisum sativum).

Aphanomyces euteiches is the most damaging soilborne pea pathogen in France. Breeding of pea resistant varieties combining a diversity of quantitative trait loci (QTL) is a promising strategy considering previous research achievements in dissecting polygenic resistance to A. euteiches. The objective of this study was to provide an overview of the diversity of QTL and marker haplotypes for resistance to A. euteiches, by integrating a novel QTL mapping study in advanced backcross (AB) populations with previous QTL analyses and genome-wide association study (GWAS) using common markers. QTL analysis was performed in two AB populations derived from the cross between the susceptible spring pea variety "Eden" and the two new sources of partial resistance "E11" and "LISA". The two AB populations were genotyped using 993 and 478 single nucleotide polymorphism (SNP) markers, respectively, and phenotyped for resistance to A. euteiches in controlled conditions and in infested fields at two locations. GWAS and QTL mapping previously reported in the pea-Aphanomyces collection and from four recombinant inbred line (RIL) populations, respectively, were updated using a total of 1,850 additional markers, including the markers used in the Eden x E11 and Eden x LISA populations analysis. A total of 29 resistance-associated SNPs and 171 resistance QTL were identified by GWAS and RIL or AB QTL analyses, respectively, which highlighted 10 consistent genetic regions confirming the previously reported QTL. No new consistent resistance QTL was detected from both Eden x E11 and Eden x LISA AB populations. However, a high diversity of resistance haplotypes was identified at 11 linkage disequilibrium (LD) blocks underlying consistent genetic regions, especially in 14 new sources of resistance from the pea-Aphanomyces collection. An accumulation of favorable haplotypes at these 11 blocks was confirmed in the most resistant pea lines of the collection. This study provides new SNP markers and rare haplotypes associated with the diversity of Aphanomyces root rot resistance QTL investigated, which will be useful for QTL pyramiding strategies to increase resistance levels in future pea varieties.

New consistent QTL in pea associated with partial resistance to Aphanomyces euteiches in multiple French and American environments.

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 complex genetic network involving a broad-spectrum locus and strain-specific loci controls resistance to different pathotypes of Aphanomyces euteiches in Medicago truncatula.

A higher understanding of genetic and genomic bases of partial resistance in plants and their diversity regarding pathogen variability is required for a more durable management of resistance genetic factors in sustainable cropping systems. In this study, we investigated the diversity of genetic factors involved in partial resistance to Aphanomyces euteiches, a very damaging pathogen on pea and alfalfa, in Medicago truncatula. A mapping population of 178 recombinant inbred lines, from the cross F83005.5 (susceptible) and DZA045.5 (resistant), was used to identify quantitative trait loci for resistance to four A. euteiches reference strains belonging to the four main pathotypes currently known on pea and alfalfa. A major broad-spectrum genomic region, previously named AER1, was localized to a reduced 440 kb interval on chromosome 3 and was involved in complete or partial resistance, depending on the A. euteiches strain. We also identified 21 additive and/or epistatic genomic regions specific to one or two strains, several of them being anchored to the M. truncatula physical map. These results show that, in M. truncatula, a complex network of genetic loci controls partial resistance to different pea and alfalfa pathotypes of A. euteiches, suggesting a diversity of molecular mechanisms underlying partial resistance.

Partial resistance of Medicago truncatula to Aphanomyces euteiches is associated with protection of the root stele and is controlled by a major QTL rich in proteasome-related genes.

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.