Single and multiple resistance QTL delay symptom appearance and slow down root colonization by Aphanomyces euteiches in pea near isogenic lines.

BACKGROUND: Understanding the effects of resistance QTL on pathogen development cycle is an important issue for the creation of QTL combination strategies to durably increase disease resistance in plants. The oomycete pathogen Aphanomyces euteiches, causing root rot disease, is one of the major factors limiting the pea crop in the main producing countries. No commercial resistant varieties are currently available in Europe. Resistance alleles at seven main QTL were recently identified and introgressed into pea agronomic lines, resulting in the creation of Near Isogenic Lines (NILs) at the QTL. This study aimed to determine the effect of main A. euteiches resistance QTL in NILs on different steps of the pathogen life cycle. RESULTS: NILs carrying resistance alleles at main QTL in susceptible genetic backgrounds were evaluated in a destructive test under controlled conditions. The development of root rot disease severity and pathogen DNA levels in the roots was measured during ten days after inoculation. Significant effects of several resistance alleles at the two major QTL Ae-Ps7.6 and Ae-Ps4.5 were observed on symptom appearance and root colonization by A. euteiches. Some resistance alleles at three other minor-effect QTL (Ae-Ps2.2, Ae-Ps3.1 and Ae-Ps5.1) significantly decreased root colonization. The combination of resistance alleles at two or three QTL including the major QTL Ae-Ps7.6 (Ae-Ps5.1/Ae-Ps7.6 or Ae-Ps2.2/Ae-Ps3.1/Ae-Ps7.6) had an increased effect on delaying symptom appearance and/or slowing down root colonization by A. euteiches and on plant resistance levels, compared to the effects of individual or no resistance alleles. CONCLUSIONS: This study demonstrated the effects of single or multiple resistance QTL on delaying symptom appearance and/or slowing down colonization by A. euteiches in pea roots, using original plant material and a precise pathogen quantification method. Our findings suggest that single resistance QTL can act on multiple or specific steps of the disease development cycle and that their actions could be pyramided to increase partial resistance in future pea varieties. Further studies are needed to investigate QTL effects on different steps of the pathogen life cycle, as well as the efficiency and durability of pyramiding strategies using QTL which appear to act on the same stage of the pathogen cycle.

Validation of QTL for resistance to Aphanomyces euteiches in different pea genetic backgrounds using near-isogenic lines.

KEY MESSAGE: Marker-assisted backcrossing was used to generate pea NILs carrying individual or combined resistance alleles at main Aphanomyces resistance QTL. The effects of several QTL were successfully validated depending on genetic backgrounds. Quantitative trait loci (QTL) validation is an important and often overlooked step before subsequent research in QTL cloning or marker-assisted breeding for disease resistance in plants. Validation of QTL controlling partial resistance to Aphanomyces root rot, one of the most damaging diseases of pea worldwide, is of major interest for the future development of resistant varieties. The aim of this study was to validate, in different genetic backgrounds, the effects of various resistance alleles at seven main resistance QTL recently identified. Five backcross-assisted selection programs were developed. In each, resistance alleles at one to three of the seven main Aphanomyces resistance QTL were transferred into three genetic backgrounds, including two agronomically important spring (Eden) and winter (Isard) pea cultivars. The subsequent near-isogenic lines (NILs) were evaluated for resistance to two reference strains of the main A. euteiches pathotypes under controlled conditions. The NILs carrying resistance alleles at the major-effect QTL Ae-Ps4.5 and Ae-Ps7.6, either individually or in combination with resistance alleles at other QTL, showed significantly reduced disease severity compared to NILs without resistance alleles. Resistance alleles at some minor-effect QTL, especially Ae-Ps2.2 and Ae-Ps5.1, were also validated for their individual or combined effects on resistance. QTL × genetic background interactions were observed, mainly for QTL Ae-Ps7.6, the effect of which increased in the winter cultivar Isard. The pea NILs are a novel and valuable resource for further understanding the mechanisms underlying QTL and their integration in breeding programs.

AER1, a major gene conferring resistance to Aphanomyces euteiches in Medicago truncatula.

Aphanomyces euteiches is a major soilborne oomycete pathogen that infects various legume species, including pea and alfalfa. The model legume Medicago truncatula has recently emerged as a valuable genetic system for understanding the genetic basis of resistance to A. euteiches in leguminous crops. The objective of this study was to identify genetic determinants of resistance to a broad host-range pea-infecting strain of A. euteiches in M. truncatula. Two M. truncatula segregating populations of 178 F(5) recombinant inbred lines and 200 F(3) families from the cross F83005.5 (susceptible) x DZA045.5 (resistant) were screened for resistance to A. euteiches. Phenotypic distributions observed suggested a dominant monogenic control of resistance. A major locus associated with resistance to A. euteiches, namely AER1, was mapped by bulk segregant analysis to a terminal end of chromosome 3 in M. truncatula and explained 88% of the phenotypic variation. AER1 was identified in a resistance-gene-rich region, where resistance gene analogs and genes associated with disease resistance phenotypes have been identified. Discovery of AER1 opens up new prospects for improving resistance to A. euteiches in cultivated legumes using a comparative genomics approach.

Candidate genes for quantitative resistance to Mycosphaerella pinodes in pea (Pisum sativum L.).

Partial resistance to Mycosphaerella pinodes in pea is quantitatively inherited. Genomic regions involved in resistance (QTLs) have been previously identified in the pea genome, but the molecular basis of the resistance is still unknown. The objective of this study was to map resistance gene analogs (RGA) and defense-related (DR) genes in the JI296 x DP RIL population that has been used for mapping QTLs for resistance to M. pinodes, and identify co-localizations between candidate genes and QTLs. Using degenerate oligonucleotide primers designed on the conserved motifs P-loop and GLPL of cloned resistance genes, we isolated and cloned 16 NBS-LRR sequences, corresponding to five distinct classes of RGAs. Specific second-generation primers were designed for each class. RGAs from two classes were located on the linkage group (LG) VII. Another set of PCR-based markers was designed for four RGA sequences previously isolated in pea and 12 previously cloned DR gene sequences available in databases. Out of the 16 sequences studied, the two RGAs RGA-G3A and RGA2.97 were located on LG VII, PsPRP4A was located on LG II, Peachi21, PsMnSOD, DRR230-b and PsDof1 were mapped on LG III and peabetaglu and DRR49a were located on LG VI. Two co-localizations between candidate genes and QTLs for resistance to M. pinodes were observed on LG III, between the putative transcription factor PsDof1 and the QTL mpIII-1 and between the pea defensin DRR230-b gene and the QTL mpIII-4. Another co-localization was observed on LG VII between a cluster of RGAs and the QTL mpVII-1. The three co-localizations appear to be located in chromosomal regions containing other disease resistance or DR genes, suggesting an important role of these genomic regions in defense responses against pathogens in pea.

Microsatellite marker polymorphism and mapping in pea (Pisum sativum L.).

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.

Consistent Quantitative Trait Loci in Pea for Partial Resistance to Aphanomyces euteiches Isolates from the United States and France.

ABSTRACT Development of pea cultivars resistant to Aphanomyces root rot, the most destructive root disease of pea worldwide, is a major disease management objective. In a previous study of a mapping population of 127 recombinant inbred lines (RILs) derived from the cross 'Puget' (susceptible) x '90-2079' (partially resistant), we identified seven genomic regions, including a major quantitative trait locus (QTL), Aph1, associated with partial resistance to Aphanomyces root rot in U.S. fields (21). The objective of the present study was to evaluate, in the same mapping population, the specificity versus consistency of Aphanomyces resistance QTL under two screening conditions (greenhouse and field, by comparison with the previous study) and with two isolates of Aphanomyces euteiches originating from the United States and France. The 127 RILs were evaluated in the greenhouse for resistance to pure culture isolates SP7 (United States) and Ae106 (France). Using the genetic map previously described, a total of 10 QTL were identified for resistance in greenhouse conditions to the two isolates. Among these were Aph1, Aph2, and Aph3, previously detected for partial field resistance in the United States. Aph1 and Aph3 were detected with both isolates and Aph2 with only the French isolate. Seven additional QTL were specifically detected with one of the two isolates and were not identified for partial field resistance in the United States. The consistency of the detected resistance QTL over two screening environments and isolates is discussed with regard to pathogen variability, and disease assessment and QTL detection methods. This study suggests the usefulness of three consistent QTL, Aph1, Aph2, and Aph3, for marker-assisted selection.

A Cluster of Major Specific Resistance Genes to Leptosphaeria maculans in Brassica napus.

ABSTRACT Two types of genetic resistance to Leptosphaeria maculans usually are distinguished in Brassica napus: qualitative, total resistance expressed at the seedling stage and quantitative, partial resistance expressed at the adult plant stage. The latter is under the control of many genetic factors that have been mapped through quantitative trait loci (QTL) studies using 'Darmor' resistance. The former usually is ascribed to race-specific resistance controlled by single resistance to L. maculans (Rlm) genes. Three B. napus-originating specific Rlm genes (Rlm1, Rlm2, and Rlm4) previously were characterized. Here, we report on the genetic identification of two novel resistance genes, Rlm3 and Rlm7, corresponding to the avirulence genes AvrLm3 and AvrLm7. The identification of a novel L. maculans- B. napus specific interaction allowed the detection of another putative new specific resistance gene, Rlm9. The resistance genes were mapped in two genomic regions on LG10 and LG16 linkage groups. A cluster of five resistance genes (Rlm1, Rlm3, Rlm4, Rlm7, and Rlm9) was strongly suggested on LG10. The relation between all these specific resistance genes and their potential role in adult-plant field resistance is discussed. These two Rlm-carrying regions do not correspond to major QTL for Darmor quantitative resistance.