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

International audienceBackground - 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

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

Diversity and combination of effects and action modes of Aphanomyces root rot resistance QTLl in pea

La connaissance des effets et modes d’action des QTL (Quantitative Trait Loci) de résistance des plantes aux maladies est un enjeu majeur pour l’exploitation durable des résistances génétiques en agriculture. Dans le pathosystème pois/Aphanomyces euteiches, présentant une importance économique majeure, l’objectif de la thèse a consisté à valider les effets de QTL de résistance, seuls ou en combinaison, et à connaitre leurs modes d’action sur les étapes du cycle de l’agent pathogène. Un total de 157 NILs (Near Isogenic Lines) de pois issues d’un programme de Back-cross Assisté par Marqueurs, porteuses d’aucun, un, deux ou trois des sept principaux QTL de résistance préalablement identifiés, a été génotypé à l’aide d’une puce 15K SNPs et évalué pour la résistance. Les NILs porteuses de QTL à effets majeurs, seuls ou en combinaison avec des QTL à effets mineurs,ont présenté des niveaux accrus de résistance partielle en conditions contrôlées et au champ par rapport aux NILs dépourvues de QTL, dans différents fonds génétiques. Certaines NILs comportant des QTL individuels ou combinés à effets mineurs ont également montré un niveau réduit de sévérité de la maladie dans l’une ou les deux conditions de test. La plupart des QTL a présenté des effets significatifs sur le ralentissement de l’infection et/ou de la quantité d’ADN pathogène ayant colonisé la racine pendant sept jours après inoculation. Cette thèse fournit des outils et éléments de choix de QTL à combiner en sélection pour augmenter l’efficacité de la résistance partielle à A. euteiches dans les futures variétés de poisKnowledge of the effects and action modes of resistance QTL to plant diseases is a major challenge for the durable use of genetic resistances in agriculture. In the pea/Aphanomyces euteiches pathosystem, which has a major economic importance, the aim of this study was to validate the single or combined effects of main resistance QTL, and study their action modes on steps of the pathogen life cycle. A total of 157 pea NILs (Near Isogenic Lines) created by Marker-Assisted Back-crossing and carrying no, one, two or three of the seven main resistance QTL previously identified, was genotyped using a 15K SNPs array and evaluated for resistance. The NILs carrying major-effect QTL, individually or in combination with minor-effect QTL, had increased levels of partial resistance in controlledconditions and in the field compared to NILs without QTL, in different genetic backgrounds. Several NILs carrying single or multiple minor-effect QTL also showed reduced levels of disease severity in one or the two test conditions. Most of the QTL had significant effects on slowing down infection and/or pathogen DNA quantity which had colonized the root for seven days after inoculation. This study gives tools and information for the choice of resistance QTL to use in pyramiding breeding strategies for increasing partial levels of resistance to A. euteiches in future pea varieties

Étude statistique et analyse sur la précision des données issues du scanner mobile VLX de NavVis pour la fourniture d'un plan d'architecture au 1/50e de bâtiments divers

The various tests carried out in this study have highlighted the importance of mobile mapping methods, with the VLX, to minimise geometric errors (especially loop closure and control points capture).Experiments were conducted in indoor and outdoor environments (open and closed), with varying degrees of detail. The digitalization of 1:50 scale plans is possible in indoor environments. The drawing of frontage plans at 1:50 scale is also possible for façades with little detail (within the range of the scanner and depending on the type of surface), but it is not recommended for Haussmannian frontispieces. The digitalization of outdoor unenclosed environments is possible as long as the mobile laser scanner has enough points of attachment for the SLAM algorithm to find its way in space, within the range of the sensors (about 30 metres high). Finally, the delivery of a 1:50 scale plan of an enclosed outdoor environment is possible as long as loop closures are performed and as measured control points (therefore known in coordinates) are incorporated in the calculation.Les différents tests menés lors de cette étude ont permis de mettre en avant l’importance de certaines techniques de cartographie dynamique au VLX, permettant de minimiser les erreurs de géométrie (notamment la fermeture de boucle et la prise de points de contrôle).Des expériences ont été conduites dans des environnements intérieurs et extérieurs (ouverts et fermés), plus ou moins riches en détails. La digitalisation de plan au 1/50e est réalisable dans un environnement intérieur. Le dessin de plans de façade au 1/50e est possible pour des façades pauvres en détails (dans la limite de la portée du scanner et suivant le type de surface), mais non recommandé pour des frontispices Haussmanniens. La numérisation d’environnements extérieurs, non clos, est possible du moment que le scanner dynamique dispose d’un nombre de points d’accroches suffisants pour que l’algorithme SLAM se repère dans l’espace, dans les limites de la portée des capteurs (environ 30 mètres de hauteur). Enfin, la livraison de plan au 1/50e d’un environnement extérieur fermé est possible à condition de réaliser une fermeture de boucle et d’incorporer des points de contrôles, connus en coordonnées, dans le calcul

Diversité et combinaison des modes d'actions des QTL de résistance à Aphanomyces euteiches chez le pois

Knowledge of the effects and action modes of resistance QTL to plant diseases is a major challenge for the durable use of genetic resistances in agriculture. In the pea/Aphanomyces euteiches pathosystem, which has a major economic importance, the aim of this study was to validate the single or combined effects of main resistance QTL, and study their action modes on steps of the pathogen life cycle. A total of 157 pea NILs (Near Isogenic Lines) created by Marker-Assisted Back-crossing and carrying no, one, two or three of the seven main resistance QTL previously identified, was genotyped using a 15K SNPs array and evaluated for resistance. The NILs carrying major-effect QTL, individually or in combination with minor-effect QTL, had increased levels of partial resistance in controlledconditions and in the field compared to NILs without QTL, in different genetic backgrounds. Several NILs carrying single or multiple minor-effect QTL also showed reduced levels of disease severity in one or the two test conditions. Most of the QTL had significant effects on slowing down infection and/or pathogen DNA quantity which had colonized the root for seven days after inoculation. This study gives tools and information for the choice of resistance QTL to use in pyramiding breeding strategies for increasing partial levels of resistance to A. euteiches in future pea varieties.La connaissance des effets et modes d’action des QTL (Quantitative Trait Loci) de résistance des plantes aux maladies est un enjeu majeur pour l’exploitation durable des résistances génétiques en agriculture. Dans le pathosystème pois/Aphanomyces euteiches, présentant une importance économique majeure, l’objectif de la thèse a consisté à valider les effets de QTL de résistance, seuls ou en combinaison, et à connaitre leurs modes d’action sur les étapes du cycle de l’agent pathogène. Un total de 157 NILs (Near Isogenic Lines) de pois issues d’un programme de Back-cross Assisté par Marqueurs, porteuses d’aucun, un, deux ou trois des sept principaux QTL de résistance préalablement identifiés, a été génotypé à l’aide d’une puce 15K SNPs et évalué pour la résistance. Les NILs porteuses de QTL à effets majeurs, seuls ou en combinaison avec des QTL à effets mineurs,ont présenté des niveaux accrus de résistance partielle en conditions contrôlées et au champ par rapport aux NILs dépourvues de QTL, dans différents fonds génétiques. Certaines NILs comportant des QTL individuels ou combinés à effets mineurs ont également montré un niveau réduit de sévérité de la maladie dans l’une ou les deux conditions de test. La plupart des QTL a présenté des effets significatifs sur le ralentissement de l’infection et/ou de la quantité d’ADN pathogène ayant colonisé la racine pendant sept jours après inoculation. Cette thèse fournit des outils et éléments de choix de QTL à combiner en sélection pour augmenter l’efficacité de la résistance partielle à A. euteiches dans les futures variétés de poi

Lutte génétique contre l'Aphanomyces du pois

National audienc

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

International audienceAphanomyces 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 x 90-2079 pea recombinant inbred line population identified Ae-Ps4.5 in a 0.8-cM confidence interval with a high effect (R-2 = 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 Cameor 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

Testing of life history traits of a soilborne pathogen in vitro: Do characteristics of oospores change according the strains of Aphanomyces euteiches and the host plant infected by the pathogen?

International audienceAphanomyces euteiches is a polyphagous, homothallic soilborne pathogen producing asexual (zoospores) and sexual (oospores) spores. Even if oospores are essential for disease development and survival, to date, no study has focused on the production rates of oospores or the quality of the offspring produced by oospores. In this study, a nonabrasive oospore extraction method from infected roots of leguminous species (pea, faba bean and vetch) was developed. This methodology includes steps of grinding and filtration. The quality of oospores (viable, dormant and dead) was assessed with tetrazolium bromide staining, and germination of oospores was tested using exudates of peas, faba bean and vetch. The average yield of the extraction method was approximately 21%. Staining revealed some differences between strains and between leguminous species. The germination percentage of oospores extracted from pea, faba bean and vetch was 25%, 62% and 70%, respectively, and a significant difference was observed according to the origin of A. euteiches‐inoculated strains. Application of exudates seems to stimulate the germination of oospores (2% for the control, 18% for pea exudates and 1% for vetch exudates). Differences observed between A. euteiches strains and leguminous species indicate that more knowledge concerning the biology of oospores is needed. This will help to better estimate evolution process of the pathogen and manage resistance and crop successions

Pathotype characterization of Aphanomyces euteiches isolates collected from pea breeding nurseries

International audienceIntroductionAphanomyces 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.MethodsWe 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.ResultsThe 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.DiscussionThese 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

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

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.[br/] 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.[br/] 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