Analysis and Identification of QTL for Resistance to Sclerotinia sclerotiorum in Pea (Pisum sativum L.).

White mold caused by Sclerotinia sclerotiorum is an important constraint to field pea (Pisum sativum L.) production worldwide. To transfer white mold resistance into an adapted background, and study the genetics of the disease, two recombinant inbred line (RIL) populations (PRIL17 and PRIL19) were developed by crossing two partially resistant plant introductions with two susceptible pea cultivars. PRIL17 (Lifter × PI240515), and PRIL19 (PI169603 × Medora) were evaluated for resistance to white mold by measuring lesion expansion inhibition (LEI) and nodal transmission inhibition (NTI) at 3, 7, and 14 days post inoculation (dpi) under controlled environmental conditions. Lesion expansion inhibition percentage (LEIP), survival rate (SR), and area under disease progress curves (AUDPC) were also calculated accordingly. Because of a positive correlation between LEI and NTI with height, short and long internode individuals of each population were analyzed separately to avoid any confounding effect of height to pathogen response. A total of 22 short genotypes demonstrated partial resistance based on at least two Porter's resistance criteria. Only two pea genotypes with partial resistance to white mold (PRIL19-18 and PRIL19-124) had both semi-leafless (afila) and short internode traits. Both the RIL populations were genotyped using genotyping by sequencing (GBS). For PRIL17 and PRIL19, genetic maps were constructed from a total of 1,967 and 1,196 single nucleotide polymorphism (SNP) and spanned over 1,494 cM and 1,415 cM representing seven and nine linkage groups, respectively. A consensus map constructed using data from both populations, had 1,486 unique SNPs over 2,461 cM belonging to seven linkage groups. Inclusive composite interval mapping (ICIM) identified thirteen quantitative trait loci (QTL) associated with white mold resistance traits in both populations. Three of them were co-located with height genes (a morphological trait that reduces infection risk and acts as disease avoidance) and the other ten QTL were associated with two forms of physiological resistance (seven for LEI and three for NTI) with LOD and r2 ranging from 3.0 to 28.5 and 5.1 to 64.3, respectively. The development of resistance lines, genetic dissection and identification of markers associated will help accelerate breeding efforts for white mold resistance using molecular breeding approaches.

Exploring the genetics of lesion and nodal resistance in pea (Pisum sativum L.) to Sclerotinia sclerotiorum using genome-wide association studies and RNA-Seq.

The disease white mold caused by the fungus Sclerotinia sclerotiorum is a significant threat to pea production, and improved resistance to this disease is needed. Nodal resistance in plants is a phenomenon where a fungal infection is prevented from passing through a node, and the infection is limited to an internode region. Nodal resistance has been observed in some pathosystems such as the pea (Pisum sativum L.)-S. sclerotiorum pathosystem. In addition to nodal resistance, different pea lines display different levels of stem lesion size restriction, referred to as lesion resistance. It is unclear whether the genetics of lesion resistance and nodal resistance are identical or different. This study applied genome-wide association studies (GWAS) and RNA-Seq to understand the genetic makeup of these two types of resistance. The time series RNA-Seq experiment consisted of two pea lines (the susceptible 'Lifter' and the partially resistant PI 240515), two treatments (mock inoculated samples and S. sclerotiorum-inoculated samples), and three time points (12, 24, and 48 hr post inoculation). Integrated results from GWAS and RNA-Seq analyses identified different redox-related transcripts for lesion and nodal resistances. A transcript encoding a glutathione S-transferase was the only shared resistance variant for both phenotypes. There were more leucine rich-repeat containing transcripts found for lesion resistance, while different candidate resistance transcripts such as a VQ motif-containing protein and a myo-inositol oxygenase were found for nodal resistance. This study demonstrated the robustness of combining GWAS and RNA-Seq for identifying white mold resistance in pea, and results suggest different genetics underlying lesion and nodal resistance.

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.

Development and characterization of 37 novel EST-SSR markers in Pisum sativum (Fabaceae).

PREMISE OF THE STUDY: Simple sequence repeat markers were developed based on expressed sequence tags (EST-SSR) and screened for polymorphism among 23 Pisum sativum individuals to assist development and refinement of pea linkage maps. In particular, the SSR markers were developed to assist in mapping of white mold disease resistance quantitative trait loci. • METHODS AND RESULTS: Primer pairs were designed for 46 SSRs identified in EST contiguous sequences assembled from a 454 pyrosequenced transcriptome of the pea cultivar, 'LIFTER'. Thirty-seven SSR markers amplified PCR products, of which 11 (30%) SSR markers produced polymorphism in 23 individuals, including parents of recombinant inbred lines, with two to four alleles. The observed and expected heterozygosities ranged from 0 to 0.43 and from 0.31 to 0.83, respectively. • CONCLUSIONS: These EST-SSR markers for pea will be useful for refinement of pea linkage maps, and will likely be useful for comparative mapping of pea and as tools for marker-based pea breeding.

Isolation and characterization of novel EST-derived genic markers in Pisum sativum (Fabaceae).

PREMISE OF THE STUDY: Novel markers were developed for pea (Pisum sativum) from pea expressed sequence tags (ESTs) having significant homology to Medicago truncatula gene sequences to investigate genetic diversity, linkage mapping, and cross-species transferability. • METHODS AND RESULTS: Seventy-seven EST-derived genic markers were developed through comparative mapping between M. truncatula and P. sativum in which 75 markers produced PCR products and 33 were polymorphic among 16 pea genotypes. • CONCLUSIONS: The novel markers described here will be useful for future genetic studies of P. sativum; their amplification in lentil (Lens culinaris) demonstrates their potential for use in closely related species.

Rapid transcriptome characterization and parsing of sequences in a non-model host-pathogen interaction; pea-Sclerotinia sclerotiorum.

BACKGROUND: White mold, caused by Sclerotinia sclerotiorum, is one of the most important diseases of pea (Pisum sativum L.), however, little is known about the genetics and biochemistry of this interaction. Identification of genes underlying resistance in the host or pathogenicity and virulence factors in the pathogen will increase our knowledge of the pea-S. sclerotiorum interaction and facilitate the introgression of new resistance genes into commercial pea varieties. Although the S. sclerotiorum genome sequence is available, no pea genome is available, due in part to its large genome size (~3500 Mb) and extensive repeated motifs. Here we present an EST data set specific to the interaction between S. sclerotiorum and pea, and a method to distinguish pathogen and host sequences without a species-specific reference genome. RESULTS: 10,158 contigs were obtained by de novo assembly of 128,720 high-quality reads generated by 454 pyrosequencing of the pea-S. sclerotiorum interactome. A method based on the tBLASTx program was modified to distinguish pea and S. sclerotiorum ESTs. To test this strategy, a mixture of known ESTs (18,490 pea and 17,198 S. sclerotiorum ESTs) from public databases were pooled and parsed; the tBLASTx method successfully separated 90.1% of the artificial EST mix with 99.9% accuracy. The tBLASTx method successfully parsed 89.4% of the 454-derived EST contigs, as validated by PCR, into pea (6,299 contigs) and S. sclerotiorum (2,780 contigs) categories. Two thousand eight hundred and forty pea ESTs and 996 S. sclerotiorum ESTs were predicted to be expressed specifically during the pea-S. sclerotiorum interaction as determined by homology search against 81,449 pea ESTs (from flowers, leaves, cotyledons, epi- and hypocotyl, and etiolated and light treated etiolated seedlings) and 57,751 S. sclerotiorum ESTs (from mycelia at neutral pH, developing apothecia and developing sclerotia). Among those ESTs specifically expressed, 277 (9.8%) pea ESTs were predicted to be involved in plant defense and response to biotic or abiotic stress, and 93 (9.3%) S. sclerotiorum ESTs were predicted to be involved in pathogenicity/virulence. Additionally, 142 S. sclerotiorum ESTs were identified as secretory/signal peptides of which only 21 were previously reported. CONCLUSIONS: We present and characterize an EST resource specific to the pea-S. sclerotiorum interaction. Additionally, the tBLASTx method used to parse S. sclerotiorum and pea ESTs was demonstrated to be a reliable and accurate method to distinguish ESTs without a reference genome.

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.