SNP marker discovery, linkage map construction and identification of QTLs for enhanced salinity tolerance in field pea (Pisum sativumL.)

Abstract

BackgroundField pea (Pisum sativum L.) is a self-pollinating, diploid, cool-season food legume. Crop production is constrained by multiple biotic and abiotic stress factors, including salinity, that cause reduced growth and yield. Recent advances in genomics have permitted the development of low-cost high-throughput genotyping systems, allowing the construction of saturated genetic linkage maps for identification of quantitative trait loci (QTLs) associated with traits of interest. Genetic markers in close linkage with the relevant genomic regions may then be implemented in varietal improvement programs.ResultsIn this study, single nucleotide polymorphism (SNP) markers associated with expressed sequence tags (ESTs) were developed and used to generate comprehensive linkage maps for field pea. From a set of 36,188 variant nucleotide positions detected through in silico analysis, 768 were selected for genotyping of a recombinant inbred line (RIL) population. A total of 705 SNPs (91.7%) successfully detected segregating polymorphisms. In addition to SNPs, genomic and EST-derived simple sequence repeats (SSRs) were assigned to the genetic map in order to obtain an evenly distributed genome-wide coverage. Sequences associated with the mapped molecular markers were used for comparative genomic analysis with other legume species. Higher levels of conserved synteny were observed with the genomes of Medicago truncatula Gaertn. and chickpea (Cicer arietinum L.) than with soybean (Glycine max [L.] Merr.), Lotus japonicus L. and pigeon pea (Cajanus cajan [L.] Millsp.). Parents and RIL progeny were screened at the seedling growth stage for responses to salinity stress, imposed by addition of NaCl in the watering solution at a concentration of 18 dS m-1. Salinity-induced symptoms showed normal distribution, and the severity of the symptoms increased over time. QTLs for salinity tolerance were identified on linkage groups Ps III and VII, with flanking SNP markers suitable for selection of resistant cultivars. Comparison of sequences underpinning these SNP markers to the M. truncatula genome defined genomic regions containing candidate genes associated with saline stress tolerance.ConclusionThe SNP assays and associated genetic linkage maps developed in this study permitted identification of salinity tolerance QTLs and candidate genes. This constitutes an important set of tools for marker-assisted selection (MAS) programs aimed at performance enhancement of field pea cultivars.

Translational Genomics in Agriculture: Some Examples in Grain Legumes

“…For instance, in the case of soybean, a total of 1,682 SNPs were identified by deep re-sequencing of the reduced representation libraries using Illumina Genome Analyzer (GA) . Similarly, 26,082 SNPs were discovered from two contrasting drought responsive genotypes in chickpea , 36,188 SNPs for two contrasting salinity genotypes in pea (Leonforte et al, 2013) and 12,141 SNPs from 10 pigeonpea genotypes representing five mapping populations segregating for Fusarium wilt and sterility mosaic disease were identified. In addition, COS markers have found wide application in cross-genome comparative studies in legume species.…”

Transcriptomic Studies in Non-Model Plants: Case of Pisum sativum L. and Medicago lupulina L.

“…SNVs detection may be executed by mapping NGS reads to an existing reference transcriptome assembly [59] or by de novo assembly of those reads [33,35,60]. In the case of existing assembly, the additional data complexity Table 1.…”

“…SNV genotyping systems are now available, varying in the number of samples and markers to be genotyped, such as GoldenGate® and Ininium from Illumina Inc., SNPStream from Beckman Coulter and GeneChip from Afymetrix [61]. Illumina GoldenGate® oligonucleotide pool assay (OPA) designed for transcriptome-discovered SNVs was used for pea salinity tolerance QTLs search [60].…”

“…Transcriptome-discovered SNVs and high-throughput genotyping systems made the construction of several highly saturated genetic maps of pea possible (see Table 1) [33,35,60].…”

SNP Discovery and Genetic Mapping Using Genotyping by Sequencing of Whole Genome Genomic DNA from a Pea RIL Population

“…Large numbers of new molecular markers are still needed to saturate pea maps and significantly improve QTL mapping both for research and breeding objectives. Although transcriptome sequencing has recently been used in pea for SNP discovery [3,15,16] and mapping [3,17,18], available genetic maps remain at low to medium density, and are mainly based on a few hundred SSRs [19] and on a few hundred [20,21] up to a few thousand [3,18,22] SNPs, usually developed through dedicated genotyping facilities. The development of larger resources is therefore required for mapping and genetic improvement purposes.…”