RNA-Seq analysis and development of SSR and KASP markers in lentil (Lens culinaris Medikus subsp. culinaris)

Lentil (Lens culinarisMedikus subsp.culinaris,2n= 14) is a cool-season legume with highproduction potential for multiple uses. However, limited molecular research has beenconducted in this species owing to its large genome, which impedes the generation ofgenome sequences and the development of molecular markers. In this study, more than 1.37billion filtered clean reads were collected by RNA-Seq of six diverse lentil accessions and217,836 transcripts and 161,095 unigenes werede novoassembled, yielding respectively 257.1and 240.6 million nucleotides. The mean transcript length was 1180 bp and the N50 and N90lengths were respectively 2075 and 479 bp. The mean length of the unigenes was 1494 bp andtheir N50 and N90 values were respectively 2203 and 714 bp. The unigenes were annotatedagainst seven databases. TheFLOWERING LOCUS T(FT) gene homolog in lentil showed highprotein sequence similarity to theFTgene homologs of pea and alfalfa. On the basis of theRNA-Seq analysis, 26,449 EST-SSR markers were designedin silico, and 276 preliminarilyscreened markers were selected to evaluate polymorphism in 94 diverse lentil accessions. Intotal, 125 (45.29%) of 276 EST-SSR markers were found to be polymorphic. A total of 130,073SNP loci were detected and 78 (61.41%) of 127 SNPs were successfully converted to KASPmarkers. Population genetic analyses of the lentil accessions with EST-SSR and KASP markersrevealed similar genetic structures, suggesting that the RNA-Seq-generated resources and thedeveloped markers are reliable for use in molecular marker-assisted breeding of lentil.