Transcriptome sequencing of lentil based on second-generation technology permits large-scale unigene assembly and SSR marker discovery

Abstract

BackgroundLentil (Lens culinaris Medik.) is a cool-season grain legume which provides a rich source of protein for human consumption. In terms of genomic resources, lentil is relatively underdeveloped, in comparison to other Fabaceae species, with limited available data. There is hence a significant need to enhance such resources in order to identify novel genes and alleles for molecular breeding to increase crop productivity and quality.ResultsTissue-specific cDNA samples from six distinct lentil genotypes were sequenced using Roche 454 GS-FLX Titanium technology, generating c. 1.38 × 106 expressed sequence tags (ESTs). De novo assembly generated a total of 15,354 contigs and 68,715 singletons. The complete unigene set was sequence-analysed against genome drafts of the model legume species Medicago truncatula and Arabidopsis thaliana to identify 12,639, and 7,476 unique matches, respectively. When compared to the genome of Glycine max, a total of 20,419 unique hits were observed corresponding to c. 31% of the known gene space. A total of 25,592 lentil unigenes were subsequently annoated from GenBank. Simple sequence repeat (SSR)-containing ESTs were identified from consensus sequences and a total of 2,393 primer pairs were designed. A subset of 192 EST-SSR markers was screened for validation across a panel 12 cultivated lentil genotypes and one wild relative species. A total of 166 primer pairs obtained successful amplification, of which 47.5% detected genetic polymorphism.ConclusionsA substantial collection of ESTs has been developed from sequence analysis of lentil genotypes using second-generation technology, permitting unigene definition across a broad range of functional categories. As well as providing resources for functional genomics studies, the unigene set has permitted significant enhancement of the number of publicly-available molecular genetic markers as tools for improvement of this species.

Diversification of indigenous gene- pool by using exotic germplasm in lentil (Lens culinaris Medikus subsp. culinaris)

“…It Fig. 1 Dendrogram showing genetic relationships among lentil genotypes based on molecular data of SSR markers (Scale indicates Jaccard's coefficient of dissimilarity) was comparatively similar to earlier study where 47.5 % SSR markers developed from lentil genome were polymorphic (Kaur et al 2011). In another study, transferrable SSR markers were also highly polymorphic in lentil (Datta et al 2011).…”

“…Among the various molecular markers, microsatellites or simple sequence repeats (SSR) have shown to be very useful, because these markers showed high polymorphism, reproducible and easy to handle (Varshney et al 2005(Varshney et al , 2009Datta et al 2011). Though in lentil, a number of SSR markers have been developed (Hamwieh et al 2005;Kaur et al 2011;Datta et al 2011), availability of polymorphic SSR markers and their use in analysis of the genetic diversity is still limited in lentil compared to other pulses such as chickpea (Hamwieh et al 2005(Hamwieh et al , 2009Kaur et al 2011;Datta et al 2011). Therefore, present investigation was aimed to assess diversification of Indian gene-pool on the basis of SSR markers and morphological traits among 21 lentil genotypes involving the exotic lines.…”

Capturing variation in Lens (Fabaceae): Development and utility of an exome capture array for lentil

“…To select the regions of interest in the genome for the array, we used several sources: (1) the coding DNA sequence from the Medicago truncatula Gaertn. genome version Mt4.0 (Tang et al., 2014); (2) Illumina RNA‐Seq reads from L. culinaris 2 × 250 MiSeq data (BioProject PRJNA434239); and (3) a collection of previously generated L. culinaris Sanger expressed sequence tags, 454 reads, and contigs (Sharpe et al., 2013 [BioProject PRJNA192531]; Kaur et al., 2011). RNA‐Seq reads were aligned to the reference genome Lc1.2 using TopHat 2.1.1 (Trapnell et al., 2009), and Cufflinks 2.2.1 (Trapnell et al., 2010) was used to determine the transcript coordinates.…”

Legume Crops Phylogeny and Genetic Diversity for Science and Breeding

“…This was a successful strategy because of the synteny between lentil and the model legume Medicago truncatula (Phan et al, 2007b). With the publication of three transcriptomes of lentil Verma et al, 2013;Kaur et al 2011), this is rapidly changing. One transcriptome targeted SNP discovery, resulting in the publication of a SNP-dense genetic linkage map .…”