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

• Published in: BMC genomics 2011-05, Vol.12 (1), p.265-265, Article 265
• Main Authors: Kaur, Sukhjiwan, Cogan, Noel O I, Pembleton, Luke W, Shinozuka, Maiko, Savin, Keith W, Materne, Michael, Forster, John W
• Format: Article
• Language: English
• Subjects: Arabidopsis thaliana; Cluster Analysis; DNA Primers - genetics; DNA, Complementary - genetics; Expressed Sequence Tags - metabolism; Gene Expression Profiling - methods; Genetic aspects; Genetic Markers - genetics; Genomes; Genotype; Lens Plant - genetics; Lens Plant - growth & development; Minisatellite Repeats - genetics; Molecular Sequence Annotation; Polymerase chain reaction; Reproducibility of Results; Sequence Analysis, DNA - methods
• ISSN: 1471-2164; 1471-2164
• DOI: 10.1186/1471-2164-12-265

Lentil (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. Tissue-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. A 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.