Using Genotyping-By-Sequencing to Identify and Annotate Single Nucleotide Polymorphisms in the ICARDA Lentil Reference Collection

Lentil (Lens culinaris Medik.) is the world’s fifth largest pulse crop. It is a self-pollinated diploid (2n=14) and has a relatively large genome size of approximately 4 Gb. This crop is produced in over 70 countries and is consumed in all the continents. Molecular markers are extremely useful in plant genetics and genomics. It is prerequisite for mapping and tagging of genes, quantitative trait loci (QTLs), segregation analysis, phylogenetic analysis, and numerous molecular biology applications. The next generation sequencing (NGS) technology has been shown to be capable of discovering and genotyping thousands of markers across a genome. The sequence-based genotyping methods are ready to replace many of the established and widely used tools such as highly-multiplex Single Nucleotide Polymorphism (SNP) platforms. Genotyping-by-sequencing (GBS), using the two-enzyme (PstI, MspI) method (as described by Poland et al.), was deployed on 130 ICARDA lentil reference collection lines plus abiotic stress responsive lines derived from diverse environments. Stacks software (version 1.19) and in-house scripts were used to call 30,786 high quality SNPs. Homology with known proteins was conducted using BLASTX (version 2.2.24) against the nr database (E-value of ≤ 1.0 e-6).  InterPro domains were assigned and associated gene ontology (GO) terms describing the SNPs were determined using BLAST2GO and in-house scripts. These technologies will be of great benefit to future genetic improvement efforts and to future understanding of the relationships between the cultivated lentil and its wild relatives.