Exploring genetic diversity in perennial ryegrass (L. perenne) populations using targeted resequencing

Association mapping studies require versatile, cost-efficient genotyping methods to generate molecular markers in large collections of plants. Depending on the extent of linkage disequilibrium within the population under study, molecular markers are either spread across the genome or targeted to specific genomic regions or candidate genes. Here, we investigated whether probe capture enrichment followed by Next Generation Sequencing is a feasible method to perform targeted resequencing of candidate genes in a large number of perennial ryegrass (Lolium perenne) individuals from diverse origins. Protein families from Brachypodium, rice, maize, sorghum and Arabidopsis served as reference to annotate 177 gene families and to identify putative L. perenne orthologs of genes with known physiological function in model organisms. We selected 539 candidate genes for probe design, using a draft genome sequence of L. perenne and transcriptome data. These candidate genes are putatively involved in the regulation of plant growth and development, plant architecture, induction of flowering, cell wall biogenesis, and phytohormone biosynthesis, signalling and response. As a proof of concept, we constructed indexed shotgun gDNA libaries of 96 L. perenne genotypes, and performed pooled probe capture followed by Illumina sequencing. This approach is usefull to create a comprehensive inventory of the sequence diversity in regulatory genes present in the genepool, to discover novel and rare molecular markers for association mapping studies, and to identify naturally occurring sequence variants that potentially disturb gene function. Genotypes carrying such rare defective alleles may be useful in reverse genetic approaches to study gene function, or for breeding.