Microsatellite Identification in Perennial Ryegrass using Next-Generation Sequencing

Microsatellite markers are potentially valuable molecular genetic markers for conservation ecology, paternity testing, pedigree reconstruction, population genetics, and linkage mapping. Traditional methods for the development of microsatellite markers can be time-consuming, laborious, and expensive. Next-generation sequencing (NGS) is a more recent and promising approach to microsatellite marker development. Perennial ryegrass (Lolium perenne L.) is an important turfgrass species with a limited set of publicly available microsatellite markers. Here we conducted Illumina NGS, as well as genotyping of a Lolium test population, to identify and characterize perennial ryegrass microsatellite markers. Sequencing and assembly results returned a microsatellite marker database containing 10,830 perfect di-and 42,718 perfect trinucleotide microsatellites with repeat unit lengths equal to or greater than six and four repeat units, respectively, as well as sufficient flanking sequence to enable polymerase chain reaction (PCR) primer design. Genotyping results from a subset of 172 di-and trinucleotide microsatellite markers indicated that 38.7% of these markers were polymorphic in perennial ryegrass, and 21.5% were transferable to and polymorphic in annual ryegrass (Lolium multiflorum Lam.). The thousands of expected polymorphic markers reported herein provide a significant new resource for studying Lolium genetic diversity, parentage analysis, linkage analysis, and germplasm in turf and forage breeding programs.