Development and implementation of a multiplexed single nucleotide polymorphism genotyping tool for differentiation of ryegrass species and cultivars

Perennial ryegrass ( Lolium perenne L.) and Italian ryegrass ( Lolium multiflorum Lam.) are important temperate forage grasses which are closely related, generating fertile interspecific hybrids. All groups are represented by multiple cultivars in the commercial pasture seeds market. Due to the close taxonomic relationship between the two species, differentiation based on morphophysiological criteria is not always readily achievable. In addition, an obligate outbreeding reproductive habit produces high levels of individual heterozygosity and intrapopulation diversity, which presents problems for discrimination between cultivars. Molecular genetic marker polymorphism provides an effective means of addressing these challenges. An iterative process of resequencing from loci distributed across the perennial ryegrass genome was used to identify single nucleotide polymorphism (SNP) markers, which were then validated and formatted in a highly multiplexed (384-plex) assay system. SNP genotyping was then performed across samples of 48–192 individuals from a total of 27 ryegrass cultivars (19 of perennial ryegrass, seven of Italian ryegrass and one hybrid cultivar). SNP markers from perennial ryegrass exhibited a high level of transfer to Italian ryegrass. Data analysis permitted quantification of intra- and inter-species diversity, as well as discrimination between cultivars within each species, including diploid and autotetraploid cultivars of perennial ryegrass. Lower levels of SNP-based diversity were detected in Italian ryegrass than in perennial ryegrass. A neighbour-joining tree based on genetic distance analysis located a hybrid cultivar to an intermediate position between the two species-specific cultivar groups. The resulting catalogue of ryegrass cultivars will provide support for the processes of cultivar accreditation and quality assurance.