Targeted genotyping-by-sequencing permits cost-effective identification and discrimination of pasture grass species and cultivars

Abstract

A targeted amplicon-based genotyping-by-sequencing approach has permitted cost-effective and accurate discrimination between ryegrass species (perennial, Italian and inter-species hybrid), and identification of cultivars based on bulked samples. Perennial ryegrass and Italian ryegrass are the most important temperate forage species for global agriculture, and are represented in the commercial pasture seed market by numerous cultivars each composed of multiple highly heterozygous individuals. Previous studies have identified difficulties in the use of morphophysiological criteria to discriminate between these two closely related taxa. Recently, a highly multiplexed single nucleotide polymorphism (SNP)-based genotyping assay has been developed that permits accurate differentiation between both species and cultivars of ryegrasses at the genetic level. This assay has since been further developed into an amplicon-based genotyping-by-sequencing (GBS) approach implemented on a second-generation sequencing platform, allowing accelerated throughput and ca. sixfold reduction in cost. Using the GBS approach, 63 cultivars of perennial, Italian and interspecific hybrid ryegrasses, as well as intergeneric Festulolium hybrids, were genotyped. The genetic relationships between cultivars were interpreted in terms of known breeding histories and indistinct species boundaries within the Lolium genus, as well as suitability of current cultivar registration methodologies. An example of applicability to quality assurance and control (QA/QC) of seed purity is also described. Rapid, low-cost genotypic assays provide new opportunities for breeders to more fully explore genetic diversity within breeding programs, allowing the combination of novel unique genetic backgrounds. Such tools also offer the potential to more accurately define cultivar identities, allowing protection of varieties in the commercial market and supporting processes of cultivar accreditation and quality assurance.

Exploitation of data from breeding programs supports rapid implementation of genomic selection for key agronomic traits in perennial ryegrass

“…A number of studies have employed different depths of sampling within populations to accurately represent the genetic diversity within a variety/population and relationships between. Most recently, Pembleton et al (2016) demonstrated that 48 or more individual samples per ryegrass variety/population are required for such studies. As a consequence, a larger number (60 or more germinants per synthetic population) were harvested and subjected to pooled RNA extraction in the present study.…”

“…The global genetic diversity of perennial ryegrass is large (Pembleton et al 2016) and so would require an extensive reference population. Diversity within a single breeding program should be lower, due both to sub-selection of founder genotypes from a broader range of germplasm, and the cyclic nature of many programs, such that individuals from pre-existing elite varieties are often chosen as parents for a next round of top-crosses.…”

Toward Genomics-Based Breeding in C3 Cool-Season Perennial Grasses

“…This method has been validated to be used in diversity (Elshire et al, 2011) and association studies (Hegarty et al, 2013; Morris et al, 2013) as well as advanced breeding application (Poland et al, 2012; Poland, 2015). A cost-effective targeted amplicons-based GBS approach was found very effective for genotyping perennial ryegrass and Italian ryegrass (Pembleton et al, 2016). Along with marker identification, GBS is a powerful tool to calculate genome-wide allele frequency of a particular locus.…”

Prospects for applications of genomic tools in registration testing and seed certification of ryegrass varieties

“…, , Pembleton et al. ). In most cases, cultivars have been effectively discriminated, although the resolution was limited for those varieties that have been derived from land races or ecotypes.…”

“…Later, based on the use of amplicon‐based genotyping‐by‐sequencing (GBS) approach on bulked seed samples, the NJ tree was extended to 63 cultivars of ryegrass, data being obtained at much lower cost (Pembleton et al. ). In both of the studies, two major cultivar‐specific groups were identified, corresponding to the two species (perennial and Italian ryegrass).…”