title: 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.
authors.affiliation: AgriBio, Centre for AgriBioscience, Biosciences Research, La Trobe University, 5 Ring Road, Bundoora, VIC, 3083, Australia.|Dairy Futures Cooperative Research Centre, AgriBio, La Trobe University, 5 Ring Road, Bundoora, VIC, 3083, Australia.|School of Applied Systems Biology, La Trobe University, Bundoora, VIC, 3086, Australia.
authors.forename: Luke W
authors.initials: LW
authors.lastname: Pembleton
authors.affiliation: AgriBio, Centre for AgriBioscience, Biosciences Research, La Trobe University, 5 Ring Road, Bundoora, VIC, 3083, Australia.|Dairy Futures Cooperative Research Centre, AgriBio, La Trobe University, 5 Ring Road, Bundoora, VIC, 3083, Australia.
authors.forename: Michelle C
authors.initials: MC
authors.lastname: Drayton
authors.affiliation: AgriBio, Centre for AgriBioscience, Biosciences Research, La Trobe University, 5 Ring Road, Bundoora, VIC, 3083, Australia.|Dairy Futures Cooperative Research Centre, AgriBio, La Trobe University, 5 Ring Road, Bundoora, VIC, 3083, Australia.
authors.forename: Melissa
authors.initials: M
authors.lastname: Bain
authors.affiliation: AgriBio, Centre for AgriBioscience, Biosciences Research, La Trobe University, 5 Ring Road, Bundoora, VIC, 3083, Australia.|Dairy Futures Cooperative Research Centre, AgriBio, La Trobe University, 5 Ring Road, Bundoora, VIC, 3083, Australia.
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Baillie
authors.affiliation: New Zealand Agriseeds, 2547 Old West Coast Road, Christchurch, 7671, New Zealand.
authors.forename: Courtney
authors.initials: C
authors.lastname: Inch
authors.affiliation: AgriBio, Centre for AgriBioscience, Biosciences Research, La Trobe University, 5 Ring Road, Bundoora, VIC, 3083, Australia.|Dairy Futures Cooperative Research Centre, AgriBio, La Trobe University, 5 Ring Road, Bundoora, VIC, 3083, Australia.|School of Applied Systems Biology, La Trobe University, Bundoora, VIC, 3086, Australia.
authors.forename: German C
authors.initials: GC
authors.lastname: Spangenberg
authors.affiliation: Hamilton Centre, Biosciences Research, Mount Napier Road, Hamilton, VIC, 3330, Australia.
authors.forename: Junping
authors.initials: J
authors.lastname: Wang
authors.affiliation: AgriBio, Centre for AgriBioscience, Biosciences Research, La Trobe University, 5 Ring Road, Bundoora, VIC, 3083, Australia. john.forster@ecodev.vic.gov.au.|Dairy Futures Cooperative Research Centre, AgriBio, La Trobe University, 5 Ring Road, Bundoora, VIC, 3083, Australia. john.forster@ecodev.vic.gov.au.|School of Applied Systems Biology, La Trobe University, Bundoora, VIC, 3086, Australia. john.forster@ecodev.vic.gov.au.
authors.forename: John W
authors.initials: JW
authors.lastname: Forster
authors.affiliation: AgriBio, Centre for AgriBioscience, Biosciences Research, La Trobe University, 5 Ring Road, Bundoora, VIC, 3083, Australia.|Dairy Futures Cooperative Research Centre, AgriBio, La Trobe University, 5 Ring Road, Bundoora, VIC, 3083, Australia.
authors.forename: Noel O I
authors.initials: NO
authors.lastname: Cogan
mesh_terms: D018744:DNA, Plant / Q000235:genetics; D015723:Gene Library; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D008129:Lolium / Q000145:classification* / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D013045:Species Specificity
chemical_list: D018744:DNA, Plant