Abstract
BackgroundSingle nucleotide polymorphisms (SNPs) provide essential tools for the advancement of research in plant genomics, and the development of SNP resources for many species has been accelerated by the capabilities of second-generation sequencing technologies. The current study aimed to develop and use a novel bioinformatic pipeline to generate a comprehensive collection of SNP markers within the agriculturally important pasture grass tall fescue; an outbreeding allopolyploid species displaying three distinct morphotypes: Continental, Mediterranean and rhizomatous.ResultsA bioinformatic pipeline was developed that successfully identified SNPs within genotypes from distinct tall fescue morphotypes, following the sequencing of 414 polymerase chain reaction (PCR) – generated amplicons using 454 GS FLX technology. Equivalent amplicon sets were derived from representative genotypes of each morphotype, including six Continental, five Mediterranean and one rhizomatous. A total of 8,584 and 2,292 SNPs were identified with high confidence within the Continental and Mediterranean morphotypes respectively. The success of the bioinformatic approach was demonstrated through validation (at a rate of 70%) of a subset of 141 SNPs using both SNaPshot™ and GoldenGate™ assay chemistries. Furthermore, the quantitative genotyping capability of the GoldenGate™ assay revealed that approximately 30% of the putative SNPs were accessible to co-dominant scoring, despite the hexaploid genome structure. The sub-genome-specific origin of each SNP validated from Continental tall fescue was predicted using a phylogenetic approach based on comparison with orthologous sequences from predicted progenitor species.ConclusionsUsing the appropriate bioinformatic approach, amplicon resequencing based on 454 GS FLX technology is an effective method for the identification of polymorphic SNPs within the genomes of Continental and Mediterranean tall fescue. The GoldenGate™ assay is capable of high-throughput co-dominant SNP allele detection, and minimises the problems associated with SNP genotyping in a polyploid by effectively reducing the complexity to a diploid system. This SNP collection may now be refined and used in applications such as cultivar identification, genetic linkage map construction, genome-wide association studies and genomic selection in tall fescue. The bioinformatic pipeline described here represents an effective general method for SNP discovery within outbreeding allopolyploid species.
Highlights
Single nucleotide polymorphisms (SNPs) provide essential tools for the advancement of research in plant genomics, and the development of single nucleotide polymorphism (SNP) resources for many species has been accelerated by the capabilities of second-generation sequencing technologies
Using the appropriate bioinformatic approach, amplicon resequencing based on 454 genomic selection (GS) FLX technology is an effective method for the identification of polymorphic SNPs within the genomes of Continental and Mediterranean tall fescue
The GoldenGateTM assay is capable of high-throughput co-dominant SNP allele detection, and minimises the problems associated with SNP genotyping in a polyploid by effectively reducing the complexity to a diploid system
Summary
Single nucleotide polymorphisms (SNPs) provide essential tools for the advancement of research in plant genomics, and the development of SNP resources for many species has been accelerated by the capabilities of second-generation sequencing technologies. Discrimination must be performed between the nucleotide variation representing differences between these sub-genomic copies (homoeologous sequence variants [HSVs]) [18] and the homologous allelic variation arising within or between individuals, that is able to be assayed as a polymorphic molecular genetic marker (SNP) [19]. This process is most challenging in outbreeding species with multiple heterozygous genomic constitutions, for which nucleotide variation within each resident sub-genome further complicates data interpretation. Quantitative genotyping methods are required to fully classify nucleotide variant dosage and verify the genotype at any given locus or homoeolocus [19]
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