Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology

Abstract

BackgroundGenetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST) information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat.ResultsBased on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM) analysis. Of these, 52 (54%) were polymorphic between parents of the Ogle1040 × TAM O-301 (OT) mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry.ConclusionsThe high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide a model for SNP discovery and genotyping in other species with complex and poorly-characterized genomes.

Oat (<i>Avena sativa</i> L.): Oil and Nutriment Compounds Valorization for Potential Use in Industrial Applications

“…Huang et al 168 dealt with the utility of genomic approaches, especially genotyping-by-sequencing GBS to provide additional information on oat breeding. In addition, the development of single nucleotide polymorphism SNP on hexaploid oats using high-throughput 454 sequencing technology 169 favours the understanding of complex genomes. Moreover, ESTs expressed sequence tags are useful tools contributing to the identification of genes from oats 170 thus improving knowledge of the full oat genome.…”

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“…Lo et al, 2010). High-throughput sequencing and HRM analysis have been combined for effective detection of SNPs in genes of crops with complex genomes (Han et al, 2011;Oliver et al, 2011). …”

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“…Transcriptome data could also be used as a resource for detecting genetic variants in many crop species (Hamilton et al, 2011;Oliver et al, 2011). NGS technologies have been routinely used to generate huge EST datasets by RNA-seq experiments, which can be used for identifying SNPs in functional genes.…”