Pattemore, JA 2011, 'Single nucleotide polymorphism (SNP) discovery and analysis for barley genotyping', PhD thesis, Southern Cross University, Lismore, NSW.
Copyright JA Pattemore 2011
Correct identification of cereal varieties is important to food quality, safety, security and authenticity, and molecular markers have recently been applied to cereal varietal identification or genotyping. Two major factors restricting the wider application of molecular markers in crop plant genotyping have been throughput and cost per data point. However in recent years the miniaturization of technology has both increased throughput and significantly reduced these costs. This study set out to identify a genotyping platform and molecular marker which could be applied to the challenge of cereal variety identification. A multiplexed Sequenom® MassARRAY™ mass spectrometry SNP assay targeting 45 loci was used to genotype 35 Australian barley varieties. Of the 45 loci screened, 33 were informative and successfully used to generate a unique profile of SNPs for each variety tested, except one variety where SNP heterogeneity prevented an unambiguous profile from being generated. The novel application of MALDI-TOF MS SNP genotyping to varietal identification has revealed significant SNP heterogeneity within some barley varieties. SNP variation was found between varieties, within varieties and within individuals of a variety. The amount of and distribution of SNP heterogeneity within two typical barley varieties was then quantified by deep sequence analysis of a transcriptome library of each variety. Intra-varietal SNP variation occurred as background heterozygosity (MAF <0.1) in 90.40% and 85.44% of the Baudin and Gairdner transcriptomes. 9.60% and 14.56% of Baudin and Gairdner transcriptome SNPs respectively, had a minor allele frequency > 0.1, but these were restricted to < 25% of the unigenes. 73.08% of the total combined unigenes had no SNPs. Variety-specific SNP parameters were defined and a set of variety-specific SNPs was generated. These results indicate that next generation sequencing technologies provide an unprecedented understanding of the levels of genetic variation remaining in some commercially released varieties of barley. SNPs are high quality; high resolution markers for measuring genetic variation within varieties, however for unambiguous varietal identification a variety-specific dataset must be generated from SNPs with known allele frequencies from local populations.