Development of a cost-effective pyrosequencing approach for SNP genotyping in barley

Abstract

An improved, efficient, reliable and cost-effective pyrosequencing protocol for single nucleotide polymorphism (SNP) genotyping in plants is described. Labelling of the PCR products, required for the single-stranded pyrosequencing assay, is carried out in a one-step PCR with a universal biotinalyted M13 primer of 19-bp. A ratio of 1 : 10 of tailed primer to M13 primer in a 15-μl reaction volume turned out to be best suited for a successful identification of different alleles based on known SNPs. This technology allows cost-effective SNP genotyping in plant genomes for loci of interest at an overall low cost in a short period of time and is therefore best suited for marker-based selection procedures.