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

Silvar, Christina; Perovic, Dragan GND; Casas, Ana M.; Igartua, Ernesto; Ordon, Frank GND

P>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-mu l 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.

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Silvar, Christina / Perovic, Dragan / Casas, Ana / et al: Development of a cost-effective pyrosequencing approach for SNP genotyping in barley. 2011.

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