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Development of a cost‐effective pyrosequencing approach for SNP genotyping in barley
Author(s) -
Silvar Cristina,
Perovic Dragan,
Casas Ana M.,
Igartua Ernesto,
Ordon Frank
Publication year - 2011
Publication title -
plant breeding
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.583
H-Index - 71
eISSN - 1439-0523
pISSN - 0179-9541
DOI - 10.1111/j.1439-0523.2011.01853.x
Subject(s) - pyrosequencing , biology , genotyping , snp genotyping , molecular inversion probe , primer (cosmetics) , snp , single nucleotide polymorphism , genetics , computational biology , genotype , gene , chemistry , organic chemistry
With 1 figure and 1 table 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.