Single nucleotide polymorphism genotyping of the barley waxy gene by polymerase chain reaction with confronting two-pair primers

A high‐throughput single nucleotide polymorphism (SNP) genotyping procedure was developed to select amylose‐free barley mutants whose waxy genes had a C‐ to T‐base substitution in exon 5, which converted Gln‐89 of the wild‐type gene into a termination codon. An F₂ population carrying an amylose‐free waxy gene was checked for segregation. Polymerase chain reaction with confronting two‐pair primers (PCR‐CTPP) produced allele‐specific PCR products that have different sizes and are inherited in a co‐dominant manner. Two alleles of the barley waxy gene with SNP were correctly identified in parental strains using the PCR‐CTPP procedure. Segregation of the SNP as detected by PCR‐CTPP in an F₂ population fitted the expected 1:2:1 ratio. The PCR‐CTPP procedure can provide a time saving and cost‐effective alternative to derived cleaved amplified polymorphic sequence in marker‐assisted selection.