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 F-2 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 F-2 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.