Development of a PCR-based allele-specific assay from an RFLP probe linked to resistance to cereal cyst nematode in wheat

The RFLP locus Xglk605 identified by the probe Tag605 maps to a proximal position on the long arm of. wheat chromosome 2B about 7 cM away from a gene conditioning resistance to cereal cyst nematode in the wheat line AUS 10894. The clone Tag605 was partially sequenced and the FCR primer set AWP1 was designed. The 292-bp product, which showed no polymorphism between varieties, was cloned and sequenced. A single base difference was found in the sequence of the AWP1 products amplified and cloned from the wheat lines AUS10894 and 'Spear'. PCR primers were designed with 3' termini that corresponded to the two alleles. A dual-PCR system was developed in which the primer sets AWP2 and AWP3 produced allele-specific amplification. The concentration of the oligonucleotide primers and the sequence of the primer-template mismatches were critical to the success of discriminatory allele amplification.