Identification of S-alleles using polymerase chain reaction-cleaved amplified polymorphic sequence of the S-locus receptor kinase in inbreeding lines of Brassica oleracea

Identification and DNA polymorphism of the S-locus receptor kinase gene (SRK) was analysed by pollen tube tests, polymerase chain reaction-cleaved amplified polymorphic sequence (PCR-CAPS) and nucleotide sequencing. SRK-specific primers that can distinguish class I and class II S haplotypes amplified single DNA fragments of 900-1050 bp. The DNA fragments of 22 inbred lines amplified with a class I SRK-specific primer pair determined seven types with HinfI and EcoRII. In addition, the DNA fragments of 17 inbred lines amplified with a class II SRK-specific primer pair determined three types with AluI. Nucleotide sequencing of the DNA fragments amplified from 10 S haplotypes showed that exons of the 3′-end in SRK are highly conserved, and that there is much variation of the introns, which produced polymorphism of the band pattern in PCR-CAPS profiles. The S haplotypes of the plants were determined by restriction analysis of PCR products and agreed with results based on pollen tube growth tests. The PCR-CAPS analysis using specific primer pairs of SRK is considered to be useful for S allele identification in breeding programmes.