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

Abstract

AbstractIdentification and DNA polymorphism of the S‐locus receptor kinasegene (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 and class II S haplotypes amplified single DNA fragments of 900‐1050 bp. The DNA fragments of 22 inbred lines amplified with a class 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 Hinf1. Nucleotide sequencing of the DNA fragments amplified from 10S 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.