Polymorphism of the kinase domain of the S-locus receptor kinase gene (SRK) in Brassica oleracea L.

Abstract

 DNA polymorphism of the S-locus receptor kinase gene (SRK) participating in self-incompatibility in Brassica was analyzed by PCR-RFLP and nucleotide sequencing. In the screening of primers for specific amplification of polymorphic DNA fragments of SRK, the best combination was that of a forward primer (PK1) having the nucleotide sequence of the second exon of S6 SRK and a reverse primer (PK4) having the complementary nucleotide sequence of the fifth exon of S6 SRK. PCR using this primer pair amplified DNA fragments of 0.9–1.0 kb from 36 S haplotypes out of 42 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonuclease(s): 25 types were found in a double digestion with MboI and AfaI. Nucleotide sequencing of the DNA fragments amplified from five S haplotypes showed that the third, fourth, and fifth exons of SRK are highly conserved, and that there are high variations of the second and third introns of SRK, which produced polymorphism of the band pattern in PCR-RFLPs. Another forward primer (PK5) having the nucleotide sequence of the second exon, which is derived from S2 SRK, amplified DNA fragments of almost the same region of SRK from 27 S haplotypes in combination with PK4. Although SRK alleles of the class-II S haplotypes were not amplified, all of the class-I S-haplotypes were amplified with a primer mixture of PK1, PK4 and PK5. The DNA fragments of both SRK alleles in S heterozygotes, or a 1 : 1 mixture of the genomic DNA of different S homozygotes, were amplified without exception, suggesting the usefulness of these primers for the identification of S heterozygotes. The DNA fragment sizes obtained by digestion with restriction endonucleases served as markers for the identification of S haplotypes.