Identification of S-haplotype Based on Polymerase Chain Reaction-Restriction Fragment Length Polymorphisms and Sequence Analysis of the S-Locus Receptor Kinase Gene in Chinese Cabbage Inbred Lines

摘要

Identification and DNA polymorphism of the kinase domain of S-locus receptor kinase (SRK) which participates in the self-incompatibility of Brassicaceae were analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism and nucleotide sequencing,. In the PCR experiment, a SRK-specific primer pair (KS 5 and KA 7) of the class I amplified a single DNA fragment of about 900 bp from 61 lines out of 134 inbred lines. Four groups were found in a double digestion with HinfI and EcoRIIusing the class I SRK-specific primer pairs. Also, the rest of the lines which were not amplified with class I SRK-specific primer pairs gave exactly one single DNA fragment from 1,050 bp with class II SRK-specific primer pair (KS 4 and KA 8). These 1,050 kinase domains of the SRK gene in Chinese cabbage were divided into four groups when they were digested in HinfI or AM, respectively These observations suggested that these primers were able to differentiate accurately between class I and II, The Chinese cabbage inbred lines used consisted of eight different S-haplotypes, and 61 lines belonging to class I consisted of four different S-haplotypes, and 7.3 lines belonging to class II consisted of four different S-haplotypes, Nucleotide sequencing of the DNA fragments amplified from eight different S-haplotypes showed that exons of 3' end in SRK were highly conserved, and that there were high variations of introns. Also, the eight different S-haplotypes could be divided into two classes (class I and II) based on nucleotide sequence similarities. The sequence similarity within class I and II S-haplotypes exhibited 82-93 and 94-99% identity, respectively While between class I and II S-haplotypes, there was a lower degree of sequence conservation with approximately 66—71% sequence identity,