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KMID : 0903620040450030119
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Journal of the Korean Society for Horticultural Science
2004 Volume.45 No. 3 p.119 ~ p.126
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Identification of S-haplotypes by PCR-RFLP and DNA Sequencing of S-Locus Receptor Kinase (SRK) in Raphanus sativus Inbred Lines
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Seo Jeong-Pal
Park Kuen-Woo
Kim Hyun-Jung
Yang Seoung-Yul
Nou Ill-Sup
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Abstract
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The S-locus-encoded S receptor kinase (SRK) is an intrinsic plasma membrane protein that is viewed as the primary stigma determinant of specificity in the self-incompatibility response of Brassica spp. From 178 inbred lines of Raphanus sativus S-homozygotes DNA fragments of the class Ⅰ and class Ⅱ S-haplotypes were amplified by PCR using specific two primer pairs. PCR with class Ⅰ and class Ⅱ primer pairs produced a single DNA fragment of 900 bp in 12 lines, and those of 1,050 bp in 166 lines, respectively. These amplified DNA fragments showed high polymorphism in electrophoresis after digestion with restriction endonucleases; and those from 12 inbred lines, amplified with class I primer pair were classified into 4 haplotypes by RFLP analysis with EcoRII＋HinfI, and those from 166 inbred lines, amplified with a class Ⅱ primer pair were classified into the 6 haplotypes by RFLP analysis with HinfI. The nucleotide sequences of the kinase domain of the above 10 different S-haplotypes were determined. Similarity of the nucleotide sequences in SRKs in class Ⅰ group was more than 85%, and that in class II group was more than 90%. Nucleotide sequences of the DNA fragments amplified from different types showed that the exons are highly conserved, but there are high variations in the introns of SRK, which produced a polymorphism of the band pattern in PCR-RFLPs. Therefore, the present class Ⅰ and class Ⅱ SRK-specific primer pairs, and endonucleases used in PCR-RFLP analysis are useful for identifying the characteristics of S-haplotypes more precisely and quickly.
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KEYWORD
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self-incompatibility, S-locus glycoprotein, S-allele, radish
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