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KMID : 0903620000410010021
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Journal of the Korean Society for Horticultural Science
2000 Volume.41 No. 1 p.21 ~ p.26
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Identification of S - haplotype by PCR - RFLP in Brassica
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박영수/Park, Young Su
노일섭/이수성/박종인/양승환/Nou, Ill Sup/Lee, Soo Seong/Park, Jong In/Yang, Seung Hwan
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Abstract
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The development of technique for the identification of S-haplotype involved in self-incompatibility in Brassica, which is based on the polymerase chain reaction (PCR) amplification of genomic DNA followed by restriction analysis is described. Polymorphism of SLG and SRK in broccoli and cabbage was analyzed by PCR-RFLP using SLG and SRK specific primer. Combination of SLG primer consists of six combination groups in class I SLG group and class II SLG group. Two combination of primers, which had a nucleotide sequence specific to a class-II SLG and universal SLG primer, respectively gave amplification of a single DNA fragment of approximately 1,300 by and 1,150 by from the genomic DNA of 21 S-homozygotes in broccoli. The 1,150 by DNA fragment from 21 S-homozygotes showed polymorphism after cleavage with the restriction endonucleases in the combi VII primers. BamHI, BgℓII, Hinfl, and HaeIII were suitable for SLG polymorphism analysis. We characterized haplotype that correspond to genes for SLG and SRK from 12 S-homozygotes of cabbage. The cleaved DNA fragments were size-fractionated by 2% agarose gel or 5% polyacrylamide gel electrophoresis and detected by silver staining. PCR using SRK primer amplified DNA fragments of 0.7 kb∼0.8 kb from 12 S-homozygotes in cabbage, and digestion of the PCR products with HinfI and subsequent polyacrylamide-gel electrophoresis revealed high polymorphism of the amplified DNA fragments. Although the used primer sequences still need improvement for the analysis of all S-homozygots, PCR-RFLP of SLG or SRK was considered to be useful for characterization in Brassica. F₁ hybrids exhibited the sum of the bands of both parents. Therefore, this method is expected to be used for a purity test of F₁ seeds. The high SLG and SRK polymorphism detected by the present investigation suggests the usefulness of the PCR-RFLP method for the identification of S-haplotype in breeding lines and for listing S-haplotype in broccoli and cabbage.
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KEYWORD
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