PCR-RFLP ofSlocus for identification of breeding lines in cruciferous vegetables

摘要

A simple method of detecting polymorphism ofSlocus glycoprotein gene,SLG, in Chinese cabbage and cabbage was developed, and used for identification of breeding lines. DNA was amplified by the polymerase chain reaction (PCR) with a pair of primers havingS6SLGsequences from inbred lines, and digested with restriction endonucleases which recognize tetranucleotide sequences. The cleaved DNA fragments were size-fractionated by polyacrylamide gel electrophoresis and detected by silver staining. PCR withS6SLGprimers amplified a fragment of ca. 1.3kb in more than half of the inbred lines tested. After digestion, polyacrylamide gel electrophoresis revealed polymorphism between the amplified 1.3kb DNA fragments. These polymorphic bands were detected by Southern hybridization using a probe ofS6SLGcDNA, suggesting that the amplified DNA wasSLG.Primers having theSLGsequences ofS2, a representative of recessiveSalleles, were used for amplification ofSLGin the lines which did not give the 1.3kb DNA fragment by the PCR withS6SLGprimers. Polymorphism of amplified DNA was found in these lines. However these primers also appeared to amplify an invariantSLR-2sequence of 1.3kb in addition to the polymorphicS2SLGrelated sequences. Although the used primer sequences still need improvement for the analysis of recessiveSalleles, PCR-RFLP ofSLGwas considered to be useful for identification of breeding lines as well as forSallele identification in cruciferous vegetables. F1hybrids exhibited the sum of the bands of both parents, and, therefore, this method is expected to be used for a purity test of F1seeds.