PCR-RFLP OF S-LOCUS FOR IDENTIFICATION OF BREEDING LINES IN CRUCIFEROUS VEGETABLES
PCR-RFLP OF S-LOCUS FOR IDENTIFICATION OF BREEDING LINES IN CRUCIFEROUS VEGETABLES
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DOI：
10.1007/bf00234508
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发表时间：
1994-07-01
影响因子：
6.2
通讯作者：
YAMAGUCHI, J
中科院分区：
文献类型：
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作者：
NISHIO, T;SAKAMOTO, K;YAMAGUCHI, J
建立了一种检测大白菜和甘蓝S基因座糖蛋白基因SLG多态性的简便方法，并将其用于大白菜和甘蓝育种品系的鉴定。以S-6自交系SLG序列为模板，通过聚合酶链式反应扩增DNA，并用识别四核苷酸序列的限制性内切酶进行酶切。用聚丙烯酰胺凝胶电泳法对裂解后的DNA片段进行大小分级，并用银染法进行检测。用S-6SLG引物进行聚合酶链式反应，在一半以上的自交系中扩增出约1.3kb的片段。经酶切后，聚丙烯酰胺凝胶电泳法显示扩增出的1.3kb DNA片段之间存在多态性。用S-6SLG基因的探针进行Southern杂交，检测到这些多态条带，表明扩增出的片段为SLG。利用具有隐性等位基因S-2的SLG序列的引物，对未产生1.3kb DNA片段的品系，用S-6SLG引物进行SLG扩增。在这些品系中发现了扩增DNA的多态性。然而，除了S-2的SLG相关序列外，这些引物似乎还扩增出一个1.3kb的不变的SLR-2序列。虽然用于S隐性等位基因分析的引物序列尚需改进，但SLG扩增限制性片段长度多态性技术可用于十字花科蔬菜育种品系的鉴定和S等位基因的鉴定。F-1杂交种表现出双亲的条带总和，因此，该方法有望用于F-1种子的纯度鉴定。
A simple method of detecting polymorphism of S locus 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 having S-6 SLG sequences 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 with S-6 SLG primers 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 of S-6 SLG cDNA, suggesting that the amplified DNA was SLG. Primers having the SLG sequences of S-2, a representative of recessive S alleles, were used for amplification of SLG in the lines which did not give the 1.3kb DNA fragment by the PCR with S-6 SLG primers. Polymorphism of amplified DNA was found in these lines. However these primers also appeared to amplify an invariant SLR-2 sequence of 1.3kb in addition to the polymorphic S-2 SLG related sequences. Although the used primer sequences still need improvement for the analysis of recessive S alleles, PCR-RFLP of SLG was considered to be useful for identification of breeding lines as well as for S allele identification in cruciferous vegetables. F-1 hybrids exhibited the sum of the bands of both parents, and, therefore, this method is expected to be used for a purity test of F-1 seeds.