芝麻高度不育材料基因组DNA提取及RAPD反应体系的建立

摘要

The genomic DNA extraction and the RAPD-PCR reaction conditions were optimized when using genomes from sesame leaves of temporary maintainer line WB51220,AB Line infertile plant 0176A,fertile plant 0176B,and Wanzhi No.1 as templates. The results indicated that the genomic DNAs extracted from sesame leaves by improved CTAB DNA extraction method satisfied the requirement of genetic diversity analysis. The best reaction condition for RAPD analysis was as follows:Taq DNA polymerase 1.5 U,dNTP 0.25 mmoL/L,Mg2+2.0 mmoL/L,and random primer 0.75μmoL/L in a total volume of 20μL. Electrophoresis results indicated that bands amplified from the genomic DNAs of four varieties had four common bands in length of 1900,1800,1000,and 900 bp,respectively. The differences were that new bands in length of 2000 bp and 450 bp were amplified from AB line infertile plant 0176A when compared with temporary maintainer line WB51220 and fertile plant 0176B,respectively. And two new bands in length of 750 bp and 1200 bp were amplified from fertile plant 0176B when compared with Wanzhi NO.1.%以临时保持系WB51220、两用系0176A不育株、可育株0176B和皖芝1号等4份材料的叶片为研究材料，对其基因组DNA的提取及对RAPD反应体系进行优化。结果表明，改良的CTAB法获得的芝麻基因组DNA片段大小经电泳检测满足RAPD等遗传多样性分析要求；筛选出RAPD最佳反应体系：20μL反应体系含有1.5 U Taq DNA聚合酶，0.25 mmoL/L dNTP，2.0 mmoL/L Mg2+，0.75μmoL/L引物；4个不同品种的叶片基因组DNA的电泳条带之间有着明显差异，他们共有的条带为1900、1800、1000、900 bp；不同的是，临时保持系WB51220比两用系0176A不育株少了1个2000 bp的条带，可育株0176B比两用系0176A不育株少了1个450 bp条带，皖芝1号比可育株0176B少了1个750 bp和1个1200 bp的条带。