通过优化影响茶树ISSR-PCR的主要参数,确立了适用于茶树的ISSR反应体系和扩增条件。结果表明在20βµl反应体系中,模板DNA、引物、Mg++、dNTP 和Taq DNA聚合酶五种主要成分的最适浓度分别为10βng、150βnmol/L、1.5βmmol/L、150βµmol/L、0.5βU。在扩增过程中,引物的适宜退火温度比其Tm值平均高4.5℃,扩增出足量产物至少需要30个热循环。利用该优化反应体系和扩增条件,对13份不同茶树种质资源进行ISSR-PCR扩增,扩增产物的多态性为77.6%。引物TRI18构建的ISSR指纹图谱,可以区分13份茶树资源中的12份,分辨率达92.3%。
The optimal ISSR-PCR reaction conditions in tea was established by studying the main parameters. Results showed that the optimum concentration of five important components i.e. template DNA, primer, Mg++, dNTP, Taq DNA polymerase in 20βµl reaction mixture was 10ng, 150βnmol/L, 1.5βmmol/L, 150βµmol/L, 0.5βU, respectively. The appropriate annealing temperature was average 4.5℃ higher than Tm of corresponding primer, and at least 30 PCR cycles should be carried out to ensure sufficient PCR products. ISSR polymorphism between thirteen tea germplasm was 77.6%, and 12 out of 13 tea germplasm could be identified by ISSR fingerprinter established with primer TRI18.
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