茶树ISSR-PCR反应体系的建立

姚明哲; 王新超; 陈亮; 杨亚军

doi:10.13305/j.cnki.jts.2004.03.005

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茶叶科学 ›› 2004, Vol. 24 ›› Issue (3) : 172-176. DOI: 10.13305/j.cnki.jts.2004.03.005

茶树ISSR-PCR反应体系的建立

姚明哲, 王新超, 陈亮, 杨亚军

作者信息 +

Establishment of ISSR-PCR Reaction Conditions in Tea Plant

YAO Ming-zhe, WANG Xin-chao, CHEN Liang, YANG Ya-jun

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文章历史 +

摘要

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%。

Abstract

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.

导出引用

姚明哲, 王新超, 陈亮, 杨亚军. 茶树ISSR-PCR反应体系的建立[J]. 茶叶科学. 2004, 24(3): 172-176 https://doi.org/10.13305/j.cnki.jts.2004.03.005

YAO Ming-zhe, WANG Xin-chao, CHEN Liang, YANG Ya-jun. Establishment of ISSR-PCR Reaction Conditions in Tea Plant[J]. Journal of Tea Science. 2004, 24(3): 172-176 https://doi.org/10.13305/j.cnki.jts.2004.03.005

中图分类号： Q523

参考文献

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