泽泻ISSR反应体系的建立与优化

Establishment and Optimization of the Method of ISSR Fingerprinting Marker on Alisma orientalis(Sam.) Juzep

以四川泽泻为实验试材，采用改进的CTAB法提取了泽泻的高质量总DNA模板，克服了泽泻中所富含的酚类物质的影响．对泽泻ISSR反应体系中各个主要影响因子进行了优化和筛选，建立了标记位点清晰、稳定、重复性好、可用于泽泻ISSR-PCR分析的最适反应体系，它们是：25μLPCR反应体系中，10×Taq酶配套缓冲液，IUTaqDNA聚合酶，1．8～2．0mmol／L MgCl2，100μmol／LdNTP，0．3μmol／L引物，DNA模板约10-20ng，退火温度在52-60％；

Alisma orientalis （Sam.） Juzep of Szeehwan is used as an experimental material. The improved method of CTAB is adopted to distill total DNA template of high quality and the infection that Alisma orientalis contains abundant kinds of hydroxybenzene has been conquered. ISSR-PCR reaction effects are tested and optimized. The optimal ISSR-PCR reaction system in Alisma orientalis is established： 25μL PCR reaction volume, 10×Taq buffer, 1.5 U Taq DNA polymerase, 1.8 - 2.0 mmol/L MgCl2 , 100 μmol/L dNTP, 0. 3 μmol/L primer and 10 - 20 ng template DNA. The appropriate annealing temperature is among 52 -60℃. This article provides elear, reliable, abundant polymorphisms molecular markers suitable for authenticating the populations of Alisma orientalis.