均匀设计在枇杷ISSR-PCR反应体系优化中的应用

本实验通过U255（5^4）均匀设计实验研究，对枇杷ISSR—PCR分子标记体系中模板DNA、dNTPs、TaqDNA聚合酶、Mg^2＋、引物5个组分的浓度进行优化。获得25μL反应体系中各成分的适宜浓度或用量分别是：模板DNA为10ng、dNTPs为0．5mmol／L、TaqDNA聚合酶为1U、Mg^2＋为2．5mmol／L，引物为0．4μmol／L。与单因素梯度优化体系相比，操作简便，简化了实验步骤，获得的实验结果可靠，从100条ISSR引物中筛选出27条扩增良好的引物，并获得了这些引物的最佳退火温度，经琼脂糖凝胶电泳获得了清晰的图谱。这一优化体系的建立为进一步利用ISSR标记技术进行枇杷种质鉴定及遗传多样性分析提供了一个标准化程序。

Optimization of ISSR-PCR Analysis and its Application in Germplasm of Loquat(E.japonica Lindl.cv) by Uniform Design

Abstract This paper presented the effect of the main reaction system elements （template DNA, dNTPs, Taq DNA polymerase, Mg2~ and primer） on ISSR-PCR which were tested by uniform design, to determine its optimal levels. A reaction system and amplified procedure suitable for loquat were established, that is, 25 p~L amplification reaction system containing 2.5 ~L 10xPCR buffer, 10 ng template DNA, 0.5 mmol/L dNTPs, 1 U Taq DNA poly- merase, 2.5 mmol/L Mg2＋ and 0.4 p~mol/L primer. Compared with single factor experiment, the exeperiment step was more simple and convenient, the result was more true and credible. 27 ISSR primers has been obtained with high stability and polymorphism simultaneity obtained for ISSR-PCR annealing temperature from 100 ISSR primers, the protocol resulted in clear and robust ISSR patterns after electrophoresis on agarose gels. This optimal system laid the standardization program of the identification and the efficient use of the loquat germplasm resource.