为了建立柿属植物目标区域扩增多态性（ TRAP）标记技术体系，对影响TRAP-PCR的Mg2＋、dNTPs、Taq DNA聚合酶、固定引物和随机引物浓度比5个因素进行了优化。确定优化的反应体系为：模板DNA 40 ng，buffer 1×，Mg2＋1．4 mmol/L，dNTPs 0．2 mmol/L，固定引物和随机引物浓度比为11∶1，Taq 酶0．5 U，反应总体积为15μL。利用柿属植物EST数据库信息设计锚定引物10条，与11条随机引物组合共110对引物。利用这些引物对柿属植物6个基因型进行TRAP-PCR扩增，其中84个引物组合能扩增出清晰条带，占引物总量的76．36％；36个引物组合表现出良好的多态性扩增，并在20份柿属植物中进行了引物验证。%The concentrations of Mg 2+, dNTPs, Taq DNA polymerase and primers which affected TRAP ( target region ampli-fied polymorphism)-PCR reaction were optimized for the establishment of TRAP molecular marker system in Diospyros spp.The optimum reaction system was acquired as follows:template DNA 40 ng, buffer 1 ×, Mg2+1.4 mmol/L, dNTPs 0.2 mmol/L, fixed primer-arbitrary primer concentration ratio 11∶1, Taq DNA polymerase 0.5 U, and total volume of reaction system 15μL.Eighty-four TRAP primer combinations which showed robust PCR amplification in 6 persimmon genotypes were screened out from 110 primer combinations (10 fixed primers times 11 arbitrary primers).Thirty-six polymorphic primer combinations were verified in 20 persimmon genotypes .
展开▼
