海南特有极小种群植物文昌锥的SCoT-PCR体系建立及有效引物评价

为了应用SCoT分子标记研究文昌锥种质资源，通过单因素试验和正交试验2种方法，分析DNA、引物、dNTPs、Taq DNA聚合酶这4种因素对文昌锥SCoT-PCR扩增结果的影响，优化建立文昌锥SCoT-PCR体系，并对文昌锥SCoT标记引物进行有效性评价。结果表明：各个因子对SCoT-PCR扩增影响大小依次为：DNA > dNTPs > 引物 > Taq DNA聚合酶；最优反应体系为：总体系为20 μL，DNA含量为2.5 ng，引物浓度为0.8 μmol·L^-1，dNTPs浓度为0.2 mmol·L^-1，Taq DNA聚合酶的含量1 U；经验证，该体系获得的扩增产物清晰、稳定；应用该体系筛选出15条多态性好且适合文昌锥扩增的引物，为今后利用SCoT分子标记技术对文昌锥及其他壳斗科植物进行相关研究提供技术支持。

Optimization of SCoT-PCR System and Screening of Effective Primers for Castanopsis wenchangensis,an Extremely Small Population Endemic to Hainan Province

The effects of DNA, primers and dNTPs, Taq DNA polymerase on the SCoT-PCR amplification results of Castanopsis wenchangensis were analyzed by single factor test and orthogonal test, and the SCoT-PCR system of C.wenchangensis was optimized. The polymorphism primer combination was screened based on the established reaction system, which provided the conditions for the study of SCoT molecular markers in the study of C.wenchangensis germplasm resources. The results showed that the influence of each factor on SCoT-PCR amplification was DNA > dNTPs > primer > Taq enzyme, and the optimum reaction system was as follows:when the total system was 20 μL, DNA content was 2.5 ng, primer concentration was 0.8 μmol·L^-1, dNTPs concentration was 0.2 mmol·L^-1, Taq content was 1 U, and the amplified products were clear and stable. In addition, 15 primers with good polymorphism and suitable for C.wenchangensis were screened out from 80 SCoT primers by this system, which provided theoretical basis and technical support for the related research of C.wenchangensis and other plants of Fagaceae by SCoT molecular marker technique in the future.