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为通过ISSR分子标记研究桃儿七遗传多样性,建立并优化桃儿七的ISSR-PCR反应体系。使用正交设计辅以单因素试验,对桃儿七ISSR-PCR反应体系中的5种主要因素(模板DNA,Mg~(2+),Taq DNA酶,d NTPs及ISSR引物)进行筛选优化,选取甘肃碌曲县西仓神山山顶桃儿七居群样本验证该反应体系。正交试验各因素显著性为引物>Taq DNA酶>DNA浓度>dNTPs>Mg~(2+),桃儿七ISSR-PCR反应的最佳体系为(25μL):2.5 mmol/L Mg~(2+),0.2 mol/L dNTP,0.4μmol/L引物,0.5 U Taq DNA酶,40 ng DNA,2.5μL 10×Buffer。该体系具有高稳定性,多态性丰富的特点,为野生桃儿七遗传多样性及保护策略研究提供了技术支持。
In order to study the genetic diversity of peach seven through ISSR molecular markers, ISSR-PCR reaction system was established and optimized. Using orthogonal design and single factor test, the five main factors (template DNA, Mg 2+, Taq DNase, d NTPs and ISSR primers) in ISSR-PCR reaction system were optimized. The samples were selected from the samples of Taoyuan seven populations in Xikunshan Mountain Peak of Luqu County, Gansu Province to verify the reaction system. The optimum conditions for the ISSR-PCR reaction were as follows: primer> Taq DNA polymerase> DNA concentration> dNTPs> Mg 2+, Taq DNA polymerase (25μL) +), 0.2 mol / L dNTP, 0.4 μmol / L primer, 0.5 U Taq DNA enzyme, 40 ng DNA, 2.5 μL 10 × Buffer. The system has the characteristics of high stability and rich polymorphism, and provides technical support for the research on genetic diversity and protection strategy of wild peach.