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以30个茶花品种为试材,采用5因素4水平正交实验以及单因素优化试验方法,研究Mg2+浓度、dNTP浓度、引物浓度、Taq DNA聚合酶浓度和模板DNA浓度对ISSR-PCR指纹图谱条带清晰度的影响,以进行茶花品种ISSR-PCR反应体系优化及引物筛选。结果表明:茶花品种ISSR-PCR最适扩增条件为25μL反应体系中,Mg2+3.0mmol/L、dNTPs 0.2mmol/L、引物0.3mmol/L、Taq DNA聚合酶0.5U、模板DNA 80ng以及52.1℃退火温度;试验从100个ISSR引物中筛选出12个适用引物;并对12个ISSR引物的多态性和稳定性进行了检验。
Using 30 varieties of Camellia sinensis as test material and orthogonal experiment of 5 factors and 4 levels and single factor optimization test method, the effects of Mg2 + concentration, dNTP concentration, primer concentration, Taq DNA polymerase concentration and template DNA concentration on ISSR-PCR fingerprinting With the clarity of the impact of Camellia species for ISSR-PCR reaction system optimization and primer screening. The results showed that the optimal amplification conditions for ISSR-PCR were as follows: Mg2 + 3.0mmol / L, dNTPs 0.2mmol / L, primer 0.3mmol / L, Taq DNA polymerase 0.5U, template DNA 80ng and 52.1 ℃ annealing temperature. Twelve primers were screened out from 100 ISSR primers. The polymorphism and stability of 12 ISSR primers were tested.