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利用正交设计L16(45)对金线莲ISSR-PCR反应体系的5因素(模板DNA、Taq酶、引物、Mg2+和dNTP)在4个水平上进行优化试验,采用直观分析法获得影响因素最佳反应水平。结果表明:最终建立的金线莲ISSR-PCR的最佳反应体系为,在25μL的反应体系中,DNA模板为40ng、Taq酶为1.60U、引物浓度为0.80mmol/L、dNTP浓度为0.96mmol/L、Mg2+浓度为2.40mmol/L。该反应体系的建立为金线莲种质资源分类、遗传多样性分析提供了更客观可靠的方法。
Five factors (template DNA, Taq enzyme, primer, Mg2 + and dNTP) of ISSR-PCR reaction system were optimized by orthogonal design L16 (45) at four levels and the most influential factors Good response level. The results showed that the optimal reaction system for ISSR-PCR was as follows: 40 ng of DNA template, 1.60 U of Taq DNA polymerase, 0.80 mmol / L of primer and 0.96 mmol of dNTP in a 25 μL reaction system / L, Mg2 + concentration 2.40mmol / L. The establishment of the reaction system provides a more objective and reliable method for the classification of the germplasm resources and the analysis of genetic diversity.