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目的:获得清晰可靠、重复性较好的ISSR-PCR扩增反应体系,应用于芋种质资源进行遗传多样性的研究中。方法:以芋的幼叶提取基因组DNA为材料,采用正交试验设计L16(45),从模板DNA浓度、引物浓度、Mg2+浓度、dNTPs浓度及TaqDNA聚合酶的用量5因素4水平出发,构建芋最佳反应体系。结果:芋ISSR-PCR的最佳反应体系为:在25μl的反应体系中,40ng DNA模板、0.4μmol/L引物浓度、2.5 mmol/L Mg2+、0.2 mmol/L dNTPs、1 U Taq DNA聚合酶。结论:利用芋种质资源对最佳反应体系的验证,结果显示该反应体系具有扩增稳定性。
OBJECTIVE: To obtain a clear and reliable ISSR-PCR amplification reaction system with good repeatability and to apply it to the genetic diversity of taro germplasm resources. Methods: The genomic DNA was extracted from young leaves of Taro, and orthogonal design was used to design L16 (45). From the template DNA concentration, primer concentration, Mg2 + concentration, dNTPs concentration and the amount of Taq DNA polymerase The best reaction system. Results: The optimal reaction system of ISSR-PCR was as follows: 40ng DNA template, 0.4μmol / L primer concentration, 2.5mmol / L Mg2 +, 0.2mmol / L dNTPs and 1U Taq DNA polymerase in 25μl reaction system. Conclusion: Utilizing the germplasm resource of Taro to validate the optimal reaction system, the results show that the reaction system has the stability of amplification.