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[目的]针对翻白草ISSR的反应特点,建立稳定可靠的ISSR分子指纹标记反应体系,为进一步研究翻白草的居群差异奠定基础。[方法]通过筛选引物并设定影响翻白草ISSR反应的诸因子的不同浓度,检测ISSR不同反应体系的扩增效果;通过分析非特异性条带的产生原因并进行条件优化,建立翻白草ISSR稳定可靠的反应体系。[结果]首次建立了可用于翻白草ISSR-PCR分析的最适宜的反应体系,确定了25lμPCR反应体系中各试剂终浓度:1×Buffer缓冲液,1 UTaq DNA聚合酶,2.5 mmol/LMg2+,0.25 mmol/L dNTP,0.4μmol/L引物,DNA模板约20~30 ng,退火温度在50~56℃;实验表明:Taq酶质量、DNA模板品质、退火温度、Mg2+浓度d、NTP浓度均对ISSR反应结果具有较大影响。[结论]所建立的翻白草ISSR反应体系具有标记位点清晰、反应系统稳定、检测多态性能力较强、重复性好等特点,可以较好地应用于翻白草的居群鉴别及居群分子生态的研究。
[Objective] The aim of the study was to establish a stable and reliable ISSR molecular fingerprinting reaction system based on the reaction characteristics of ISSR, which lays the foundation for further study on the population differences of. [Method] The amplification efficiency of different ISSR reaction system was tested by screening primers and setting different concentrations of various factors that affect ISSR reaction. By analyzing the causes of non-specific bands and optimizing the conditions, ISSR stable and reliable reaction system. [Result] The optimal reaction system for ISSR-PCR analysis was established for the first time. The final concentration of each reagent in 25μlμPCR reaction system was determined: 1 × Buffer, 1 UTaq DNA polymerase, 2.5 mmol / L Mg2 + 0.25 mmol / L dNTP and 0.4 μmol / L primer, the DNA template was about 20-30 ng and the annealing temperature was 50-56 ℃. The results showed that the quality of Taq DNA, DNA template quality, annealing temperature, Mg 2+ concentration d and NTP concentration ISSR reaction results have a greater impact. [Conclusion] The established ISSR reaction system had the characteristics of clear marker site, stable reaction system, strong polymorphism detection ability and good repeatability, which could be applied to the population identification of Study on population molecular ecology.