马褂木ISSR-PCR扩增体系的优化

【目的】建立稳定的马褂木ISSR-PCR扩增体系,为马褂木种群ISSR多样性分析提供技术支持。【方法】采用单因素试验对ISSR-PCR扩增体系中的MgCl2、dNTPs、引物浓度、TaqDNA聚合酶、DNA模板用量和退火温度等主要因素进行筛选优化。【结果】采用试剂盒方法提取马褂木叶片DNA的效果优于改良CTAB法,最优PCR反应体系为：总体积20.0μL中含有10×Buffer2.0μL、MgCl21.8mmol/L、dNTPs0.2mmol/L、引物0.5μmol/L、TaqDNA聚合酶1.00U、DNA模板60ng、退火温度59.1℃。【结论】优化后的马褂木ISSR-PCR扩增体系能够扩增获得清晰、稳定的条带,可满足马褂木种群ISSR多样性分析的要求。

Optimization of ISSR reaction system of Liriodendron chinese

Objective]A stable ISSR reaction system suitable for Liriodendron chinese was established to provide techni- cal support for ISSR diversity analysis of LJfiodendron chinese population. Method]In the single factor experiment, main fac- tors of ISSR reaction system including MgCl2, dNTPs, primer concentration, Taq DNA polymerase, DNA template amount and annealing temperature were optimized and screened. Result]Using kit to extract DNA in Liriodendron chinese leaves was better than using the improved CTAB method. The optimal PCR reaction system was： 20 p~L reaction volume containing 2.0 I~L10x buffer, 1.8 mmol/L MgCI2, 0.2 mmol/L dNTP, 0.5 txmol/L primer, 1U Taq polymerase,60 ng template DNA, and 59.1℃ anneal- ing temperature. Conclusion]Through the optimized ISSR-PCR reaction system, clear and stable stripes could be attained, thus it＇s proper for ISSR diversity analysis of Liffodendron chinese population.