红景天种内遗传多样性分析AFLP方法建立

Method of Intraspecific Genetic Diversity Analysis of Rhodiola L. Based on AFLP Markers

研究建立一种用于药用植物红景天种内遗传多样性分析的AFLP分子标记方法。以提取红景天(Rhodiola.roseaL)种基因组DNA为模板,25μL酶切体系中采用两步双酶切(Mse I、EcoR I),在20μL连接体系中采用T4连接酶,22℃连接过夜,50μL体系预扩增,TaqPlus酶2.5 U,dNTP 160μM,对应引物0.5μM,10×PCR Buffer(含Mg2+)4.5μL,25μL体系选择性扩增,TaqPlus酶1.5 U,dNTP 80μM,对应引物0.25μM,10×PCR Buffer(含Mg2+)2.5μL。AFLP分子标记技术是一种快速、准确、稳定的药用植物红景天的种内遗传多样性分析手段。

The level and change of intraspecific genetic diversity of Rhodiola L. were analyzed to establish a method of amplified fragment length polymorphism （ AFLP ）. Genome DNA used as template was digested with EcoR Ⅰ and Mse Ⅰ by two steps in 25μL reaction system. Ligation was done with T4 DNA ligase at 22℃ for overnight. The 50μL pre - amplification reaction mix contains 2.5 U Taq plus DNA polymerase, 160μM dNTP, 0.5 μM pre - amplification primers, 4.5μL 10 × PCR Buffer（ Mg^2＋ ）. Selective amplification is carried out using 1.5 U Taq plus DNA polymerase, 80μM dNTP, 0.25μM pre - amplification primers, 2.5μL 10 × PCR Buffer（Mg^2＋） and added dd H2O to a 25μL volume. The conclusion was drawn that AFLP molecular marker technology is a prompt, accurate and efficient method to analyze the change of intra - specific genetic diversity of Rhodiola L.