SRAP分子标记分析体系的建立及白簕资源亲缘关系分析

本实验对影响SRAP-PCR体系中的Mg2＋、dNTPs和引物浓度等因子进行了体系优化,建立了一套适合白簕SRAP检测的25μL反应体系：1.5mmol/LMg2＋,1.5mmol/LdNTPs,1μmol/L引物,10ng模板DNA,1.5UTaqDNA聚合酶;进一步以白梗簕菜为模板,利用优化的体系进行多态性标记引物组合分析,共筛选出17对引物;利用这些引物对7个白簕品种进行SRAP遗传多样性分析,共扩增出461条谱带。其中多态性谱带155条,多态性谱带比率为33.6%,白簕品种间的相似系数为0.7077~0.9474。经UPGMA聚类分析结果显示,所检测的7个白簕品种分为两大类,其中亲缘关系较近的青梗簕菜和细叶密刺簕菜聚成了一组;而其余的4个种,包括白梗簕菜、细叶小刺簕菜、紫柄簕菜、大叶大刺簕菜和红梗簕菜,聚成另一组。

Optimization of SRAP-PCR System for Phylogenetic Analysis of Acanthopanax trifoliatus Species

The genomic DNA of Acanthopanax trifoliatus was extracted by using modified CTAB method. The uniform design was used to optimize the sequence-related amplified polymorphism （SRAP） reaction system for phylogenetic analysis of Acanthopanax trifoliatus. The present study showed that an optimal SRAP-PCR system was established. The reaction volume of the system is 25 μL which consists of 1.5 mmol/L Mg2＋, 1.5 mmol/L dNTPs, 1 μmol/L primer, 10 ng template DNA and 1.5 U Taq DNA polymerase. SRAP-PCR markers were used to study the genetic diversity of 7 Acanthopanax trifoliatus resources. Using Bai-geng Acanthopanax trifoliatus as template to screen polymorphic primer combinations and 17 SRAP primers were selected for the present study. Total bands amplified were 461. Of these bands, 155 were polymorphic, and the percentage of polymorphic bands was 33.6%. The similarity coefficient among the seven species of Acanthopanax trifoliatus are 0.707 7~0.947 4. The result of UPGMA analysis showed that seven species of Acanthopanax trifoliatus were clustered into two groups. Qing-geng Acanthopanax trifoliatus and Xi-ye-mi-ci Acanthopanax trifoliatus have close relationship and clustered into one group. And other five species including Bai-geng Acanthopanax trifoliatus, Xi-ye-xiao-ci Acanthopanax trifoliatus, Zi-bing Acanthopanax trifoliatus, Da-ye-da-ci Acanthopanax trifoliatus and Hong-geng Acanthopanax trifoliatus clustere into another group.