硬叶兜兰SRAP-PCR体系建立及亲缘关系研究  

Genetic Relationship Analysis of Paphiopedilum micranthum Based on SRAP Technology

作　　者：李宗艳[1] 李静[1] 郭荣[2] 秦艳玲[1] 

机构地区：[1]西南林业大学园林学院,昆明650224 [2]西南林业大学基础部,昆明650224

出　　处：《基因组学与应用生物学》2014年第1期168-173,共6页Genomics and Applied Biology

基　　金：园林植物与观赏园艺省级重点学科和重点实验室(项目编号:500974)资助

摘　　要：采用CTAB法对硬叶兜兰叶片总DNA进行提取,通过正交实验设计,用SRAP正向引物me1（5＇-TGAGTCCAAACCGGATA-3＇）和反向引物em2（5＇-ACACACACACACACACT-3＇）对反应体系进行优化;并应用其进行种群遗传差异分析。结果表明：优化的20μL的PCR反应体系中DNA模板为90 ng,Taq DNA聚合酶1.6 U,dNTPs 0.30 mmol/L,MgCl22.0 mmol/L和引物各0.50μmol/L,经重复电泳验证适合进行硬叶兜兰的SRAP分析;利用该体系对7个不同种源的167份硬叶兜兰进行SRAP-PCR扩增,对筛选的10个引物扩增,共得到288条条带,其中多样性条带234条,多态位点百分率（PPB）为81.25%;种群的遗传距离值在0.065 9～0.191 6,UPGMA聚类结果显示供试种群在遗传相似度为0.863时,可以分为2支,即第一分支由马固、斗咀和杨柳井居群组成,第二支由古林箐、夹寒箐、小坝子和田坝居群组成。SRAP标记可较好地适用于硬叶兜兰种群遗传差异的鉴定。Genetic diversity was detected in the seven populations of Paphiopedilum micranthum by a SRAP marker. Total DNA ofPaphiopedilum rnicranthum was extracted from fresh leaves by a modified CTAB method. An orthogonal design was used to optimize an amplification system with a pair of the primer combination （mel sequence： 5＇-TGAGTCCAAACCGGATA-3＇; cm^2 sequence： 5＇-ACACACACACACACACT-3＇）. The results indicated that a suitable amplification system contained 90 ng DNA template, 0.30 mmol/L dNTPs, 2.0 mmol/L Mgcl2, 1.6 U Taq DNA polymerase and 0.5 ixmol/L each primer in the 20 μL PCR volume, with which the distinct, reproducible, well-resolved bands could be produced by the repeated gel electrophoresis. The optimization reaction system could be used in the research on P. micranthurn. 167 samples from seven populations were drawn to detect its genetic diversity with ten primer pairs screened by this optimization reaction system. A total of 288 amplified bands were obtained, among which 234 bands were polymorphic and accounted for 81.25%. Genetic distance a- mong the seven populations varied from 0.065 9 to 0.191 6. Based on the UPGMA cluster dendrogram, the seven populations were divided into two groups （group A and group B） at genetic similarity of 0.863, which the group A included Magu, Douzui and Yangliujing population and the group B contained Jiahanqing, Gulinqing, Tianba, and Xiaobazi. SRAP molecular marker could be suitable to detect genetic diversity among populations of Paphiopedilum micranthurn.

关 键 词：硬叶兜兰 SRAP-PCR反应体系 遗传变异 亲缘关系 

分 类 号：S682.31[农业科学—观赏园艺]