华顶杜鹃ISSR反应体系的优化及亲缘关系的初步分析

Optimization of ISSR reaction conditions and analysis of phylogenetic relationship for Rhododendron huadingense

关键词:  华顶杜鹃  ISSR  优化  亲缘关系

Key words:  Rhododendron huadingense  ISSR  optimization  phylogenetic relationship

摘要:

以华顶杜鹃为材料,利用单因素试验确立适合华顶杜鹃ISSR-PCR的优化反应体系:在25 μL反应体系中,Mg²⁺1.5或2.0 mmol/L,dNTPs 0.3 mmol/L,引物0.40 μmol/L,Taq DNA聚合酶lU,模板DNA 40 ng; 分析ISSR-PCR反应体系中Mg²⁺、dNTP、引物、模板DNA、Taq DNA聚合酶浓度及退火温度对ISSR-PCR反应的影响。PCR扩增程序:预变性94 ℃ 3 min; 变性94 ℃ 45 s; 退火(Tm+2)℃(根据引物不同设定)75 s; 延伸72 ℃ 1.5 min; 40个循环; 继续延伸72 ℃ 10 min; 1.5%琼脂糖电泳分离PCR产物。并以优化好的体系初步分析华顶杜鹃及5个近缘种(茶绒杜鹃、Rhododendron mayebrae、Rh.sataense、南平杜鹃和映山红)的亲缘关系,6个种明显地聚为2大类:A类含有映山红、南平杜鹃; B类含有茶绒杜鹃、Rh.sataense、Rh.mayebrae与华顶杜鹃,为明确华顶杜鹃的系统地位提供可借鉴的依据。

Abstract:

Inter-Simple Sequence Repeat(ISSR)is a good molecular marker for revealing genetic diversity,and the reaction system differed in different species. In order to study the genetic diversity of Rhododendron huadingense,a protocol of reproducible ISSR was established and phylogenetic relationship was analyzed. The main factors influencing ISSR-PCR including the concentration of Mg²⁺,dNTP,primer,template DNA,Taq polymerase,and annealing temperature,PCR cycles were investigated. The optimal reaction conditions were as follow in 25 μL reaction system:1×buffer,Mg²⁺1.5 or 2.0 mmol/L,dNTP 0.3 mmol/L,primer 0.40 μmol/L,Taq polymerase1U,template DNA 40 ng. The optimal amplification program was:3 min initial denaturation step(94 ℃),followed by 40 cycles of 45 s(94 ℃),75s 〖(Tm+2)℃,according to different primer〗,and 1.5min(72 ℃). The reactions were completed by a final extension step of 10 min(72 ℃). The PCR products were analyzed by electrophoresis using a 1.5% agarose gel. According to cluster analysis,the populations of 6 species were classified into two large groups,the populations of Rh.simsii and Rh.nanpinggense were divided into one group,and the populations of Rh.rufulum,Rh.sataense,Rh.mayebrae and Rh.huadingense were divided into another group. These results would supplement the information for protection and utilization of Rh.huadingense and also provide further data for the study of genetic variation and species differentiation of Rh.huadingense.