广西植物2012，Vol.32Issue(5)：593-598,6.DOI:10.3969/j.issn.1000-3142.2012.05.005
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
摘要
Abstract
Inter-Simple Sequence Repeat (ISSR) is a good molecular marker for revealing genetic diversity,and the re-action 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 influen-cing 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 p.h 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. Ac-cording 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. may-ebrae and Rh. huadingense were divided into another group. These results would supplement the information for pro- tection and utilization of Rh. huadingense and also provide further data for the study of genetic variation and species differentiation of Rh. huadingense.关键词
华顶杜鹃/ISSR/优化/亲缘关系Key words
Rhododendron huadingense/ISSR/optimization/phylogenetic relationship分类
农业科技引用本文复制引用
颜士辉,郑蔚虹,丁炳扬..华顶杜鹃ISSR反应体系的优化及亲缘关系的初步分析[J].广西植物,2012,32(5):593-598,6.基金项目
国家自然科学基金(30370106) （30370106）
温州大学课题(2005L013) （2005L013）
