杜鹃花属植物SRAP-PCR反应体系的优化及其应用

Optimization of SRAP-PCR Reaction System in Rhododendron and Its Application

采用改良CTAB法提取杜鹃花属植物基因组DNA,对影响SRAP-PCR反应体系中的Mg2+、dNTPs和引物浓度进行优化,建立了杜鹃花属植物SRAP分子标记的扩增体系:20μl的PCR体系中含有模板DNA50 ng、10×PCR buffer(不含Mg2+)、Mg2+2.50 mmol/L、dNTPs 0.20 mmol/L、引物0.40μmol/L、TaqDNA聚合酶1.0 U。并对10种杜鹃花属植物群体进行扩增验证,共获得261条扩增条带,251个多态性位点,多态性位点比率为96.17%,结果表明供试材料间差异明显、多态性较高,该体系适合杜鹃花属植物种间差异性分析,为今后杜鹃花属植物分子生物学的深入研究奠定了基础。

The genomic DNA of Rhododendron was extracted by the revised CTAB method.The concentrations of Mg2＋,dNTPs and primers which affected the SRAP-PCR reactions were optimized,and the SRAP molecular marker system in Rhododendron was established.The optimum system of 20 μl was as follows： template DNA 50 ng,10×PCR buffer（without Mg2＋）,Mg2＋ 2.50 mmol / L,dNTPs 0.20 mmol / L,primers 0.40 μmol / L,Taq DNA polymerase 1.0 U.Ten genotypes of Rhododendron were amplified and detected under the optimum system.SRAP fingerprinting amplified by 16 pairs of primers revealed that 261 unambiguous bands were obtained,of which 251 sites were polymorphic,and the polymorphism frequency was 96.17%.The results indicated that the system was steady and reliable,which would be helpful to study molecular biology of Rhododendron.