辣椒RAPD扩增体系的建立

目的]为辣椒的品种资源鉴定与遗传多样性分析奠定基础。方法]采用改良的CTAB法对辣椒叶片基因组DNA进行提取,并采用正交试验设计法对辣椒RAPD-PCR反应进行优化筛选。结果]采用改良的CTAB法获得了高质量的辣椒基因组DNA,用Tris-平衡酚∶氯仿∶异戊醇（25∶24∶1,V∶V∶V）进行抽提,最后再用氯仿抽提以除去其中含有的酚,避免了对后续PCR扩增的影响。基于一般的RAPD反应程序和调整试验,优化了PCR反应体系。结果表明,在模板20 ng 1.5μl、引物（20μmol/L）1.5μl、dNTPs（2.5 mmol/L）2.2μl、10×Buffer缓冲液（含Mg2＋）2.5μl、Taq（5 U/μl）0.35μl条件下扩增效果较好。结论]建立了最适的辣椒RAPD扩增体系。

Research on the Establishment of RAPD Amplification Program of Hot Pepper

Objective] The basis of the identification of hot pepper(Capsicum annuum) germplasm resource and the analysis of its diversity was laid.Method] The technique of the RAPD-PCR reaction to the high-quality DNA,extracted from hot pepper with the improved CTAB method,was optimized with the method of orthogonal experimental design.Result] The hot pepper DNA was extracted in the solution of Tris-balanced phenol : chloroform : isoamyl alcohol(25∶24∶1,V∶V∶V)and then the phenol was removed with chloroform,which efficiency would be avoided of in the following PCR amplification.The PCR system was optimized through the adjustment experiment based on the common RAPD reaction program.The result indicated that the good amplification efficiency was under the condition: the genomic DNA 20 ng,1.5 μl;the primer(20 μmol/L),1.5 μl;the dNTPs(2.5 mmol/L),2.2 μl;the 10×PCR Buffer,2.5 μl(containing Ma2+),2.5 μl;and the Taq polymerse(5 U/μl),0.35 μl.Conclusion] The optimal RAPD amplification system of hot pepper was established.