春兰ISSR-PCR反应体系的建立和优化

目的]为进一步从分子水平上研究春兰种群的遗传多样性奠定基础。方法]以春兰基因组DNA为模板,通过单因子试验研究ISSR反应体系中主要成分对扩增结果的影响,以寻找适合春兰ISSR分析的反应体系。结果]春兰的ISSR反应体系较适宜的扩增条件为:25lμPCR反应体积中,15.78μl双蒸水,2.5μl 1×CR buffer,1.1 UTaqDNA聚合酶,100 ng基因组DNA,2.0 mmol/L MgCl2,150μmol/LdNTPs,2μmol/L引物。最佳扩增程序为:94℃预变性5 min,然后进行40个循环;94℃变性45 s,复性温度比各引物的TM值略低1~2℃,45 s,72℃延伸75 s,循环结束后72℃延伸7 min,4℃保存。结论]该优化系统的建立为进行春兰鉴定及种质遗传多样性分析提供了一个标准化程序。

Establishment and Optimization of ISSR-PCR Reaction System for Cybidium goeringii

Objective]The aim was to lay the foundation for the further study of population genetic diversity of Cybidium goeringii from the molecular level.Method]With the genomic DNA of C.goeringii as template,the influence of the main components in the ISSR reaction system on the amplification result was studied by single factor experiment to search for the reaction system suitable for ISSR analysis of C.goeringii.Result]The suitable amplification conditions of ISSR reaction system in C.goeringii was as 15.78 μl double distilled water,2.5 μl 1×PCR buffer,1.1 U Taq DNA polymerase,100 ng genomic DNA,2.0 mmol/L MgCl2,150 μmol/L dNTPs,2 μmol/L primer in the 25 μl PCR reaction volume.The optimum amplification programme was that after pre-denaturing of 5 min at 94 ℃,40 cycles were performed with denaturing of 45 s at 94 ℃,denaturing temperature of 1～2 ℃ lower than the different primer's TM value,annealing of 45 s,extension of 75 s at 72 ℃,a final extension step of 7 min at 72 ℃ and preservation at 4 ℃.Conclusion] The establishment of the optimization system laid the standardization program for the identification of C.goeringii and the analysis of its gemplasm genetic diversity.