三角叶黄连ISSR反应体系的建立与优化

Establishment and optimization of ISSR reaction system for Coptis deltoidea

目的针对三角叶黄连ISSR的反应特点,建立稳定可靠的ISSR-PCR分子标记反应体系,为进一步研究三角叶黄连的种质资源遗传多样性奠定基础。方法通过筛选引物并设定影响三角叶黄连ISSR-PCR反应诸因子的不同梯度,检测其不同反应体系的扩增效果,分析非特异性条带的产生原因并进行条件优化,建立三角叶黄连ISSR-PCR稳定可靠的反应体系。结果建立了可用于三角叶黄连ISSR-PCR分析的最适宜的反应体系:25μLPCR反应体系中,内含10×PCR Buffer 2.5μL,1.5mmol/L Mg2+,200μmol/L dNTP,0.3μmol/L引物,40ng模板,1.0U Taq DNA聚合酶。扩增程序为94℃预变性5min,然后进行35个循环:94℃变性30s,(据不同引物的退火温度)复性60s,72℃延伸90s,循环结束后72℃延伸7min,4℃保存。结论所建立的三角叶黄连ISSR反应体系具有标记位点清晰、反应系统稳定、检测多态性能力强、重复性好等特点,可以较好地应用于三角叶黄连的种质资源遗传多样性及居群鉴别的研究。

Objective In order to study the genetic diversity of Coptis deltoidea germplasm, ISSR-PGR system of C. deltoide was established and optimized according to the characters of it. Methods The effect of ISSR-PCR was examined by screening primers and designing the different concentration of the factors in the ISSR. The reliable system for C. deltoide populations researching was established by analyzing the reasons for occurrence of differential bands and optimizing the reaction conditions. Results The optimal ISSR-PCR system in C. deltoide was established for the first time, that is, 25 μL amplification reactions system containing 10×PCR Buffer 2.5 μL, 1.5 mmol/L Mg^2＋ , 200μmol/L dNTP, 0. 3 vmol/L primer, 40 ng template, 1.0 U Taq DNA polymerase. The optimal amplified procedure was as follows： after a pre- denaturing of 5 min at 94℃, 35 cycles were performed with denaturing of 30 s at 94℃, annealing of 1 min due to denaturing temperature of different primer, extension of 1.5 min at 72 ℃, a final extension step of 7 min at 72℃ and kept at 4℃. Conclusion The ISSR-PCR systems established for studying of C. deltoide show the characters of clear marker site, stable reaction system, reliable abundant polymorphisms, and better repeatability. It is suitable for the study on genetic diversity of germplasm resource and identifi- cation of C. deltoide population.