摘要
旨在构建胡椒(Piper nigrum L.)基因组DNA的ISSR-PCR反应体系,以便为海南胡椒属植物的遗传多样性分析研究打下基础。利用单因素随机试验设计,对胡椒ISSR-PCR反应体系中各组分(TaqDNA聚合酶、dNTPs、模板DNA、引物和Mg2+)的浓度进行优化,同时筛选ISSR-PCR反应的循环数和最适退火温度。确定了最优的ISSR-PCR反应体系为:总体积20μL,其中Taq DNA聚合酶1.0 U,dNTPs 0.8 mmol/L,引物0.2 mmol/L,Mg2+1.8 mmol/L,模板DNA 50 ng。同时通过梯度PCR试验,确定引物最佳退火温度及最佳循环数。胡椒ISSR-PCR最佳反应程序为:95℃预变性5 min;94℃变性30 s,54.9℃(引物834)退火30 s,72℃延伸45 s,共计35个循环,循环结束后72℃延伸7 min,4℃保存。这一反应体系可成功地应用于海南胡椒属植物的遗传多样性分析。
The conditions of ISSR-PCR reaction were optimized using the method of single-factor experiment respectively.The optimal ISSR-PCR amplification was established as follows： in a 20 μl reaction mixtures containing 1.0 U Taq DNA polymerase,0.8 mM of each dNTP,0.2 mM primer,1.8 mM Mg2＋,and 50 ng template DNA for 35 thermal cycles.Amplifications were performed in an Biometra TGradient Thermal Cycler using the following conditions： in 20 μl reaction volumes for each primer,initial denaturation at 95℃ for 5min;denaturation at 94℃ for 30 s;annealing at the optimum anneal temperature selected for 30 s;extension at 72℃ for 45 s;35 cycles;the last cycle followed by 7 min extension at 72℃.This reaction system can be successfully applied to the analysis of genetic diversity of Piper plants in Hainan Island.
出处
《热带农业科学》
2012年第8期26-30,78,共6页
Chinese Journal of Tropical Agriculture
基金
国家自然科学基金项目(No.31060204)
关键词
胡椒
ISSR
分子标记
PCR扩增
black pepper
ISSR
molecular marker
PCR amplification
