天然山地樟子松遗传多样性研究的ISSR-PCR反应体系的优化  

Optimization of the ISSR-PCR Reaction System for Genetic Diversity Research of Natural Pinus sylvestris var. mongolica

作　　者：刘彤[1] 郝雨[1] 周志强[1] 

机构地区：[1]东北林业大学,哈尔滨150040

出　　处：《东北林业大学学报》2006年第5期20-22,共3页Journal of Northeast Forestry University

基　　金：黑龙江省青年基金(QC03C23)资助项目。

摘　　要：以樟子松基因组DNA为模板,研究了ISSR的影响因素,建立了一套适宜于樟子松的ISSR优化反应体系及程序,以期为进行樟子松种群间遗传分化的研究奠定基础。研究结果表明,20μL反应体系中,模板DNA、引物、Mg2+、dNTP和TaqDNA聚合酶5种主要成分的最适浓度分别为35ng、150nmol/L、1.5mmol/L、200μmol/L、1.5U。反应程序为:94℃预变性5min,之后进行34个循环,每个循环包括94℃变性30s、56℃退火45s、72℃延伸2min,最后于72℃延伸7min。在扩增过程中,引物的适宜退火温度比其Tm值平均高4℃,扩增出足量产物至少需要35个热循环。A set of optimized reaction system and program was set up in terms of the influencing factors of ISSR using genome DNA of Pinus sylvestris var. mongolica （ Mongolian Scotch pine） as the template. Results showed that the optimum concentrations of five important components, i.e. template DNA, primer, Mg^2＋ , dNTP and TaqDNA polymerase were 35 ng, 150 nmol/L, 1.5 mmol/L, 200 p.mol/L and 1.5 U in 20 p.L reaction mixture, respectively. The reaction program was devised for 5 minutes of predenaturalization at 94℃, 34 cycles of 30 seconds for denaturalization at 94 ℃ , 45 seconds of anheal at 56 ℃, 2 minutes of extension at 72 ℃, and 7 minutes of extension at 72 ℃ in the final cycle. The appropriate anheal temperature was 4 ℃ higher than Tm of corresponding primer in average, and at least 35 PCR cycles should be conducted to ensure sufficient PCR products. The optimal ISSR reaction system can be applied to the study of genetic differentiation between Mongolian Scotch pine populations.

关 键 词：樟子松 ISSR影响因子 最佳反应体系 

分 类 号：S791.259[农业科学—林木遗传育种]