锥栗自然居群ISSR-PCR分析技术的建立

Establishment of inter -simple sequence repeat reaction system in Castanea henryi

提取野生锥栗的基因组DNA作为ISSR-PCR模板,对反应体系中的模板用量、Mg2+浓度、dNTPs浓度、引物浓度和TaqDNA聚合酶用量5个主要影响因素进行梯度试验,并对扩增程序中的退火温度与循环次数进行了探讨,初步确立了适合野生锥栗的ISSR-PCR分析技术体系,即25μL反应体系中,模板DNA40或50ng、Mg2+2.25mmol.L-1、dNTPs0.2mmol.L-1、引物0.2μmol.L-1、TaqDNA聚合酶1.0U;PCR扩增程序为:94℃预变性3min;然后94℃变性45s,(Tm+2～4℃)退火45s,72℃延伸2min,共32个循环;最后72℃充分延伸5min;1.5%琼脂糖凝胶电泳分离扩增产物。

In order to investigate the genetic diversity and relationship within and among Castanea henryi, Zhejiang and Huangshan natural C. henryi were subjected to inter-simple sequence repeat （ISSR-PCR） analysis. Genomic DNA of C. henryi was extracted from leaves as the template, the mainly influencing factors of ISSR were studied and the experiment parameters were optimized. By adjusting the concentration of template DNA, Mg^2＋, dNTPs, primer and the contents of Taq DNA polymerase, the PCR amplification system were optimized; Then we also optimized the amplified program, including the annealing temperature and PCR cycles, the PCR amplification conditions were established, finally. The optimal experiment conditions were as follow： 25 μL reaction system containing 40 or 50 ng template DNA, 2.25 mmol ·L^-1 Mg^2＋, 0.2 mmol ·L^-1 dNTPs, 1.0 U Taq DNA polymerase, 0.2 μmol·L^-1 primer; with a ABI thermal cycle, the optimal amplification program was an initial denature for 3 min at 94 ℃, followed by 32 cycles of 45 s at 94 ℃, 45 s at Tm＋2～4 ℃, 2 min at 72 ℃, and a final extension for 5 rain at 72 ℃.