毛白杨ISSR反应体系的建立及优化

Establishment and optimization of an ISSR reaction system for Populus tomentosa Carr.

为应用ISSR技术开展毛白杨遗传变异分析、辅助育种、品种鉴定、系统进化等研究,以(GTG)6为引物,通过单因子实验分别研究了退火温度、引物浓度d、NTP浓度、Mg2+浓度、Taq DNA聚合酶用量对毛白杨ISSR-PCR反应的影响,建立并优化了适宜于毛白杨ISSR分析的扩增体系:20μL PCR反应体系,1×Taq DNA酶缓冲液(10 mmol/L Tris--HCl,50 mmol/L KCl,0.1%Trion X-100,pH 9.0),1.5 mmol/L MgCl2,1.0 U Taq酶,10 ng模板DNA,0.2μmol/L引物,0.2 mmol/L dNTP.引物(GTG)6的最适退火温度为61℃.

In order to use ISSR markers to study genetic variation, assistant breeding, cultivar identification and phylogenesis of Populus tomentosa, the annealing temperature, concentration of primer, dNTP, Mg^2＋ and Taq DNA polymerase dosage on ISSR-PCR amplications were tested to determine their optimal levels and establish the following optimal reaction system for ISSR analysis in P. tomentosa. The results showed that the optimal conditions for ISSR-PCR of P. tomentosa were as follows： PCR reaction volume of 20 μL, 1 ×Taq buffer （10 mmol/L Tris-HCl, 50 mmol/L KCl, 0.1% Triton X-100, pH 9.0）, 1.0 U Taq DNA Polymerase, 0.2 μmol/ L primer, 0.2 mmol/L dNTP, 1.5 mmol/L MgC12 and 10 ng template DNA. The optimized annealing temperature was 61℃ for primer （GTG）6.