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

摘要: 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℃.

Abstract: 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,Mg²⁺ 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 MgCl-2 and 10 ng template DNA.The optimized annealing temperature was 61℃ for primer(GTG)-6.