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目的:建立头花蓼ISSR的反应体系。方法:通过正交试验,研究了Mg2+浓度、dNTP浓度、Taq DNA聚合酶浓度、引物浓度这4个因素在3个水平上对ISSR-PCR的影响。结果:确立了适合于头花蓼ISSR PCR的优化体系:25μL PCR反应体系中含有1×buffer缓冲液(10 mmol.L-1 KC1,8 mmol.L-1(NH4)2SO4,10 mmol.L-1 Tris.HC1,pH 8.0),3.0 mmol.L-1 MgC12,d NTP 200μmol.L-1,2.0 U.25μL-1 Taq酶,0.25 mmol.L-1引物,40 ng.L-1模板DNA。利用温度梯度PCR,确定了最适宜的退火温度为48℃。结论:该优化体系的建立为进一步对头花蓼进行种质资源的遗传多样性分析奠定了基础。
Objective: To establish the ISSR reaction system of Polygonum capitatum. Methods: The effects of Mg2 + concentration, dNTP concentration, Taq DNA polymerase concentration and primer concentration on ISSR-PCR at three levels were studied by orthogonal test. Results: The optimal system of ISSR PCR for Polygonum capitatum was established: 25μL PCR reaction contained 1 × buffer (10 mmol.L-1 KC1, 8 mmol.L-1 (NH4) 2SO4, 10 mmol.L -1 Tris.HC1, pH 8.0), 3.0 mmol.L-1 MgC12, dNTP 200 μmol.L-1, 2.0 U.25 μL-1 Taqzyme, 0.25 mmol.L-1 primer, 40 ng.L-1 template DNA. Using temperature gradient PCR, the optimum annealing temperature was 48 ℃. Conclusion: The establishment of this optimized system lays a foundation for further analysis of the genetic diversity of Polygonum capitatum germplasm resources.