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通过对多花黄精ISSR-PCR反应中的Taq酶、buffer(Mg2+)、模板DNA、dNTP以及引物5个关键因素进行了5因素4水平的正交设计试验,确立了适合多花黄精基因组DNA的稳定性强、扩增最多数量谱带的ISSR-PCR最优反应体系,即25μL的反应体系中含有1.0 U Taq DNA聚合酶,3.5 mmol.L-1buffer(Mg2+),80 ng模板DNA,0.08 mmol.L-1dNTP和0.16μmol.L-1引物。采用建立的多花黄精ISSR-PCR最佳反应体系进行退火温度梯度试验,确定了引物UBC841的最适退火温度为54.8℃,且最适退火温度因引物而异。这一优化的ISSR-PCR反应体系的建立为今后利用ISSR技术进行黄精种质资源分类、遗传图谱构建和基因定位奠定了良好的技术基础。
Through 5 factors and 4 orthogonal design tests of Taq enzyme, buffer (Mg2 +), template DNA, dNTP and primer in the ISSR-PCR reaction, The optimal reaction system for ISSR-PCR was to amplify the most abundant bands, that is, 25 μL of reaction system contained 1.0 U Taq DNA polymerase, 3.5 mmol.L-1buffer (Mg2 +), 80 ng template DNA, 0.08 mmol L-1dNTP and 0.16 μmol.L-1 primers. The optimized annealing temperature gradient test was used to establish the optimal reaction system of the polygonatum ISSR-PCR, and the optimal annealing temperature of the primer UBC841 was 54.8 ℃, and the optimum annealing temperature was different due to the primer. The establishment of this optimized ISSR-PCR reaction system laid a good technical foundation for the future use of ISSR technology for the classification of yellow germplasm resources, genetic map construction and gene mapping.