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为建立成熟可靠的红毛丹SRAP-PCR扩增检测技术体系,本研究首先采用单因素实验设计,对反应体系中的DNA模板、Mg2+、d NTPs、Taq DNA聚合酶和引物浓度等5个主要影响因素,设置不同的水平梯度,筛选出适宜的因子范围;在此基础上,进一步采用L16(45)正交设计,建立了红毛丹SRAP-PCR最佳反应体系:20μL体系中包含DNA模板20 ng、d NTPs 0.25 mmol/L、引物0.6μmol/L、r Taq酶1.0 U、Mg2+2.5 mmol/L。并利用优化的反应体系,从116对SRAP引物组合中筛选出37对扩增条带清晰、产物多态性较好的引物。本研究建立的SRAP-PCR体系及筛选的引物,将为红毛丹从分子水平进行种质资源遗传多样性分析、品种指纹图谱构建等研究提供基础。
In order to establish a reliable and reliable rapamycin SRAP-PCR amplification detection system, we first used a single factor experimental design to determine the DNA template, Mg2 +, dNTPs, Taq DNA polymerase and primer concentration in the reaction system Based on this, the orthogonal design of L16 (45) was used to establish the optimal SRAP-PCR reaction system for rambutan. The 20μL system contained the DNA template 20 ng, d NTPs 0.25 mmol / L, primer 0.6 μmol / L, r Taq enzyme 1.0 U, Mg2 + 2.5 mmol / L. Using the optimized reaction system, 37 pairs of SRAP primer combinations were screened for a good primer pair with a clear amplification band and good product polymorphism. The SRAP-PCR system and primers screened in this study will provide the basis for rambutan molecular genetic diversity analysis and fingerprinting of cultivars from the molecular level.