摘要： 为建立菜用大黄最佳的SRAP反应体系，进一步对菜用大黄品种进行遗传多样性分析，以菜用大黄基因组DNA为模板，采用单因素与正交设计相结合的方法，对SRAP-PCR扩增体系中的模板DNA、Mg²⁺、dNTPs、引物和 Taq DNA聚合酶浓度5个因素进行优化，并确立菜用大黄SRAP-PCR的最佳体系。结果表明：菜用大黄各因素对反应体系影响大小依次为：引物浓度>Mg²⁺浓度>dNTPs浓度> Taq DNA聚合酶浓度>DNA用量；最佳反应体系为：25 μL反应体系中含10×PCR Buffer 2.50 μL、模板DNA 30 ng、Mg²⁺ 2.50 mmol/L、dNTPs 0.25 mmol/L、Taq DNA聚合酶0.04 U/μL、上下游引物各0.5 μmol/L。用8个菜用大黄品种DNA样品对优化的反应体系进行验证，均获得了条带清晰且多态性较好的扩增图谱，证实了该体系的稳定性和可靠性，可用于菜用大黄的遗传多样性分析。

关键词: 菜用大黄, SRAP-PCR, 体系优化

Abstract: The purpose of this study was to determine the best system of SRAP reaction and provide a foundation for further studying the genetic diversity of culinary rhubarb.Using the genome DNA as template,and combining the single-factor experiment with the orthogonal design,five factors including the concentrations of template DNA,Mg²⁺,dNTPs, Taq DNA polymerase and primers in SRAP-PCR reaction were optimized,and then the optimal SRAP-PCR reaction system for culinary rhubarb was established.The results indicated that the order of each factor affecting the result of PCR was:primer>Mg²⁺>dNTPs> Taq DNA polymerase dosage>DNA.The optimal SRAP-PCR reaction for culinary rhubarb in a 25 μL reaction system was 10×PCR Buffer 2.50 μL,template DNA 30 ng,Mg²⁺ 2.50 mmol/L,dNTPs 0.25 mmol/L, Taq DNA polymerase dosage 0.04 U/μL and each primer 0.5 μmol/L.The optimized system was verified by the genomic DNA samples from eight cultivars of culinary rhubarb,and the clear bands and abundant polymorphism were obtained.It was concluded that the SRAP-PCR reaction system was stable and reliable,and was suitable to analyze the genetic diversity of culinary rhubarb.

Key words: Culinary rhubarb, SRAP-PCR, Optimization

中图分类号: 

• S644.9