菜用大黄SRAP-PCR反应体系的优化及验证  

Optimization and Verification of SRAP- PCR System for Culinary Rhubarb

作　　者：郭小菲[1] 姜立娜[1] 蔡祖国[1] 任文娟[1] 赵一鹏[1] 

机构地区：[1]河南科技学院园艺园林学院,河南新乡453003

出　　处：《华北农学报》2014年第4期105-110,共6页Acta Agriculturae Boreali-Sinica

基　　金：河南省科技厅基础前沿项目(122300410134)

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:25μL反应体系中含10×PCR Buffer 2.50μL、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0.5μmol/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。The purpose of this study was to determine the best system of SRAP reaction and provide a founda -tion for further studying the genetic diversity of culinary rhubarb .Using the genome DNA as template ,and combi-ning 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 25μL 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 0.5μmol/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 .

关 键 词：菜用大黄 SRAP-PCR 体系优化 

分 类 号：S644.9[农业科学—蔬菜学]