• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

福建省玉米大斑病菌ISSR-PCR反应体系的优化和引物的筛选

ISSR-PCR Optimization and Primer Selection for Analyzing Exserohilum turcicum Isolates Collected in Fujian

  • 摘要:
      目的  明确适用于福建省玉米大斑病菌ISSR分子标记的反应体系和引物。
      方法  采用单因素水平优化法对ISSR-PCR扩增反应中的Taq聚合酶用量、dNTPs浓度、Mg2+浓度、模板DNA浓度、PCR反应循环数以及引物的最佳退火温度等重要参数进行优化。
      结果  适合福建省玉米大斑病菌群体遗传多样性分析的ISSR-PCR反应体系(25 μL)为:Taq聚合酶0.55 U、dNTPs 0.30 mmol·L-1、Mg2+ 1.30 mmol·L-1、DNA模板100 ng、引物10 pmol。ISSR-PCR扩增程序为:94℃预变性4 min;94℃变性45 s,51.2~56.0℃退火45 s,72℃延伸1.5 min,35个循环;72℃延伸10 min。利用优化的反应体系从56条ISSR引物中筛选出稳定性好、多态性高的引物10条:UBC117、UBC118、UBC808、UBC835、UBC847、UBC855、UBC856、UBC857、UBC866和UBC887,其最佳退火温度分别为55.6、53.1、51.2、51.2、56.0、53.1、53.1、51.2、51.2和51.2℃。利用优化的ISSR-PCR反应体系对21株供试菌株进行PCR扩增,结果表明,相同地理来源以及不同地理来源菌株间的DNA多态性均不同,表明福建省玉米大斑病菌群体存在丰富的遗传多样性。
      结论  本研究优化的ISSR-PCR反应体系和筛选的引物可用于福建省玉米大斑病菌群体遗传多样性和遗传结构的研究。

     

    Abstract:
      Objective  To determine the optimal reaction conditions and primers for the inter-simple sequence repeat (ISSR) PCR in analyzing isolates of Exserohilum turcicum, a pathogen of the northern corn leaf blight in Fujian.
      Method  Conditions for the ISSR-PCR amplification including Taq polymerase dosage, concentrations of dNTPs, Mg2+ and template DNA and reaction cycle as well as annealing temperatures for selected primers were optimized using a single-factor method.
      Result  For a 25 μL ISSR-PCR genetic diversity analysis on E. turcicum, the following conditions were applied:0.55 U of Taq polymerase, 0.30 mmoL·L-1 of dNTPs, 1.30 mmoL·L-1 of Mg2+, 100 ng of template DNA, and 10 pmol of primer under 94℃ for 4 min followed by 35 cycles of 45 s at 94℃, 45 s at 51.2-56.0℃, and 1.5 min at 72℃, and finally, at 72℃ for 10 min. Ten stable, polymorphic primers, i.e., UBC117, UBC118, UBC808, UBC835, UBC847, UBC855, UBC856, UBC857, UBC866, and UBC887, were selected from 56 ISSR primers, with their optimized annealing temperatures determined to be 55.6, 53.1, 51.2, 51.2, 56.0, 53.1, 53.1, 51.2, 51.2, and 51.2℃, respectively. Regardless of their geographical origins, the 21 E. turcicum isolates differed on DNA polymorphism indicating an abundance on genetic diversity among them.
      Conclusion  The optimized ISSR-PCR reaction conditions and primers could be applied for genetic studies on E. turcicum in Fujian.

     

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