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对通用引物PCR(UP-PCR)扩增反应中的dNTP、引物、MgCl2、DNA聚合酶和模板浓度以及退火温度(Tm)、PCR反应循环数等重要参数进行摸索和优化,建立适合玉米大斑病菌(Exserohilum turcicum)的UP-PCR反应体系,并筛选出7条多态性较好且稳定的引物。对来自辽宁、吉林、黑龙江玉米主产区的24株病菌进行UP-PCR分析,共扩增出75条谱带,大小介于250～2 000 bp,多态性比率为72.0%,说明UP-PCR标记在玉米大斑病菌中存在较高的多态性。聚类分析显示,在阈值为0.796处菌株被分为8个类群。东北地区玉米大斑病菌群体存在明显的种内遗传分化现象,菌株的遗传多样性和地理来源无明显的相关性,和生理小种组成有一定的相关性,但并不是遗传谱系就等于生理小种的简单对应关系。UP-PCR技术可以充分展现玉米大斑病菌菌株间的亲缘关系及差异性,可为有效地开展玉米大斑病菌的遗传进化及探讨其致病性的生理分化提供一种新的方法。
The key parameters of dNTPs, primers, MgCl2, DNA polymerase and template concentration, as well as the annealing temperature (Tm) and the number of cycles of PCR reaction in universal primer PCR (UP-PCR) amplification reaction were explored and optimized. (Exserohilum turcicum) UP-PCR reaction system, and selected seven polymorphic better and stable primers. A total of 75 bands were amplified by UP-PCR from 24 strains of main maize producing areas in Liaoning, Jilin and Heilongjiang provinces. The bands ranged from 250 to 2 000 bp and the percentage of polymorphisms was 72.0% PCR markers showed high polymorphism in S. turcica. Cluster analysis showed that at a threshold of 0.796 strains were divided into 8 taxa. There was obvious intraspecific genetic differentiation in the populations of S. turcica in northeastern China. There was no significant correlation between the genetic diversity and geographical origin of the isolates, and there was some correlation between the genetic diversity and physiological races. However, Kinds of simple correspondence. UP-PCR technology can fully demonstrate the genetic relationship and differences between the isolates of S. turcica and provide a new method for effectively carrying out the genetic evolution of S. turcica and exploring its pathogenic physiological differentiation.