棉花黄萎病菌ISSR反应体系优化及其遗传多样性分析  

Optimization of ISSR reaction and analysis of genetic diversity of Verticillium dahliae in cotton

作　　者：刘燕霞[1] 侯丽娟[1] 李卫[1] 杨家荣[1] 肖蕊[1] 

机构地区：[1]西北农林科技大学植物保护学院,陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《植物保护学报》2010年第5期425-430,共6页Journal of Plant Protection

基　　金：国家公益性行业(农业)科研专项(3-19);高等学校学科创新引智计划项目(B07049)

摘　　要：为给棉花黄萎病菌分子变异及遗传多样性研究提供可靠的检测方法,以棉花黄萎病菌基因组DNA为模板,采用正交优化方法对PCR体系中DNA聚合酶、引物、dNTPs、DNA模板、Mg2＋及10×Buffer等重要参数进行6因素4水平优化,建立了棉花黄萎病菌ISSR-PCR优化反应体系,并从20条ISSR通用引物中筛选出多态性较好的10条引物。采用该优化反应体系和10条ISSR引物对采自陕西棉花主产区的21个棉花黄萎病菌菌株和3个参照菌株进行ISSR分析。结果显示,10条ISSR引物共扩增出87条谱带,条带分子量均在250~2 000 bp之间,平均每条引物扩增出8.7个条带,其中58条为多态性条带,占65.2%。聚类分析结果显示,在相似系数0.59处,供试菌株分为2个遗传类型。表明棉花黄萎病菌菌株间的亲缘关系与地理来源存在一定的相关性,而与其病害症状类型无相关性。In order to offer the reliable molecular analysis technique for studying the molecular variation and genetic diversity of Verticillium dahliae in cotton,the orthogonal design was adopted using genomic DNA of V.dahliae as the template to optimize ISSR amplification system for V.dahliae in accordance with six factors（Mg2＋,Taq polymerase,dNTP,primer,DNA template,10×Buffer） at four levels respectively.The optimized ISSR-PCR amplification system was established and the 10 polymorphic primers screened from 20 general ISSR primers were obtained.The optimized ISSR-PCR amplification system and 10 polymorphic primers were applied to study the genetic diversity of 21 strains of V.dahliae in cotton collected from Shaanxi main cotton growing regions and attachment of 3 strains of V.dahliae as the comparison.The results showed that there were 87 fragments obtained by amplification of ISSR with 10 polymorphic primers and each primer averagely generated 8.7 DNA fragments which molecular weight was between 250-2000bp.Among them,58 fragments were polymorphic and the polymorphic rate reached 65.2%.Based on the clustered analysis,21 strains of V.dahliae were clustered into 2 genetic lineages at 0.59 genetic similarity and it indicated that there were some relations of genetic lineages of V.dahliae to its geographical origin,but there was no relation to its phenotype of disease symptoms.

关 键 词：棉花黄萎病菌 ISSR 正交试验 遗传多样性 

分 类 号：S435.62[农业科学—农业昆虫与害虫防治]