烟草靶斑病菌(Rhizoctonia solani)ISSR反应体系的优化及遗传多样性分析  

Optimization of ISSR Reaction System and Genetic Diversity Analysis of Rhizoctonia solani from Tobacco Target Spot

作　　者：赵秀香[1] 苏燕妮[1] 伏颖[2] 赵艳琴[3] 孙宏伟 孙剑萍 吴元华[1] 

机构地区：[1]沈阳农业大学植物保护学院,沈阳110161 [2]沈阳市疾病预防控制中心,沈阳110031 [3]内蒙古民族大学农学院,内蒙古通辽028000 [4]黑龙江省烟草公司牡丹江烟草科学研究所,黑龙江牡丹江157011

出　　处：《沈阳农业大学学报》2016年第1期97-102,共6页Journal of Shenyang Agricultural University

基　　金：国家烟草专卖局科技项目[国烟办综(2010)182号];辽宁省烟草专卖局科技攻关项目[辽烟计(2010)86号];黑龙江省烟草专卖局科技攻关项目(HN201301号)

摘　　要：以烟草靶斑病菌(Rhizoctonia solani)基因组DNA为模板,采用单因素和正交设计L_(16)(4~5)相结合的方法对影响ISSR-PCR反应的主要因子进行优化,并对采自东北三省烟区的20个烟草靶斑病菌菌株进行ISSR分析,建立了适宜于病菌的ISSR分子标记的最佳反应体系,即20μL的反应体系中含有模板DNA 30ng,Mg^(2+)浓度2.0mmol·L^(-1),d NTP浓度0.20 mmol·L^(-1),Taq酶1.0U,引物浓度0.4μmol·L^(-1)。利用优化的反应体系从20个ISSR引物中筛选出13个多态性和重复性好的引物,对20个烟草靶斑病菌菌株进行ISSR分析,共扩增出132条带,多态条带比率为73.48%,在相似系数0.74处将其划分为3个类群,其中LTL-7、HLK-1、HBX-2、HBX-4、LFC-9、LTL-5、LTL-9和JLH-1共8个菌株被划分在IGⅠ中;IGⅡ中包括LTL-6、LKD-8、HLK-3、LTL-115、LTL-2、LFC-4和LTL-8共7个菌株;其余5个菌株分别为LTL-66、LTL-111、HLK-4、JLH-3和LTL-22被划分到IGⅢ中。分析结果表明ISSR遗传聚类组群与菌株的地理来源具有一定的相关性,而与菌株的致病性无明显的相关性。The main factors affecting ISSR-PCR system were optimized by the methods of single factor and orthogonal experiments using DNA of Rhizoctonia solani as template. Inter-simple sequence repeat(ISSR) analysis was used to investigate the genetic diversity of 20 isolates which were isolated from three Northeast provinces of China. The total volume of the optimal reaction system was 20μL, including 30 ng template DNA, 2.0 mmol·L^(-1) Mg^(2+), 0.20mmol·L^(-1)d NTP, 1.0U Taq DNA polymerase and 0.4μmol·L^(-1) primer. 13 of 20 ISSR primers with good polymorphism and repeatability were screened for the study of the genetic diversity of 20 isolates of R. solani.132 scored bands were obtained using the optimal reaction system, the ratio of polymorphic bands was 73.48% and these isolates were classified into 3 groups at similarity coefficient 0.74, IG Ⅰ included LJT-7 and other 7 isolates; IG Ⅱincluded 7 isolates; and the rest 5 isolates were divided into IG Ⅲ. The result showed that the ISSR genetic cluster groups had certain correlation with geographic origin of the isolates, but no significant correlation with pathogenicity.

关 键 词：烟草靶斑病 立枯丝核菌 ISSR反应体系 遗传相似系数 聚类分析 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]