苹果炭疽病病原菌ISSR-PCR反应体系的优化及遗传多样性分析  

Optimization of ISSR-PCR reaction system and genetic diversity analysis of Colletotrichum species causing bitter rot of apple

作　　者：符丹丹[1,2] 庄杰丽[1] 张荣[1] 孙广宇[1] 

机构地区：[1]西北农林科技大学植物保护学院,旱区作物逆境生物学国家重点实验室,陕西杨凌712100 [2]河南科技大学食品与生物工程学院,洛阳471023

出　　处：《植物保护学报》2013年第3期231-236,共6页Journal of Plant Protection

基　　金：国家自然科学基金(31171797)

摘　　要：为了从分子水平探讨苹果炭疽病病原菌的群体遗传多样性,采用L16(45)正交设计对ISSR-PCR反应体系中的Mg2+浓度、dNTP浓度、Taq DNA聚合酶用量、引物浓度和DNA含量进行优化,并利用优化的体系进行引物筛选及苹果炭疽病菌遗传多样性分析。确立最优反应体系为2.0mmol/L Mg2+、0.2 mmol/L dNTP、2 U Taq DNA聚合酶、1μmol/L引物和DNA 100 ng。筛选获得的10条引物对供试菌株共扩增出42条谱带,均为多态性条带。供试菌株在相似系数0.50处分为2个类群,分别与根据形态学鉴定的胶孢刺盘孢Colletotrichum gloeosporioides和尖孢刺盘孢C.acuta-tum 2个类群相一致,在相似系数0.74处,分为4个亚群,表明苹果炭疽病菌存在明显的种间及种内遗传分化。To understand the population genetic diversity of pathogens associated with apple bitter rot at the molecular level, the orthogonal design of L16（45） was used to optimize ISSR-PCR reaction related to factors including Mg2＋, dNTP, Taq DNA polymerase, primer and DNA. ISSR primers were screened based on optimized ISSR-PCR system, and applied to analyze the genetic diversity of Colletotrichum spp. causing bitter rot of apple. The optimized ISSR-PCR system was as follows： 2.0 mmol/L Mg2＋, 0.2 mmol/L dNTP, 2 U Taq DNA polymerase, 1 μmol/L primer and 100 ng DNA. Forty-two polymorphic bands were amplified with 10 screened primers. Clustering analysis revealed that tested strains were clustered into 2 groups at 0.50 genetic similarity coefficient, which corresponded with morphological identification to C.gloeosporioides and C.acutatum, and 4 subgroups at 0.74 level. It indicated that there were obvious interspecies and intraspecies genetic variations among the pathogens causing apple bitter rot.

关 键 词：苹果炭疽病 胶孢刺盘孢 尖孢刺盘孢 种群 

分 类 号：S436.611[农业科学—农业昆虫与害虫防治]