福建省玉米大斑病菌ISSR-PCR反应体系的优化和引物的筛选

doi:10.19303/j.issn.1008-0384.2019.07.009

福建农业学报 ›› 2019, Vol. 34 ›› Issue (7): 810-817.doi: 10.19303/j.issn.1008-0384.2019.07.009

福建省玉米大斑病菌ISSR-PCR反应体系的优化和引物的筛选

代玉立¹, 甘林¹, 阮宏椿¹, 杨静民², 石妞妞¹, 杜宜新¹, 陈福如¹, 杨秀娟¹

1. 福建省农业科学院植物保护研究所/福建省作物有害生物监测与治理重点实验室, 福建福州 350013;
2. 建瓯市农业农村局, 福建建瓯 353100

收稿日期:2019-04-09 修回日期:2019-07-03 出版日期:2019-07-20 发布日期:2019-10-14
通讯作者: 杨秀娟(1972-),女,硕士,研究员,研究方向:植物病理学(E-mail:yxjzb@126.com) E-mail:yxjzb@126.com
作者简介:代玉立(1984-),男,博士,助理研究员,研究方向:真菌学及植物真菌病害(E-mail:dai841225@126.com)
基金资助:
福建省科技计划项目——省属公益类科研院所基本科研专项（2017R1025-3、2018R1025-1）；国家重点研发计划（2018YFD0200706）；福建省农业科学院青年科技英才百人计划项目（YC2016-4）；福建省农业科学院青年自由探索项目（AA2018-14）；福建省财政专项——福建省农业科学院植物保护创新团队建设项目（STIT2017-1-8）

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

DAI Yu-li¹, GAN Lin¹, RUAN Hong-chun¹, YANG Jing-min², SHI Niu-niu¹, DU Yi-xin¹, CHEN Fu-ru¹, YANG Xiu-juan¹

1. Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Institute of Plant Protection, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350013, China;
2. Jian'ou Municipal Bureau of Agriculture and Rural Affairs, Jian'ou, Fujian 353100, China

Received:2019-04-09 Revised:2019-07-03 Online:2019-07-20 Published:2019-10-14

摘要/Abstract

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。

关键词: 玉米大斑病菌, 反应体系, 简单序列重复区间扩增多态性, 玉米大斑病, 遗传多样性

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.

Key words: Exserohilum turcicum, reaction conditions, inter-simple sequence repeat, northern corn leaf blight, genetic diversity

中图分类号:

S432.4

代玉立, 甘林, 阮宏椿, 杨静民, 石妞妞, 杜宜新, 陈福如, 杨秀娟. 福建省玉米大斑病菌ISSR-PCR反应体系的优化和引物的筛选[J]. 福建农业学报, 2019, 34(7): 810-817.

DAI Yu-li, GAN Lin, RUAN Hong-chun, YANG Jing-min, SHI Niu-niu, DU Yi-xin, CHEN Fu-ru, YANG Xiu-juan. ISSR-PCR Optimization and Primer Selection for Analyzing Exserohilum turcicum Isolates Collected in Fujian[J]. Fujian Journal of Agricultural Sciences, 2019, 34(7): 810-817.

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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

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