三种苹果重要葡萄座腔菌DNA条形码基因的筛选

doi:10.14088/j.cnki.issn0439-8114.2018.02.031

湖北农业科学 ›› 2018, Vol. 57 ›› Issue (2): 126-131.doi: 10.14088/j.cnki.issn0439-8114.2018.02.031

三种苹果重要葡萄座腔菌DNA条形码基因的筛选

陈战¹, 沈宏¹, 许萍萍², 王振华³, 吴翠萍⁴, 李彬⁴

1.南京农业大学生命科学学院,南京 210001;
2.南京出入境检验检疫局,南京 210011;
3.湖北出入境检验检疫局,武汉 430050;
4.江苏出入境检验检疫局,南京 210019

收稿日期:2017-08-15 出版日期:2018-01-25 发布日期:2018-01-10
通讯作者: 李彬,（电子信箱）libin@jsciq.gov.cn。
作者简介:陈战（1992-）,男,安徽亳州人,硕士,研究方向为生物信息学,（电话）15996270529（电子信箱）chenzhan0909@163.com
基金资助:
国家质量监督检验检疫总局科技计划项目（2015IK157）

Screening DNA Barcode of Three Pathogenic Fungi within Genus Botryosphaeria in Apple

CHEN Zhan¹, SHEN Hong¹, XU Ping-ping², WANG Zhen-hua³, WU Cui-ping⁴, LI Bin⁴

1.College of Life Sciences, Nanjing Agricultural University,Nanjing 210001,China;
2.Nanjing Entry-Exit Inspection and Quarantine Bureau, Nanjing 210011,China;
3.Hubei Entry-Exit Inspection and Quarantine Bureau,Wuhan 430050,China;
4.Jiangsu Entry-Exit Inspection and Quarantine Bureau,Nanjing 210019, China

Received:2017-08-15 Online:2018-01-25 Published:2018-01-10

摘要/Abstract

摘要： 葡萄座腔菌属（Botryosphaeria）真菌是导致多种水果溃烂的重要致病菌,从形态上很难区分。选取了4个DNA片段（ITS、LSU、SSU、EF-1α）作为3种重要苹果致病菌（Botryosphaeria stevensii、Botryosphaeria dothidea、Botryosphaeria obtusa）的候选条形码进行PCR扩增、Sanger测序和序列分析。结果表明,PCR扩成功率ITS>SSU>LSU=EF-1α,ITS的PCR扩增成功率达到100%;序列分析发现ITS和EF-1α基因种内序列保守,种间差异明显,而且B. dothidea菌种在EF-1α基因中存在一段100 bp左右的特异片段,可以特异区分该菌株,但核糖体基因LSU和SSU种间、种内没有明显差异;结合GenBank中的葡萄座腔属真菌ITS序列进行系统发育分析,显示3种葡萄座腔菌属菌株分别位于3个独立的分支,分析结果与形态鉴定结果一致。推荐使用ITS和EF-1α两个基因共同作为这3种葡萄座腔属真菌的条形码。

关键词: 葡萄座腔菌（Botryosphaeria）, 苹果病原菌, 条形码, EF-1α, ITS

Abstract: Species of Botryosphaeria are the important pathogenic fungi on many kinds of fruits. They are very always difficult to distinguish from each other based on morphological characters. In this study, to identificate of three important apple-pathogens Botryosphaeria stevensii, Botryosphaeria dothidea, Botryosphaeria obtuse. 4 gene fragments were selected, which encoded ITS、LSU、SSU、EF-1α,to be the candidate DNA barcodes. By using of PCR amplification, Sanger sequencing and data analysis,we aimed to find the molecular markers in the identification of these species. The results showed that, based on the PCR and sequencing success,it was ranked as follow：ITS>SSU>LSU=EF-1α,and ITS primer worked best（100%）. According to the acquired sequences,ITS and EF-1α were quite conserved among different strains of the same species,and have the significant differences between the species. Meantime,a region of EF-1α （approx. 100 bp） was exclusively existed in the investigated strains of B. dothidea,which therefore could be used for the distinction of this species. However,the sequences of LSU and SSU have the same variation within and between the species. Combining the available ITS sequences in GeneBank,we constructed maximum likelihood tree to identify the phylogenetic relationships of Botryosphaeria. The results showed that three investigated species belonged to three monophyletic clades. These results were congruent with the taxonomic units based on the morphological characters. Accordingly, we recommended the usage of ITS and EF-1α to be the efficient molecular makers in the identification of B. stevensii、B. dothidea、B. obtuse.

Key words: Botryosphaeria, apple pathogen, barcode, EF-1α;, ITS

中图分类号:

S432.4

陈战, 沈宏, 许萍萍, 王振华, 吴翠萍, 李彬. 三种苹果重要葡萄座腔菌DNA条形码基因的筛选[J]. 湖北农业科学, 2018, 57(2): 126-131.

CHEN Zhan, SHEN Hong, XU Ping-ping, WANG Zhen-hua, WU Cui-ping, LI Bin. Screening DNA Barcode of Three Pathogenic Fungi within Genus Botryosphaeria in Apple[J]. HUBEI AGRICULTURAL SCIENCES, 2018, 57(2): 126-131.

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三种苹果重要葡萄座腔菌DNA条形码基因的筛选

Screening DNA Barcode of Three Pathogenic Fungi within Genus Botryosphaeria in Apple

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