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摘要：烟草花叶病毒属病毒可侵染多种植物，给农业生产带来严重危害。为建立该病毒属的条形码鉴定技术，以该属22个确定种的标准序列为凭证标本序列，分析其保守区域，设计6对候选条形码引物（Tobamo1-Tobamo6），其扩增片段长度为200～1 240 bp。以田间样本对引物进行筛选，通过比较各引物的扩增效率、测序成功率、获得序列的亲缘关系对引物的通用性和物种分辨率进行评价。结果表明，Tobamo6的通用性最高，扩增率和测序成功率均为100%；物种分辨率良好，获得序列的种内种间变异可有效鉴定实际样本中的辣椒轻斑驳病毒、烟草花叶病毒、番茄花叶病毒和黄瓜绿斑驳花叶病毒；可作为烟草花叶病毒属的首选条形码引物。

关键词：烟草花叶病毒属（Tobamovirus）；条形码技术；鉴定

中图分类号：S463.85 文献标识码：A 文章编号：0439-8114（2015）11-2624-04

DOI：10.14088/j.cnki.issn0439-8114.2015.11.016

Development of Barcoding Technique for Identification of the Genus Tobamovirus

ZHANG Yong-jiang1，MA Rong-qun2，XIN Yan-yan1，HUANG Yue2

（1.Chinese Academy of Inspection and Quarantine， Beijing 100029， China； 2.Qingdao Institute of Agricultural Science Research， Qingdao 266100， Shandong， China）

Abstract： Plant viruses of Tobamovirus can infect many plants and is one of the most devastating threats to agricultural production. To develop barcoding technique for identification of genus Tobamovirus， the standard nucleotide sequences of twenty-two type species used as voucher specimen were analyzed by using the software Omiga 2.0， and six pair candidate barcoding primers Tobamo1-Tobamo6 were designed， which amplified 200～1 240 bp bands. These primers were tested by using field samples. Their universality and species resolution were compared during RT-PCR amplification rate， sequencing success rate and sequence phylogenetic relationship. The results demonstrated that Tobamo6 had the highest universality with 100% PCR amplification rate and sequencing success rate； and better species resolution， which can successfully identify four Tobamovirus viruses including Cucumber green mottle mosaic virus，Pepper mild mottle virus，Tobacco mosaic virus and Tomato mosaic virus from field samples. All this suggested Tobamo6 as the primary barcode for the identification of Tobamovirus viruses.

Key words： Tobamovirus； barcoding technique； identification

烟草花叶病毒属（Tobamovirus）为芜菁黄花叶病毒目（Tymovirales）帚状病毒科（Virgaviridae）成员；根据国际病毒分类委员会（International Committeeon Taxonomy of Viruses， ICTV）于2012年发布的最新病毒分类第九次报告，该病毒属包含曼陀罗轻斑驳病毒（Brugmansia mild mottle virus， BrMMV）等25个确定种和苘麻黄花叶病毒（Abutilon yellow mosaic virus， AbYMV）、甜椒斑驳病毒（Bell pepper mottle virus， BPMoV）、仙人掌轻斑驳病毒（Cactus mild mottle virus， CMMoV）、黄瓜斑驳病毒（Cucumber mottle virus， CuMoV）、鸡蛋果花叶病毒（Maracuja mosaic virus， MarMV）及Tropical soda apple mosaic virus （TSAMV）等6个暂定种[1]。同时，该属内新的病毒种类还有增加的趋势；而且该属病毒的寄主范围相当广泛，给农业生产带来重大损失；因此，加强对该属病毒的检测鉴定具有重要意义[2]。国内外采用直接观测法、电镜法、生物学测定法、血清学检测及分子生物学检测等方法[3]对该属病毒进行鉴定研究。……