烟草花叶病毒属条形码鉴定方法的建立

Development of Barcoding Technique for Identification of the Genus Tobamovirus

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。

Plant viruses of Tobamovirus can infect many plants and is one of the most devastating threats to agricultural pro- duction. 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 Tobamol-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.