基于高通量测序的技术检测梨树病毒

Detection of Pear Virus by High-Throughput Sequencing Technology

【目的】研究运用基于高通量测序的技术检测梨树病毒,为梨树病毒的检测提供新方法。【方法】于2014年、2017年、2018年4月中旬采集库尔勒香梨花朵若干,分别进行转录组测序,将得到的序列经过转录本拼接、层次聚类和基因功能注释,筛选出注释为植物病毒的序列作为候选病毒序列。利用RT-PCR方法检测随机采集的香梨枝条样品,验证高通量测序结果的可靠性。【结果】根据3组转录组测序数据的生物信息学分析结果,分别对注释为来源于植物病毒的66、202和921条基因序列进行分析。3种梨树中已报道的病毒,分别是苹果茎痘病毒、苹果茎沟病毒和苹果褪绿叶斑病毒,以及梨树中未报道的芸薹黄化病毒。采用设计的特异性引物,通过RT-PCR技术对筛选出的4种病毒进行扩增验证,结果扩增出苹果茎痘病毒和苹果茎沟病毒的目的片段。【结论】高通量测序技术可作为检测梨树病毒的一种快速、有效手段。

【Objective】To provide a new method for the detection of pear virus.【Methods】Several flowers of'Korla pear'were collected in mid-April 2014,2017 and 2018,and the transcriptome was sequenced.The obtained sequences were transcribed,hierarchically clustered and functionally annotated.The sequence annotated as a plant virus was used as a candidate viral sequence.The randomly collected samples of pear branches were detected by RT-PCR to verify the reliability of high-throughput sequencing results.【Results】According to the bioinformatics analysis of the three groups of transcriptome sequencing data,a total of 66 gene sequences,202 gene sequences and 921 gene sequences were selected from the gene sequences annotated as plant viruses.Three kinds of viruses were reported to infect pear trees,namely,apple stem pitting virus,apple stem groove virus and apple chlorotic leaf spot virus.According to the designed specific primers,the four kinds of viruses were screened by RT-PCR,and only apple stem pitting virus and apple stem groove virus amplified the target fragment.【Conclusion】High-throughput sequencing technology can be used as a rapid and effective means for detecting the pear virus.