两种高通量测序技术鉴定苹果病毒效果评价及两种新病毒的鉴定  

Evaluation of the Effectiveness of Two High-Throughput Sequencing Techniques in Identifying Apple Viruses and Identification of Two Novel Viruses

作　　者：潘媛 王德 刘楠 孟祥龙 戴蓬博 李波 胡同乐[1] 王树桐[1] 曹克强[1] 王亚南[1] PAN Yuan;WANG De;LIU Nan;MENG XiangLong;DAI PengBo;LI Bo;HU TongLe;WANG ShuTong;CAO KeQiang;WANG YaNan(College of Plant Protection,Hebei Agricultural University,Baoding 071001,Hebei)

机构地区：[1]河北农业大学植物保护学院,河北保定071001

出　　处：《中国农业科学》2025年第2期266-280,共15页Scientia Agricultura Sinica

基　　金：河北省重点研发计划(21326506D);河北省自然科学基金(C2022204196);国家现代农业(苹果)产业技术体系(CARS-27);河北省引进国外智力项目;河北省现代农业产业技术体系(HBCT2024150208)。

摘　　要：【目的】宏转录组测序(macro-transcriptome sequencing)与小RNA测序(small RNA sequencing)是病毒鉴定中常用的高通量测序技术(high-throughput sequencing technique)。本文旨在评价两种高通量测序方法检测不同类型的组织样本对苹果新发病毒鉴定的效率,为苹果病毒病的准确诊断提供依据。【方法】2022年8月自衡水深州市采集带有新病毒症状的‘鲁丽’苹果果皮和枝皮,提取总RNA后,分别构建宏转录组文库和小RNA文库进行高通量测序,利用生物信息学技术和软件分析数据。首先,比较高通量测序结果的各项指标,然后,采用层次分析法(analytic hierarchy process,AHP)和5级分级评分法综合比较各项指标的加权值,评价不同测序方法的效果。最后,利用RT-PCR方法验证高通量测序结果,对新发病毒进行基因组特性和系统进化关系分析。【结果】从拼接效果来讲,采用相同的组织材料,宏转录组优于小RNA测序技术,采用相同的技术,果皮的拼接效果优于枝皮。从检出的病毒种类数量来讲,宏转录组测序技术检测枝皮检出的病毒种类最多,包括苹果褪绿叶斑病毒(apple chlorotic leaf spot virus,ACLSV)、苹果茎痘病毒(apple stem pitting virus,ASPV)、苹果茎沟病毒(apple stem grooving virus,ASGV)、苹果坏死花叶病毒(apple necrotic mosaic virus,ApNMV)、苹果胶质木病毒-2(apple rubbery wood virus 2,ARWV-2)、苹果绿皱果相关病毒(apple green crinkle associated virus,AGCaV)、柑橘囊胶相关病毒(citrus concave gum-associated virus,CCGaV)、柑橘碎叶病毒(citrus tatter leaf virus,CTLV)8种病毒;小RNA测序技术检测枝皮,检出病毒种类最少,小RNA测序技术检测果皮与枝皮病毒种类存在差异,采用果皮作为检测对象,两种方法检测出病毒种类数量相同。比较各项指标的综合评分,宏转录组检测枝皮分数最高。高通量测序结果与RT-PCR检测结果一致。ARWV-2、CCGaV在河北省为首次发现,分别�【Objective】Macro-transcriptome sequencing and small RNA sequencing are commonly used high-throughput sequencing techniques in virus identification.The objective of this study is to explore the application efficiency of macro-transcriptome sequencing and small RNA sequencing in the identification of emerging viruses in apples,analyze the impact of different tissue types on the identification results,and to provide a basis for the accurate diagnosis of apple virus diseases.【Method】The samples of apple peel and branch bark were collected from‘Luli’apple trees exhibiting novel viral symptoms in Shenzhou County of Hengshui City in August 2022.Total RNA was extracted,and macro-transcriptome libraries and small RNA libraries were constructed for high-throughput sequencing.Bioinformatic techniques and software were utilized to analyze and evaluate the sequencing data.Initially,the indicators from the high-throughput sequencing technique results were compared.Subsequently,a comprehensive evaluation of the effectiveness of each sequencing method was conducted using the analytic hierarchy process(AHP)and a 5-level grading system to calculate the weighted values of these indicators.Finally,RT-PCR was employed to validate the high-throughput sequencing results,and the genomic characteristics and phylogenetic relationships of the emerging viruses were analyzed.【Result】In terms of splicing effect,using the same tissue material,the macro-transcriptome sequencing outperformed small RNA sequencing.When the same technique was applied,the splicing effect for fruit peel tissue was better than that of branch bark.In terms of the number of virus species detected,macro-transcriptome sequencing identified the highest number of virus species in branch bark,including eight viruses:apple chlorotic leaf spot virus(ACLSV),apple stem pitting virus(ASPV),apple stem grooving virus(ASGV),apple necrotic mosaic virus(ApNMV),apple rubbery wood virus 2(ARWV-2),apple green crinkle associated virus(AGCaV),citrus concave gum-associated v

关 键 词：宏转录组测序 小RNA测序 苹果病毒 鉴定 河北省 

分 类 号：S85[农业科学—兽医学]