南方农业学报2025，Vol.56Issue(4)：1158-1170,13.DOI:10.3969/j.issn.2095-1191.2025.04.014
基于小RNA深度测序的广西牛大力花叶病病原分子鉴定及其全基因组序列分析
Molecular identification of pathogens infecting Millettia speciosa Champ.mosaic disease in Guangxi and complete genome sequence analysis based on small RNA deep sequencing
摘要
Abstract
[Objective]To clarify the viral pathogens which caused Millettia speciosa Champ.exhibiting mottled mo-saic symptom in Nanning and Qinzhou of Guangxi,which could provide scientific basis for prevention and control of Mil-lettia speciosa Champ.mosaic disease.[Method]During 2022-2024,leaf samples of Millettia speciosa Champ.suspected infected by viruses were collected from Nanning and Qinzhou.The methods of small RNA deep sequencing(RNA-Seq),reverse transcription polymerase chain reaction(RT-PCR),segmental cloning,sequence analysis and alignment,phylo-genetic tree construction were applied for virus identification and phylogenetic analysis on suspected infectious samples.[Result]The results showed that 49 contigs obtained by RNA-seq were annotated as viral genome,of which 25 contigs were annotated as cowpea mild mottle virus(CpMMV),2 contigs were annotated as cucurbit chlorotic yellows virus(CCYV)and 22 contigs were annotated as lettuce chlorosis virus(LCV).These results above indicated that the Millettia speciosa Champ.samples might infected by the 3 viruses:CpMMV,CCYV and LCV.The RT-PCR test results showed that only the target band of CpMMV could be amplified in the samples,while LCV and CCYV failed to amplify the target band,confirming that the collected Millettia speciosa Champ.samples were infected by CpMMV.One sample of Millettia speciosa Champ.from Nanning with positive CpMMV detection was randomly selected.A CpMMV complete genome se-quence with a total length of 8194 bp was successfully obtained by the method of segmental cloning and named CpMMV_GX_NiuDaLi.BLASTn analysis of the sequence in GenBank showed that the sequence had a high nucleotide similarity with each isolate of CpMMV registered in GenBank,nucleotide similarity of CpMMV_NJ_JS(GenBank acces-sion number:OK625819.1)and CpMMV_JS_NJ(GenBank accession number:MW354944.1)was the highest(97.46%).Analysis of the phylogenetic evolutionary tree constructed based on the complete genome sequence of CpMMV and the sequence of the CP gene showed that the isolates used for the analysis were divided into 2 large branches,the isolates from China in a big branch and all foreign isolates,except for the New Delhi isolate from New Delhi,India(GenBank accession number:OQ791180.1),were in another major branch.It indicated that these isolates have regional distribution characteristics.CpMMV_GX_NiuDaLi was in a large branch with various isolates from China,and was also in a small branch with CpMMV_NJ_JS and CpMMV_JS_NJ,indicating that there was close genetic rela-tionship among the 3 isolates.[Conclusion]The samples of Millettia speciosa Champ.with mottled leaves in Nanning and Qinzhou of Guangxi are infected by CpMMV.关键词
牛大力/花叶病/小RNA深度测序/豇豆轻斑驳病毒/广西Key words
Millettia speciosa Champ./mosaic disease/small RNA deep sequencing/cowpea mild mottle virus/Guangxi分类
农业科技引用本文复制引用
李祐聪,莫翠萍,谢慧婷,陈锦清,秦碧霞,崔丽贤,蔡健和,李界秋,李战彪..基于小RNA深度测序的广西牛大力花叶病病原分子鉴定及其全基因组序列分析[J].南方农业学报,2025,56(4):1158-1170,13.基金项目
广西自然科学基金项目(2020GXNSFAA259003) （2020GXNSFAA259003）
广西重点研发计划项目(桂科AB23026068) （桂科AB23026068）
广西农业科学院基本科研业务专项(桂农科2021YT071,桂农科2024ZX08) Guangxi Natural Science Foundation(2020GXNSFAA259003) （桂农科2021YT071,桂农科2024ZX08）
Guangxi Key Research and De-velopment Plan Project(Guike AB23026068) （Guike AB23026068）
Basic Scientific Research Project of Guangxi Academy of Agricultural Sciences(Guinongke 2021YT071,Guinongke 2024ZX08) （Guinongke 2021YT071,Guinongke 2024ZX08）
