新德里番茄曲叶病毒西瓜分离物的全基因组序列测定及分析

Whole genome sequencing and analysis of isolates of tomato leaf curl New Delhi virus collected from watermelon

从上海采集到表现卷叶和黄化症状的西瓜病叶,为明确其病原类型,本研究提取病叶RNA并进行小RNA高通量测序,经序列拼接比对和斑点酶联免疫吸附试验(dot-enzyme linked immunosorbent assay,Dot-ELISA),证实病样感染新德里番茄曲叶病毒(tomato leaf curl New Delhi virus,ToLCNDV)。进一步通过滚环复制(rolling circle replication,RCR)富集DNA,利用背靠背引物进行聚合酶链反应(polymerase chain reaction,PCR)扩增,获得2个ToLCNDV西瓜分离物的DNA-A和DNA-B全长序列,暂命名为ToLCNDV SH-WM1和ToLCNDV SH-WM2。使用MEGA 11.0、SDT v1.2等软件进行系统进化分析,结果表明,ToLCNDV SH-WM1与ToLCNDV SH-WM2及已报道的ToLCNDV其他中国分离物的亲缘关系较近,其全核苷酸序列相似度为98.99%~99.70%。全基因组多态性分析及群体变异分析显示,ToLCNDV中国分离物的单核苷酸变异率小于3%,其中,同义突变位点46个,非同义突变位点29个,不存在插入或缺失突变,各个编码蛋白中AC4蛋白氨基酸序列最为保守。本研究揭示ToLCNDV中国分离物具有较低的种内遗传变异。

Watermelon diseased leaves exhibiting leaf curl and yellowing symptoms were collected from Shanghai.To clarify the pathogenic type,small RNA high-throughput sequencing of the diseased leaves was performed,and the diseased samples were confirmed to be infected by tomato leaf curl New Delhi virus(ToLCNDV)through sequence splicing and dot-enzyme linked immunosorbent assay(Dot-ELISA).The DNA was further enriched by rolling circle replication(RCR)and was amplified by polymerase chain reaction(PCR)using back-to-back primers to obtain the full-length DNA-A and DNA-B sequences of two ToLCNDV watermelon isolates,which were tentatively named as ToLCNDV SH-WM1 and ToLCNDV SH-WM2.Phylogenetic analysis using MEGA 11.0 and SDT v1.2 softwares showed that ToLCNDV SH-WM1 was closely related to ToLCNDV SH-WM2 and other reported ToLCNDV isolates from China,with a total nucleotide sequence similarity of 98.99%-99.70%.Whole genome polymorphism analysis and population variation analysis showed that the single nucleotide variation rate of ToLCNDV isolates from China was less than 3%,with a total of 46 synonymous mutation sites and 29 non-synonymous mutation sites.There were no insertion or deletion mutations,and the amino acid sequence of AC4 protein was the most conservative among all encoded proteins.This study reveals that the ToLCNDV isolates from China have low intraspecific genetic variation.