【摘要】【目的】明确大麦黄条点花叶病毒(Barley yellow striate mosaic virus,BYSMV)在中国北方小麦主产区的分布及其种群遗传多样性,为病害流行预警和防控提供理论依据。【方法】2008—2016年,在河北、山东、江苏、安徽、河南、陕西和山西等7个省66个县/市/区田间,采集了864份疑似病毒病症状的植物样品。提取样品总RNA,利用一步法三重RT-PCR技术检测样品中的BYSMV、水稻黑条矮缩病毒(Rice black-streaked dwarf virus,RBSDV)和北方禾谷花叶病毒(Northern cereal mosaic virus,NCMV)。利用RT-PCR扩增获得BYSMV的L和N基因片段,克隆并测定核苷酸序列,应用MEGA、Dna SP和PAML等软件分析BYSMV分离物的系统进化和遗传多样性特征。【结果】从48个县/市/区采集的336份样品中检测到BYSMV,检出率为38.89%,该病毒主要分布于陕西、河北、山西和山东,另外,河南及安徽北部亦有分布,江苏徐州和邳州仅局部发生。基于BYSMV的L、N基因序列构建的系统发育树均可将分离物划分为2个亚组,亚组I中的分离物其来源涉及全部7个省份,而亚组II中的分离物仅来自陕西和山西2个省,基于L基因序列系统发育分析表明亚组II分离物与伊朗的分离物亲缘关系较近,BYSMV的遗传分化与分离物的地理来源相关,而与寄主植物、发生时间无明显相关性。运用RDP程序包的7个软件进行基因重组分析显示没有支持重组的证据。选择压力分析显示,亚组内和亚组间的ω(d N/d S)值(0.02—0.19)远小于1,表明群体正承受净化选择。L和N基因的单倍型多样性(Hd)值(0.90909和0.99524)均大于0.5、核苷酸多样性(π)值(0.01324和0.01224)均高于0.005,表明中国BYSMV群体遗传多样性丰富。基于L和N基因片段的遗传分化研究显示,东部和西部群体的遗传分化系数(FST)值(0.32201和0.37326)均大于0.25,且统计检验差异显著,表明东部和西部的BYSMV群体严重分化;基因流(Nm)值(0.53和0.42)均小于1,说明有限的基因流是促使群体发生遗传分化的主要原因。【结论】BYSMV在中国北方小麦主产区分布广泛,河北、山东、江苏、安徽、河南、陕西和山西等地均有不同程度的发生。BYSMV群体具有丰富的遗传多样性,且东部和西部群体之间存在严重的遗传分化。
【基金资助】 国家“973”计划(2014CB138400); 植物病虫害生物学国家重点实验室开放基金(SKLOF201614);
Distribution and Genetic Diversity of Barley yellow striate mosaic virus in Northern China
【Abstract】[Objective] The objective of this study is to clarify the distribution and genetic diversity of Barley yellow striate mosaic virus (BYSMV) in major wheat production areas in northern China, and to provide a theoretical basis for the early warning, prevention and control of epidemic diseases. [Method] During 2008–2016, about 864 suspected virus-infected samples were collected from 66 districts in Hebei, Shandong, Jiangsu, Anhui, Henan, Shaanxi and Shanxi provinces and all the three viruses including BYSMV, Rice black-streaked dwarf virus (RBSDV) and Northern cereal mosaic virus (NCMV) were identified using one-step multiplex RT-PCR. L and N gene fragments of BYSMV were obtained by RT-PCR amplification and cloned, and then determined by nucleotide sequence analysis. The sequences were analyzed by softwares of MEGA, DnaSP, and PAML to elucidate the phylogenesis and genetic diversity of BYSMV isolates. [Result] A total of 336 samples collected from 48 districts in seven provinces were detected with BYSMV and the detection rate was 38.89%. The virus was mainly distributed in Shaanxi, Hebei, Shanxi and Shandong. In addition, it was also distributed in Henan and northern Anhui. Xuzhou and Pizhou in Jiangsu Province were only localized. The phylogenetic analysis showed that the population could be divided into two subgroups (I and II) based on fragments of L and N genes. The isolates in subgroup I were derived from all seven provinces, but the isolates in subgroup II were only from Shaanxi and Shanxi provinces. It was indicated that Iran isolate was related to the subgroup II isolates based on the phylogenesis of L gene sequence. The clustering of the isolates was related to their geographical origins, and not to the host plants or sampling dates. The genetic analysis by using 7 softwares of the RDP package showed that there was no evidence supporting the recombination. The selection pressure analysis showed that the ω (dN/dS) values varied from 0.02 to 0.19 which were far less than 1 within or between subgroups. It was indicated that the population was undergoing purifying selection. The haplotype diversity (Hd) values (0.909 09 and 0.995 24) of L and N gene fragments were greater than 0.5 and the nucleotide diversity (π) values (0.013 24 and 0.012 24) were higher than 0.005, indicating that there was a high level of genetic diversity in the population of BYSMV in China. The genetic differentiation based on L and N gene fragments showed that the fixation indices FST (0.322 01 and 0.373 26) of eastern and western subpopulations were greater than 0.25. The difference of statistical test was significant, which indicated that the BYSMV population in eastern and western regions was seriously differentiated. The Nm values (0.53 and 0.42) were less than 1, which indicated that the limited gene flow was the main reason of genetic differentiation. [Conclusion] BYSMV was widely distributed in the wheat production areas of northern China, and occurred in Hebei, Shandong, Jiangsu, Anhui, Henan, Shaanxi and Shanxi provinces on different levels. The population of BYSMV had a high level of genetic diversity in China, and there was a severe genetic differentiation between the eastern and western subpopulations.
【Keywords】 Barley yellow striate mosaic virus (BYSMV); phylogenesis; genetic diversity; selection pressure; genetic differentiation;
【Funds】 National Basic Research Program of China (973 Program) (2014CB138400); Open Fund of State Key Laboratory for Biology of Plant Diseases and Insect Pests (SKLOF201614);
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