Li et al. Virology Journal (2017) 14:165 DOI 10.1186/s12985-017-0832-3 RESEARCH Open Access The genetic structure of Turnip mosaic virus population reveals the rapid expansion of a new emergent lineage in China Xiangdong Li1†, Tiansheng Zhu2†, Xiao Yin1†, Chengling Zhang4, Jia Chen1, Yanping Tian1* and Jinliang Liu3* Abstract Background: Turnip mosaic virus (TuMV) is one of the most widespread and economically important virus infecting both crop and ornamental species of the family Brassicaceae. TuMV isolates can be classified to five phylogenetic lineages, basal-B, basal-BR, Asian-BR, world-B and Orchis. Results: To understand the genetic structure of TuMV from radish in China, the 3′-terminal genome of 90 TuMV isolates were determined and analyzed with other available Chinese isolates. The results showed that the Chinese TuMV isolates from radish formed three groups: Asian-BR, basal-BR and world-B. More than half of these isolates (52.54%) were clustered to basal-BR group, and could be further divided into three sub-groups. The TuMV basal-BR isolates in the sub-groups I and II were genetically homologous with Japanese ones, while those in sub-group III formed a distinct lineage. Sub-populations of TuMV basal-BR II and III were new emergent and in a state of expansion. The Chinese TuMV radish populations were under negative selection. Gene flow between TuMV populations from Tai’an, Weifang and Changchun was frequent. Conclusions: The genetic structure of Turnip mosaic virus population reveals the rapid expansion of a new emergent lineage in China. Keywords: Turnip mosaic virus, Potyvirus, Genetic structure, Population, China Background has flexuous filamental particles of 700–750 nm long Due to the error-prone nature of their RNA-dependent and can be transmitted by 40–50 species of aphids in a RNA polymerases, populations of plant RNA viruses are non-persistent manner [4, 5]. The TuMV genome con- genetically heterogeneous and the genetic structure may sists of one single-stranded positive sense RNA molecule change with time and environment [1, 2]. Studies of the of approximately 9830 nucleotides (nt) and contains a genetic structure of viruses will provide information large open reading frame (ORF) [6]. The genomic RNA about the mechanisms and factors driving their evolu- is translated into a large polyprotein and a frame-shift tion and help us to understand the molecular evolution- protein. The large polyprotein are subsequently proc- ary history of viruses in relation to their dispersion and essed by the action of three viral-encoded proteinases emergence of new epidemics [3]. (Pl, HC-Pro and NIa-Pro) into ten mature functional Turnip mosaic virus (TuMV) is a species of the largest products [7, 8]. A frame-shift protein, P3N-PIPO, was plant virus genus Potyvirus (family Potyviridae). TuMV reported to be involved in the pathogenesis and move- ment of TuMV [9, 10]. TuMV can infect plants of 300 species in 43 families, * Correspondence: [email protected]; [email protected] †Equal contributors and is probably the most widespread and economically 1Laboratory of Plant Virology, Department of Plant Pathology, College of important virus infecting both crop and ornamental spe- ’ Plant Protection, Shandong Agricultural University, Tai an, Shandong 271018, cies of family Brassicaceae [11, 12]. In an extensive survey China 3College of Plant Sciences, Jilin University, Changchun 130062, China conducted in 28 countries, TuMV ranked second for crop Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. Virology Journal (2017) 14:165 Page 2 of 10 yield losses [4]. TuMV is a highly variable and has many Recombination analysis biological and serological strains [13–16]. According to its The sequences of 101 TuMV isolates and other 28 ob- host range, TuMV isolates can be classified to two patho- tained from the GenBank database were subjected to re- types, B (mainly infects plants of the genus Brassica)and combination analyses using the software package RDP3, BR (infects plants of both Brassica and Raphanus). The which assembled programs RDP [30], GENECONV [31], brassica-infecting TuMV isolates were categorized into BOOTSCAN [32], MAXCHI [33], CHIMEARA [34] and four phylogenetic lineages, basal-B, basal-BR, Asian-BR SISCAN. The sequences were analyzed using the default and world-B, which correlated well with their differences settings for different detection programs and a Bonferroni- in pathogenicity and geographical origin [17]. Most corrected P-value cut off of 0.05. The potential recombi- recently, a monophyletic sister lineage called ‘Orchis nants identified by the programs in RDP3 were re-checked group’ was detected from wild orchids-infecting TuMV using PHYLPRO [35]. The RDP, BOOTSCAN and SIS- isolates, which are more likely the ancestor of TuMV [18]. CAN programs were based on phylogenetic methods, As in other potyviruses [19], recombination is a frequent whereas GENECONV, MAXCHI and CHIMAERA pro- event in the evolution of TuMV. Intra- and inter-lineage grams were substitution methods, and the PHYLPRO pro- recombinants are common in natural populations of gram was a distance comparison method. Only those TuMV and can be detected throughout the genome sequences with recombination supported by at least three [6, 20–22]. The Chinese and Japanese TuMV isolates programs or two kinds of methods and with P-value − are part of the same population but are a discrete <1.0 × 10 6 were regarded as ‘clear’ recombinants; other- lineage [22, 23]. The gene flow between sub- wise, they were called as ‘tentative’ recombinants [23, 25]. populations of TuMV from Vietnam, Japan and China are frequent [20]. The basal-BR isolates have occurred Phylogenetic analysis of the TuMV population over the whole Japanese islands and have evolved into Sequence alignments were performed using the CLUS- four sub-lineages [23–25]. TAL W program (Thompson et al., 1994). Phylogenetic Previous studies showed that the TuMV isolates of tree of TuMV isolates excluding the recombinant ones China can be clustered to world-B and Asian-BR groups was constructed using methods including Maximum [10, 17, 24, 26, 27]. However, we have detected the exist- Likelihood (ML) method that are packaged in the ence of basal-BR isolates in China and reported the MEGA6.0 [36]. The CP gene of one Narcissus yellow complete genomic sequences of two basal-BR isolates that stripe virus (NYSV) isolate was used as outgroup [37]. represented two novel recombination patterns [6, 28]. Bootstrap analysis was repeated 1000 times to evaluate Here, we studied the genetic structure of TuMV popula- the significance of the internal branches. tion in China and found that the basal-BR group of TuMV was expanding in China. Sequences diversity and population demography analysis DnaSP version 5.10 was used to calculate the values of nu- cleotide diversity, Tajima’sD,FuandLi’s D and F tests, Methods haplotype diversity and nucleotide diversity [38–40]. Taji- Virus samples, RNA extraction and sequencing ma’sD,FuandLi’s D and F tests hypothesize that all mu- Leaf samples of radish from Heilongjiang, Jilin and tations are selectively neutral. Tajima’s D test depends on Shandong provinces from 2005 to 2010 were collected. the differences between the numbers of segregating sites All the samples were biologically purified by three cycles and the average number of nucleotide differences. Fu and of single lesion isolation in Chenopodium amaranticolor Li’s D test is related the differences between the number and propagated in B. rapa. Inoculated plants were main- of singletons (mutations appearing only once among the tained in a glasshouse at 25 °C. sequences) and the total numbers of mutations. Fu and Total RNAs were extracted from 100 mg TuMV-infected Li’s F test is based on the differences between the numbers B. rapa leaves with the Invitrogen Trizol Kit following in- of singletons and the average number of nucleotide differ- structions of the manufacturer. The 3-terminus of TuMV ences among all pairs of sequences. Haplotype diversity (~1.1 kb) were amplified with RT-PCR using primers CP-F refers to the frequency and number of haplotypes in the (5′-ATC TTC GAA GAT TAC GAA GA-3′)andCP-R population. Nucleotide diversity estimates the average (5′-CCT TGC TTC CTA TCA AAT G-3′)[29].Thefrag- pairwise differences among sequences. The nucleotide ments were cloned into pMD18-T vector (TaKaRa Biotech- diversities were calculated within and between groups. nology Dalian Co, Ltd) and sequenced by a ABI PRISM™ DnaSP version 5.10 [40] was also used to estimate the fre- 377 DNA Sequencer. For each isolate, at least four clones quency distribution of the number of pairwise differences from two separate PCR were sequenced. In case of any in- among all sequences. Mismatch distribution of all popula- consistence, at least two more clones will be sequenced to tions were estimated on all pairs of haplotypes present in obtain the consensus sequence. a population [40]. Mismatch distribution analysis was Li et al. Virology Journal (2017) 14:165 Page 3 of 10 based on 1000 simulated samples and used to evaluate while absolute value of Fst <0.33orNm > 1 suggests fre- whether a population had undergone sudden expansion or quent gene flow.