Laboratory of Plant Virology, Faculty of Agriculture, Saga University, Saga 840-8502, Japan1
Laboratory of Plant Pathology, Faculty of Agriculture & Life Sciences, Hirosaki University, Hirosaki 036-8561, Japan2
Tohoku Seed Co. Ltd, Utsunomiya 321-3232, Japan3
Plant Pathology & Microbiology Department, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK4
Crop & Food Research, Private Bag 4704, Christchurch, New Zealand5
Faculty of Life Science, Zhenjiang University, Hangzhou 310029, PR China6
Department of Virology, Agriculture Research Organization, The Volcani Centre, Bet Dagan 50250, Israel7
Faculty of Science, Australian National University, Canberra, ACT 2601, Australia8
Author for correspondence: Kazusato Ohshima. Fax +81 952 28 8709. e-mail ohshimak{at}cc.saga-u.ac.jp
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Abstract |
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Introduction |
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The nucleotide (nt) sequences of the genes of many different species of plant viruses have been determined, and their phylogenetic relationships have been inferred (Van der Vlugt et al., 1993 ; Bateson et al., 1994
; Tordo et al., 1995
; Aleman-Verdaguer et al., 1997
; Revers et al., 1997
; Roossinck et al., 1999
; Bousalem et al., 2000
). In some of the studies several isolates of a single species have been studied, and these have shown strain differences that correlate with geographical origins or with strain specialization. We have used this approach in order to understand whether gene sequence analysis can reveal more of the factors that influence worldwide variation within a single virus species, Turnip mosaic virus (TuMV).
TuMV has an RNA genome and infects a wide range of plant species, mostly, but not exclusively, from the family Brassicaceae. It is probably the most widespread and important virus infecting both crop and ornamental species of this family, and occurs in many parts of the world including the temperate and tropical regions of Africa, Asia, Europe, Oceania and North/South America (Provvidenti, 1996 ). TuMV belongs to the genus Potyvirus. This is the largest genus of the largest family of plant viruses, the Potyviridae (Ward et al., 1995
), which itself belongs to the picorna-like supergroup of viruses of animals and plants. TuMV, like other potyviruses, is transmitted by aphids in the non-persistent manner (Shukla et al., 1994
). All potyviruses have flexuous filamentous particles 700750 nm long, each of which contains a single copy of the genome, which is a single-stranded positive sense RNA molecule about 10000 nt long. The genomes of potyviruses have a single open reading frame that is translated into a single large polyprotein, which is hydrolysed, after translation, into several proteins by virus-encoded proteinases (Riechmann et al., 1992
). The genomes of the Canadian (Ca) (Nicolas & Laliberté, 1992
) and Japanese (1J) (Ohshima et al., 1996
) isolates of TuMV are 9830 and 9833 nt in length and have single open reading frames which encode polyproteins of 3163 and 3164 amino acids, respectively. Of all potyviral genes, that encoding the coat protein (CP) and situated at the 3'-end of the genome has been most frequently studied for its genetic diversity, whereas there have been fewer studies on the diversity of the first protein (P1) gene, which is the most variable potyviral gene and situated at the 5'-end of the genome (Shukla et al., 1994
).
In the study reported here, 76 isolates of TuMV were collected from naturally infected plants throughout the world, and their P1 and CP genes sequenced. Comparisons of these sequences show correlations between their genetic variation and geographical origins, show that some lineages are adapted to particular crop species, and that recombination is a significant generator of the genetic diversity in populations of this virus.
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Methods |
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Cloning of P1 and CP genes.
Viral RNAs were extracted directly from TuMV-infected B. rapa or N. benthamiana leaves. Complementary DNAs (cDNAs) of the P1 and CP genes of several isolates could not be amplified by RTPCR, and others did not attain sufficient concentration in RTPCR for direct sequencing, so their P1 and CP genes were cloned by two methods. In the first, the viral RNA was reverse transcribed and amplified using the Titan One Tube RTPCR Kit (Roche Diagnostics). The amplified cDNAs were hydrolysed with restriction enzymes using the sites incorporated by the PCR primers, and then separated by electrophoresis in agarose gels. The bands of interest were excised with a razor blade and purified by using the QIAquick Gel Extraction Kit (Qiagen). The eluted DNAs were ligated into alkaline phosphatase-treated pBluescript II SK(+) plasmid vectors (Stratagene), hydrolysed with the appropriate restriction enzymes and used to transform Escherichia coli XL-1 Blue (Stratagene). Alternatively, the viral RNAs which could not be amplified by RTPCR were cloned using random hexamer primers and the TimeSaver cDNA Synthesis Kit (Amersham Pharmacia Biotech), based on the Gubler & Hoffman (1983 ) method. Synthesized cDNAs were also cloned into pBluescript II SK(+) plasmid vectors that had been hydrolysed by appropriate restriction enzymes.
DNA sequencing.
Nucleotide sequences of the P1 and CP genes of each isolate were determined using three to six cDNA clones. Each cDNA clone was sequenced by primer walking in both directions using the Dye Terminator Cycle Sequencing FS Ready Kit (Applied Biosystems) and an Applied Biosystems DNA Sequencer model 373A. At most we found 2 nt differing between the clones of any one isolate and, when any difference was found, we then sequenced its RTPCR products directly to determine which was most common. Sequence data were assembled using DNASIS version 3.5 computer program (Hitachi).
Phylogenetic analyses.
The nucleotide sequences were aligned using CLUSTAL W or X (Thompson et al., 1994 ; Jeanmougin et al., 1998
) with default parameters. Their phylogenetic relationships were determined by several methods using the neighbour-joining (NJ) and maximum-likelihood (ML) algorithms of PHYLIP (Version 3.5; Felsenstein, 1993
), the maximum parsimony (P) algorithm of PAUP 4.0 beta Version 8 (Swofford, 1998
) and the TREE-PUZZLE (Strimmer & von Haeseler, 1996
; Strimmer et al., 1997
) packages. For NJ analyses, distance matrices were calculated by DNADIST with the Kimura two-parameter option (Kimura, 1980
), and trees constructed from these matrices by the NJ method (Saito & Nei, 1987
). The homologous regions of the genome of two isolates (mild and j1) of Japanese yam mosaic virus (JYMV) (Fuji & Nakamae, 1999
) were used as the outgroup for these analyses, as BLAST searches had shown them to be the sequences in the international sequence databases most closely related to those of TuMV. The calculated trees were displayed by TREEVIEW (Page, 1996
). The sequences and sub-sequences of them were checked for incongruent relationships that might have resulted from recombination using SISCAN Version 2 (Gibbs et al., 2000
; http://www.anu.edu.au/BoZo/software/), PHYLPRO (Weiller, 1998
) and DIPLOMO (Weiller & Gibbs, 1995
). The distance relationships of various sets of sequences were also compared by the DIPLOMO method for evidence of time-dependent and lineage-dependent sequence differences.
For some analyses the aligned P1 and CP genes of each isolate were joined to form a P1/CP sequence 1940 nt long. Then the corresponding parts of two JYMV genomes were aligned with the TuMV sequences by inserting appropriate gaps, and, finally, every position in the aligned sequences with a gap in any sequence was removed. This produced aligned sequences that were 1830 nt long with the P1 sequence represented by the 5'-terminal 969 nt (originally 1086 nt) and the CP sequence by the 3'-terminal 861 nt (originally 864 nt). Trees were calculated by NJ, ML and P methods from nt 3001450 of these sequences. A bootstrap value for each internal node of the NJ trees was calculated using 1000 random resamplings with SEQBOOT (Felsenstein, 1985 ) and these values are shown in Fig. 1
.
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Results |
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Gene sequences
In all the TuMV isolates sequenced in this study the P1 gene was 1086 nt in length and the CP gene 864 nt in length. Only a CP gene of an isolate (GK1) previously sequenced by Lehmann et al. (1997 ) was 861 nt. The nucleotide sequence identities for P1 and CP genes between all 78 isolates were 67100% and 84100%, respectively. The P1 and CP gene sequences of the 1J isolate (Ohshima et al., 1996
), together with their encoded amino acid sequences, were used to search the GenBank database using the BLAST program. The TuMV sequences were found to be consistently more similar to the homologous parts of JYMV than those of any other potyvirus, and when NJ trees were calculated from these sequences, the JYMV sequences formed a group that was itself a sister to all TuMV sequences.
Recombinants
The relationships of the aligned CP genes and of the P1 genes of the TuMV isolates were calculated separately using the NJ, ML and P methods. The resulting trees were similar but showed many inconsistencies in the relative positions of several isolates, and had poor bootstrap support for some lineages (data not shown). These inconsistencies were shown more clearly by plotting the patristic distances between the isolates in the P1 gene tree against their patristic distances in the CP gene tree (Fig. 2A); patristic distances are the distances between taxa within a tree, as distinct from the observed distances between taxa from which the tree was calculated. It can be seen that although the distances between most pairs of P1 genes are twice as much as between their CP genes, there are many pairs that deviate from the mode. For example, in Fig. 2(A)
the P1/CP ratio for the NDJ:GBR7 comparison is around 10 but for the NDJ:OD14J comparison less than 1.
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Many other sequences had phylogenetically inconsistent regions. Some, namely GBR7, HZ5, ITA7, PV0104, Rn98 and St48, had CRSs when examined by SiScan using synonymous differences, but it was not possible to determine which sequence was the recombinant, and which were the sequences closest to the parents, as they had CRSs involving all three pairs of sequences. Others, including GK1, ITA1, NPL4, NZ158, USA4 and UZB1, gave CRSs only when non-synonymous differences were compared, which may indicate that they have sequence regions with convergent similarities resulting from differential selection. When these sequences were omitted from the comparisons of P1 and CP patristic distances, the P1/CP correlation further increased to 0·889.
Comparisons were also made of trees calculated from successive slices of the P1/CP sequences taken along their length and showed that the region between nt 300 and 1450 had the greatest variation (Fig. 5), but was phylogenetically most consistent.
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Next largest was the least diverse group. All 22 isolates came from brassicas, mostly Raphanus, all from east Asia mostly Japan, and all are BR-pathotype, so we call this group the Asian-BR group. The other two groupings have the longest branches. One, of eight B-pathotype isolates from both brassicas and non-brassicas from Eurasia, was not monophyletic and, in most trees, was linked directly with the outgroup, so we call it basal-B cluster. Closest to it in most trees, and similarly variable but monophyletic, was the basal-BR group of seven BR pathotype Eurasian isolates. The affinities of isolate Rn98 are uncertain as, although a B-pathotype isolate, it was placed between the basal-B and basal-BR clusters in NJ, ML and P trees with no clear bootstrap support for inclusion in either of the clusters. It is possible that Rn98 may represent a lineage that included the B-pathotype progenitor of the basal-BR group.
The between-group relationships are more variable than the within-group relationships as shown by the cluster of points in Fig. 6 near the centre of the graph (i.e. comparisons between isolates in different groups) being broader than the cluster closer to the intersection of the axes (i.e. comparisons between isolates in the same group). Nonetheless the between-group cluster shows a clear correlation between the ML and NJ relationships, and the few outgroups mostly involve isolates with the longest basal branches, such as IS1 and UZB1.
There was a clear geographical pattern within most groups; Chinese, Japanese and New Zealand isolates formed separate groups and these groupings had high bootstrap support (Fig. 1). It was also clear that many of the isolates with deepest branches in the trees were from the Old World, whereas the Asian and Antipodean isolates were in a small number of phylogenetically distinct branches. Similarly, the B pathotype isolates from Brassica spp. were phylogenetically more diverse and geographically more dispersed than those from Raphanus. The simplest explanation of these patterns is that TuMV probably originated, like its brassica hosts, in Europe or the Mediterranean region whereas many of the Asian and especially the BR pathotype isolates were from geographically restricted and genetically distinct populations derived from small founding populations that had spread from the centre of origin. A DIPLOMO analysis failed to detect any correlation between the relationships between isolates and the year in which they were isolated (data not shown).
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Discussion |
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Specifically designed programs, such as SISCAN, are required to detect the sort of phylogenetic anomalies in sequences that can result from recombination or selection, as the tree-building methods, combined with bootstrapping, that are widely used for phylogenetic analysis may fail to detect recombination (Worobey, 2000 ). Using such programs we have shown that TuMV populations, like those of an increasing number of other viruses (Bousalem et al., 2000
, Padidam et al., 2000
, Sanz et al., 2000
, Smith et al., 2000
), contain recombinants. We found that five of the 78 P1/CP TuMV sequences were clear recombinants (Table 2
), and at least another ten may also have been recombinant. However, as the P1/CP sequence represents only about one-fifth of the TuMV genome, the true proportion of recombinants may be much greater, but the true extent of recombination in the world TuMV population will only be known when the full genomic sequences of a representative set of isolates are known.
Our phylogenetic analyses by the NJ, ML and P methods indicate that TuMV, like its principal host family, the Brassicaceae, probably originated and has its major centre of diversity in the EuropeMediterraneanAsia Minor region. The lineages with the greatest branch lengths (i.e. the basal-B and basal-BR) are probably the oldest, and include many isolates from that region. These groups also include isolates from non-brassica hosts. This may indicate that isolates, optimally adapted to crops of brassicas, spread worldwide in the footsteps of modern agriculture more readily than those adapted to other species, although it could also indicate that the older populations of TuMV are more variable, and hence contain more variants able to infect non-brassica species.
Our phylogenetic analyses also indicate that the original TuMV population was probably B pathotype, but that BR pathotype isolates have evolved from the B pathotype on several occasions; the basal-BR cluster and the Asian-BR group are only of BR pathotype isolates, but there are also BR isolates, all from Japan, in three separate parts of the world-B group. It would be interesting to know whether these solitary BR isolates differ from their nearest B-pathotype relatives in their recombinant status, as this could indicate which part of the genome determines the pathotype.
The fact that R. sativus is not susceptible to B pathotype isolates, whereas Brassica spp. and non-brassica species are susceptible to both pathotypes suggests that all BRxB recombinants found in R. sativus were initially generated in mixedly infected Brassica spp. but were then transmitted to R. sativus.
Our analyses of the genetic variation of two genes of the world population of TuMV have revealed its possible region of origin, and indicated the sort of evolutionary changes that may have occurred during its migratory spread, host adaptation and pathogenic segregation, but knowledge of the complete genomic sequences of a very large and representative collection of isolates will be required to confirm whether those conclusions are correct or result from special features of the sample of isolates, and genes, that we studied.
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Acknowledgments |
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Footnotes |
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References |
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Received 7 December 2001;
accepted 11 February 2002.