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Genetic Evolution Analysis on Wild Isolates of Citrus Tristeza Virus Originated in China Based on Coat Protein Genes Sequences
Agricultural Sciences in China
November 2010
2010, 9(11): 1623-1629
Genetic Evolution Analysis on Wild Isolates of Citrus Tristeza Virus Originated in China Based on Coat Protein Genes Sequences YI Long1, 2, ZHOU Chang-yong1, ZHOU Yan1 and LI Zhong-an1 Citrus Research Institute, Southwest University/Citrus Research Institute, Chinese Academy of Agricultural Sciences, Chongqing 400712, P.R.China 2 Jiangxi Navel Orange Engineering and Technology Research Center, Gannan Normal University, Ganzhou 341000, P.R.China 1
Abstract The coat protein (CP) genes were cloned and sequenced from viral particles of 11 isolates of citrus tristeza virus (CTV) collected from wild citrus plants in China and 4 Chinese isolates from cultivated sweet orange and pummelo varieties, respectively. By analyzing and comparing the nucleotide and amino acid sequences of CP genes, the 11 wild CTV isolates were found over 92% identical with 4 Chinese CTV isolates and 21 exotic CTV isolates from cultivated citrus. From 91 to 100% of the CTV CP gene sequences in wild type citrus plants were generally well conserved. Genetic evolution analysis indicated that the GC% of the CP gene was less than AT%, and more transition were found in the CP genes than transversion with the transition/transversion ratio ranging from 6.3 to 7.0 among species. The substitution frequency was the highest at the third codon, followed by the first and second codon. The ratio of non-synonymous mutations (dN) to synonymous mutations (dS) was far lower than 1, suggesting that the CP gene might have experienced purifying selection in the evolution. Phylogenetic analysis revealed that the 11 CTV isolates in Chinese wild type citrus belonged to different phylogenetic clusters, and shared higher homology and closer relationships with other cultivated citrus CTV isolates from different countries, which indicated complicated genetic relationships among the CTV isolates. In addition, CTV isolates with similar biological characteristics usually located into the same clusters. Therefore, the conclusion was drawn that pathogenicity was critical to evolution and origin of CTV. Key words: wild type citrus, citrus tristeza virus, sequence comparison, phylogenetic evolution
INTRODUCTION Citrus tristeza virus (CTV), the causal agent causing one of the most destructive diseases of citrus, is a member of genus Closterovirus, belonging to Closteroviridae family. CTV virions are flexuous filamentous of around 2 000 × 11 nm, with a genomic RNA (gRNA) molecule and two coat proteins (CP and CPm) covering 95 and 5% of the particle length respectively (Bar-Joseph et al. 1989; Pappu et al. 1994; Febres et al. 1996). Received 8 April, 2010
The CTV gRNA is a single-stranded, positive-sense RNA molecule of approximately 20 kb, organized in 12 open reading frames (ORFs) encoding at least 19 proteins (Karasev et al. 1995; Yang et al. 1999). CTV is readily propagated by infected buds of citrus and locally spread by aphids in a semi-persistent manner (Yokomi et al. 1994). CTV was initially identified by disease symptoms both in the field plants and in the indicator plants of graft-inoculated citrus. The isolates and strains were divided into two categories, mild and severe, using symptom variety. Mild isolates cause
Accepted 24 June, 2010
YI Long, Ph D, Tel: +86-797-8393068, E-mail: [email protected]; Correspondence ZHOU Chang-yong, Professor, Tel: +86-23-68349007, E-mail: [email protected] © 2010, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(09)60259-5
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only mild or no symptoms in sensitive citrus indicator hosts and usually result in no economic loss, and severe isolates cause decline, stem pitting, or both and may vary in intensity (Garnsey et al. 1987). Recently, these two types were found to be associated with serological or molecular genetic characters (Nikolaeva et al. 1998; Niblett et al. 2000; Ayllon et al. 2001). To date, the complete genome nucleotide sequences of the nine isolates of CTV have been published: T30, a mild isolate from Florida (Albiach-Martí et al. 2000); T36, a decline isolate from Florida (Karasev et al. 1995); VT, a stem-pitting isolate from Israel (Mawassi et al. 1996); SY568, a stem-pitting isolate from California (Yang et al. 1999); T385, a mild isolate from Spain (Vives et al. 1999); NuagA, a seeding yellow isolate from Japan (Suastika et al. 2001); T318A, a stem-pitting isolate from Spain (Ruiz-Ruiz et al. 2006); Qaha, a severe isolate from Egypt (GenBank accession no. AY340974); Mexico-ctv, a quick decline and stem-pitting isolate from Mexico (DQ272579). Nucleotide sequence analysis is the most accurate procedure for CTV differentiation and estimation of molecular and genetic variation. Results from recent study suggested that aphid transmission and host passage may significantly alter the composition of CTV populations, and may play an important role in their evolution (Albiach-Martí et al. 2000; Sentandreu et al. 2006). Recent studies on CTV isolates from wild type plants have been reported in China using molecular analysis (Yi et al. 2007). However, study on genetic diversity and difference between the CTV isolates from wild type and cultivated citrus has not been reported. In this study, the differences were estimated among the CTV isolates from both wild type citrus and cultivated citrus by sequence analysis, and theoretical data for analyzing relationship between genetic variation and evolution of CTV isolates is provided.
MATERIALS AND METHODS
YI Long et al.
C. medica in Mengla, Yunnan; CT-W6 and CT-W7 from wild C. ichangensis in Linshui, Sichuan; CT-W8, CTW9 from wild C. ichangensis and C. aurantium respectively in Yizhang, Hunan; CT-W10 from wild C. reticulata in Chongyi, Jiangxi; CT-W11 from wild C. reticulata in Hezhou, Guangxi (Yi et al. 2007). The severe isolate CT3 and the mild isolate CT9, from cultivated pummelo (Zhao et al. 2002), together with the severe isolate CT4 (Zhao et al. 2002) and the mild isolate CT21 (Zhou et al. 2007), from cultivated sweet orange, were kindly provided by Citrus Research Institute, Chinese Academy of Agricultural Sciences as representatives of CTV isolates found in China. CTV isolates were collected from field trees, and were maintained in sweet orange [Citrus sinensis (L.) Osb.] plants in insect-proof greenhouses.
Nucleic acid extraction and RT-PCR Total nucleic acids of the samples, 11 wild CTV isolates and 4 orchard CTV isolates from China, were extracted according to the method proposed by Zhou et al. (2001). The primer T36CP was used as an internal positive control for amplifying the CP gene (672 bp) (Gillings et al. 1993), the sense primer was CP1: 5´-ATGGACGACGA- AACAAAG-3´ , and the anti-sense primer was CP3: 5´ -TCAACGTGTGTTGAATTT-3´. 5 μL cDNA of CTV was amplified and performed in a 25-μL reaction volume containing a 1 × concentration reaction buffer (Promega, Madison, USA), 0.2 mmol L-1 dNTPs, 1.5 mmol L-1 MgCl2, 0.5 μmol L-1 of each primer and 0.625 U of Taq DNA polymerase (TaKaRa, Dalian, China). Amplification profiles were as follows: 94°C for 2 min; 30 cycles and each cycle was 94°C for 30 s, 52°C for 30 s and 72°C for 45 s; an extension step at 72°C for 7 min. PCR products were separated by electrophoresis in a 1.2% agarose gel and detected by ethidium bromide staining.
Sequence alignment and statistical analysis Virus isolates The wild CTV isolates CT-W1, CT-W2 and CT-W3 were collected from wild Poncirus polyandra in Fumin, Yunnan; CT-W4 from wild Citrus medica in Yangbi, Yunnan; CT-W5 from wild hybridism of C. grandis and
The PCR products of approximately 672 bp for the complete CP gene were gel purified and the nucleotide sequences were determined in both directions with an ABI Prism DNA sequencer 377 (Sangon Biological Engineering & Technology and Service, Shanghai, China). © 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Genetic Evolution Analysis on Wild Isolates of Citrus Tristeza Virus Originated in China Based on Coat Protein Genes Sequences
Other CTV nucleotide sequences used in our analysis were obtained from the GenBank entries: American isolates T36, T30, SY568, and T3 (DQ355053); Israel isolate VT; Spain isolates T385 and T318A; Japanese isolate NuagA; Egypt isolate Qaha; Mexico isolate Mexic-ctv, Portuguese isolates 13C (AF184113) and 28C(AF184118); Australia isolates PB61 (AJ297702); Korea isolates Cheju-1 (AF249279) and Cheju-2 (AF339088); New Zealand isolates NZRB-3 (AY896563), NZRB-5 (AY896561), NZRB-6 (AY896568), and NZRB3 (AJ297702); Iran isolates M2 (AY190048); and Indian isolates Bangalore (AF501867) and Pune (AF501869). The nucleotide sequences (636 bp), except the sequences of primers, were aligned with CLUSTAL W (ver. 1.4) (Thompson et al. 1994). The MEGA ver. 4.0 (Kumar et al. 2008) was used to estimate the base content, the transition and transversion, and the Ts/Tv ratio. The rates of non-synonymous (dN) and synonymous (dS) substitutions, and their ratios were determined by the Nei & Gojobori methods (Kumar et al. 2008) , and the differentiation statistic was done using Z-test (P < 0.05) (Kumar et al. 2008). Based on the maximum parsimony method (MP) and neighbor-joining method (NJ), phylogenetic relationships were inferred, taking the sequences of coat protein gene of beet yellow virus (BYV) (NC-001598) as the out-group, and reliability of the clusters was evaluated by bootstrap analysis based on 1000 repetitions, using MEGA 4.0.
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deposited in the GenBank database under accession numbers CT3 (FJ998187), CT4 (FJ998188), CT9 (FJ998189), CT21 (FJ998190), and CT-W1-11 (FJ998191-FJ998201).
RESULTS Sequence identity analysis The nucleotide sequences and amino acid sequences of the CP genes from the 11 wild CTV isolates were found similarity ranging from 92.1 to 99.5% and 94.8 to 100% respectively. The identity of nucleotide sequence ranged from 91.3 to 99.5% and 91.3 to 99.6% when compared with 4 Chinese CTV isolates of cultivated citrus and 21 exotic CTV isolates of cultivated citrus, whereas the identity of amino acid sequences ranged from 95.2 to 100% and 94.3 to 100%, respectively (Table 1). Based on the sequence alignments of all the analyzed CTV isolates it was found that the identity between nucleotide sequences and amino acid sequences of the CTV CP gene was over 90%. The CP gene sequences of wild type citrus plants were generally well conserved, and the CP gene was very important for preserving the characters of the CTV virus during the development. No significant difference was shown between the CTV CP gene sequences of wild type citrus plants and cultivated citrus, either in the nucleotide sequences or amino acid sequences.
Nucleotide sequence accession numbers
Base content analysis
The nucleotide sequence data reported in this paper were
In this investigation, GC% was less than AT%, and base
Table 1 Sequence identity of the CP genes of the wild CTV isolates from different geographical origins Wild CTV isolates CT-W1-11
Comparison CTV isolates (geographical origin) CT-W1-11 (China) CT3, T9, CT4, CT21 (China) T36, T30, SY568, T3 (USA) NuagA (Japan) T385, T318A (Spain) VT (Israel) Qaha (Egypt) Mexico-ctv (Mexico) PB61 (Australia) M2 (Iran) Bangalore, Pune (India) 13C, 28C (Portugal) Cheju-1, Cheju-2 (Korea) NZRB-3, NZRB-5, NZRB-6 (New Zealand)
Identity of nucleotide sequences (%) 92.1-99.5 91.3-99.5 91.6-99.0 92.9-98.8 92.1-98.5 92.4-97.3 91.6-95.2 91.6-95.2 92.2-98.4 92.9-98.7 91.6-99.5 92.6-99.3 91.3-98.8 92.4-99.6
Identity of amino acid sequences (%) 94.8-100 95.2-100 94.3-100 96.2-100 94.8-99.0 95.2-97.1 94.3-96.6 94.3-96.6 95.7-99.0 96.2-99.5 96.2-99.5 95.2-99.0 94.3-98.1 96.2-99.5
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
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YI Long et al.
A content was the highest among the four bases in the CP genes of the 11 CTV isolates from wild type citrus, and also in CTV isolates from cultivated citrus (Table 2).
Transition and transversion, non-synonymous and synonymous substitutions The percentage of variable sites of nucleotide sequences and amino acid sequences in the CP genes of the 36 analyzed CTV isolates were 25.2 and 17.5%, respectively. More transitions than transversions were found in the CP genes, with the transition/transversion ratio ranging from 6.3 to 7.0 among species. The substitution frequency was the highest at the third codon, followed by the first and second codon (Table 3). The significant difference was found among the rates of non-synonymous mutations (d N) and synonymous mutations (d S) in the CP genes (P = 0.000 < 0.05). The dN/d S was far lower than 1, suggesting that the CP gene was purely selected during the evolution.
Phylogenetic analysis Taking beet yellow virus (BYV) as the out-group, the
phylogenetic trees of nucleotide sequences of the CP genes were constructed using maximum parsimony method (MP) and neighbor-joining method (NJ). Both methods produced consistent phyogenetic trees with equivalent topology, indicating that the same CTV isolate shared the same grouping in the phylogenetic trees (Fig.). Further analysis indicated that the 11 CTV isolates from Chinese wild type citrus were located in different phylogenetic clusters regardless of their geographical origins. For example, CT-W1, CT-W2, CTW3, CT-W4, and CT-W5 were from Yunnan Province in China, but were located into different clusters. The similar case was observed with the isolates CT-W6 and CT-W7 from Sichuan Province and isolates CT-W8 and CT-W9 from Hunan Province. The investigation revealed that the 11 CTV isolates in Chinese wild type citrus shared a higher homology and closer relationships with foreign CTV isolates, and the CTV isolates classified into one cluster shared a higher homology than those classified into different clusters in phylogenetic trees. The isolates were divided into six distinct groups according to the phylogenetic analysis. Interestingly, there was a trend that the isolates with similar biological characteristics were usually dropped into the same
Table 2 Percentage of the base composition in the CP genes of 3 CTV source Source 1) T
C
A
G
T1
C1
27.4 27.7 27.7
18.8 18.4 18.4
27.9 27.9 27.7
25.9 26.0 26.2
20.9 21.7 21.1
15.4 14.6 15.0
Percentage of the base composition (%) A1 G1 T2 C2 A2
G2
T3
C3
A3
G3
28.3 28.3 28.5
17.7 17.7 17.7
34.4 34.6 35.0
20.9 20.3 20.2
19.9 20.2 19.4
24.7 25.0 25.4
35.4 35.4 35.4
26.9 26.9 26.9
20.0 20.2 20.1
35.4 35.3 35.3
11 CTV isolates from wild type citrus; , 4 CTV isolates from Chinese cultivated citrus; , 21 exotic CTV isolates of cultivated citrus. 1, the base at the first location of codon; 2, the base at the second location of codon; 3, the base at the third location of codon. The same as below.
Table 3 Transition and tranversion, non-synonymous, and synonymous substitutions in the CP genes of 3 CTV sources Source and location of codon1) P1 P2 P3 P1 P2 P3 P1 P2 P3 1)
Ts
Tv
R
dN
dS
dN /dS
P
29 4 0 25 40 5 0 34 33 5 1 26
5 1 1 3 6 2 0 4 5 1 1 3
6.3 3.5 0.3 8.9 6.3 3.2 1.0 8.1 7.0 5.0 1.6 9.0
0.012 ± 0.003
0.153 ± 0.016
0.078
0.000
0.016 ± 0.005
0.215 ± 0.024
0.074
0.000
0.019 ± 0.004
0.160 ± 0.016
0.119
0.000
P1, P2 and P3 indicate the first, second, and third codon. Ts, transition; Tv, transversion; R, Ts/Tv; dN, the rates of non-synonymous substitutions; d S, the rates of synonymous substitutions.
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Genetic Evolution Analysis on Wild Isolates of Citrus Tristeza Virus Originated in China Based on Coat Protein Genes Sequences
A
93 86 92 75
75
96 99
67
62 79
69 99 63
86
78
62 81 99
CT-W10 NuagA CT21 CT-W1 13C CT-W3 I group Cheju-1 CT-W5 CT-W11 SY568 T318A M2 NZRB-5 II group VT 28C T30 T385 III group CT-W4 CT9 NZRB-3 CT4 Pune T3 IV group CT-W8 CT-W6 Bangalore NZRB-6 Cheju-2 CT-W2 V group CT-W9 CT-W7 PB61 CT3 Qaha VI group T36 Mexico-ctv BYV Outgroup
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CT-W3 Cheju-1 CT-W5 95 CT-W11 CT-W1 91 13C I group NuagA CT21 SY568 T318A 82 75 M2 52 CT-W10 71 0.01 VT 0.01 II group 98 0.01 28C NZRB-5 56 0.01 T30 56 0.03 86 T385 III group 96 CT-W4 0.05 CT9 NZRB-3 0.03 CT-W8 0.01 96 Bangalore 58 NZRB-6 IV group CT-W6 T3 56 CT4 Pune 68 0.02 Cheju-2 0.02 0.01 PB61 75 0.01 CT-W7 V group CT-W2 88 CT-W9 CT3 Qaha VI group 72 0.01 T36 90 Mexico-ctv 0.97 BYV Outgroup
B
0.01 change
Fig. Phyogenetic tree of CP gene nucleotide sequences of the 36 CTV isolates. A, the strict consensus tree of 49 maximum parsimony (MP) trees (taxon separation = 21 pixels, tree width = 400 pixels). The figures associated with branches are bootstrap percentages above 50%. B, the neighbor-joining (NJ) tree (Taxon separation = 21 pixels, branch length = 752 pixels/0.1, tree width = 7293 pixels). Numbers above branches indicates the mean distance above 0.01; numbers below branches indicates bootstrap values above 50%.
group, such as severe isolates NuagA, SY568 and T318A cluster into group I, the Qaha, T36, Mexicoctv, and CT3 isolates cluster into group VI, and mild isolates T30, T385 and CT9 cluster into group III (the phylogenetic tree with 70% or greater boot-strap support), and the mild isolate CT9 isolated from Chinese pummelo may be different from all other isolates analyzed because of the far distance (0.05) in the NJ tree.
DISCUSSION Comparative analysis of the nucleotide and deduced amino acid sequences of the CP genes showed a high degree of homology among different strains of CTV. A correlation was found between the biological characteristics and the CP genes sequences (Pappu et al. 1993),
which were conserved at 3´ terminal of the CTV isolates genomes with a nucleotide sequence identity of 90% (Hilf et al. 2005) . In pairwise comparisons between the CP genes of the 11 CTV isolates of wild citrus plants from China, 4 Chinese isolates from cultivated oranges and pummelos and the 21 exotic isolates from cultivated citrus in this study, the identity of nucleotide sequences and deduced amino acid sequences of the CP genes of the CTV isolates in the wild type citrus were over 90%, indicating the CP genes may be very conserved in the CTV genome and functionally important to keep the fundamentality in process of the CTV virus particle assembly, and some of the variants produced by mutation may be eliminated by negative selection. In this study, the GC% was less than AT% in the CP genes of the wild CTV isolates. The similar result was found in those of Chinese CTV isolates and exotic CTV
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isolates from cultivated citrus where lower content of bases GC indicated less variability of sequences. The ratio of transition/transversion was 6.3 in the CTV isolates from wild type citrus, 6.3 was found in the CTV isolates from Chinese cultivated citrus and no difference was found between the CTV isolates of wild type citrus and Chinese cultivated citrus. However, the ratio of 7.0 was found in the exotic CTV isolates from cultivated citrus, with more transversions and this showed they have more mutations than the Chinese CTV isolates. The rates of non-synonymous mutations (dN) and synonymous mutations (dS) in the CTV from wild type citrus was 0.078, those from Chinese cultivated citrus was 0.074, but 0.119 was in the exotic CTV isolates of cultivated citrus. This result suggested that the CP genes of exotic CTV isolates have more non-synonymous mutations than those of Chinese CTV isolates from the wild type citrus and cultivated citrus. When one gene is important to the organism, the nonsynonymous mutations can easily be eliminated by natural selection (Nei et al. 2000; Yang et al. 2000; Hanada et al. 2006). The results suggested that the CP genes of the CTV isolates from the China were more conserved than those of exotic CTV isolates. Phylogenetic analysis showed that 11 CTV isolates in Chinese wild type citrus were located in different phylogenetic clusters, regardless of their geographical origins. They were CT-W1, CT-W2, CT-W3, CT-W4, and CT-W5 from Yunnan which was much lower than that between those and the CTV isolates from different geographical origins. The similar results were found in the Chinese CTV isolates and exotic CTV isolates from cultivated citrus, which indicated complicated genetic relationships among the CTV isolates. Phylogenetic relationship and statistical analysis indicated that the homology among the 11 wild CTV isolates from the same area was much lower than that among other CTV isolates of cultivated citrus from different geographical origins. In addition, the CTV isolates with similar biological characteristics usually located into the same clusters, suggesting that pathogenicity was critical to the evolution and origin of CTV.
Acknowledgements This work was supported by the National Natural Science Foundation of China (30900977), the Key Project
YI Long et al.
210111 of Chinese Ministry of Education and Natural Science Foundation Project of CQ CSTC (2009BB1310).
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Genetic Evolution Analysis on Wild Isolates of Citrus Tristeza Virus Originated in China Based on Coat Protein Genes Sequences
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November 2010
2010, 9(11): 1623-1629
Genetic Evolution Analysis on Wild Isolates of Citrus Tristeza Virus Originated in China Based on Coat Protein Genes Sequences YI Long1, 2, ZHOU Chang-yong1, ZHOU Yan1 and LI Zhong-an1 Citrus Research Institute, Southwest University/Citrus Research Institute, Chinese Academy of Agricultural Sciences, Chongqing 400712, P.R.China 2 Jiangxi Navel Orange Engineering and Technology Research Center, Gannan Normal University, Ganzhou 341000, P.R.China 1
Abstract The coat protein (CP) genes were cloned and sequenced from viral particles of 11 isolates of citrus tristeza virus (CTV) collected from wild citrus plants in China and 4 Chinese isolates from cultivated sweet orange and pummelo varieties, respectively. By analyzing and comparing the nucleotide and amino acid sequences of CP genes, the 11 wild CTV isolates were found over 92% identical with 4 Chinese CTV isolates and 21 exotic CTV isolates from cultivated citrus. From 91 to 100% of the CTV CP gene sequences in wild type citrus plants were generally well conserved. Genetic evolution analysis indicated that the GC% of the CP gene was less than AT%, and more transition were found in the CP genes than transversion with the transition/transversion ratio ranging from 6.3 to 7.0 among species. The substitution frequency was the highest at the third codon, followed by the first and second codon. The ratio of non-synonymous mutations (dN) to synonymous mutations (dS) was far lower than 1, suggesting that the CP gene might have experienced purifying selection in the evolution. Phylogenetic analysis revealed that the 11 CTV isolates in Chinese wild type citrus belonged to different phylogenetic clusters, and shared higher homology and closer relationships with other cultivated citrus CTV isolates from different countries, which indicated complicated genetic relationships among the CTV isolates. In addition, CTV isolates with similar biological characteristics usually located into the same clusters. Therefore, the conclusion was drawn that pathogenicity was critical to evolution and origin of CTV. Key words: wild type citrus, citrus tristeza virus, sequence comparison, phylogenetic evolution
INTRODUCTION Citrus tristeza virus (CTV), the causal agent causing one of the most destructive diseases of citrus, is a member of genus Closterovirus, belonging to Closteroviridae family. CTV virions are flexuous filamentous of around 2 000 × 11 nm, with a genomic RNA (gRNA) molecule and two coat proteins (CP and CPm) covering 95 and 5% of the particle length respectively (Bar-Joseph et al. 1989; Pappu et al. 1994; Febres et al. 1996). Received 8 April, 2010
The CTV gRNA is a single-stranded, positive-sense RNA molecule of approximately 20 kb, organized in 12 open reading frames (ORFs) encoding at least 19 proteins (Karasev et al. 1995; Yang et al. 1999). CTV is readily propagated by infected buds of citrus and locally spread by aphids in a semi-persistent manner (Yokomi et al. 1994). CTV was initially identified by disease symptoms both in the field plants and in the indicator plants of graft-inoculated citrus. The isolates and strains were divided into two categories, mild and severe, using symptom variety. Mild isolates cause
Accepted 24 June, 2010
YI Long, Ph D, Tel: +86-797-8393068, E-mail: [email protected]; Correspondence ZHOU Chang-yong, Professor, Tel: +86-23-68349007, E-mail: [email protected] © 2010, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(09)60259-5
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only mild or no symptoms in sensitive citrus indicator hosts and usually result in no economic loss, and severe isolates cause decline, stem pitting, or both and may vary in intensity (Garnsey et al. 1987). Recently, these two types were found to be associated with serological or molecular genetic characters (Nikolaeva et al. 1998; Niblett et al. 2000; Ayllon et al. 2001). To date, the complete genome nucleotide sequences of the nine isolates of CTV have been published: T30, a mild isolate from Florida (Albiach-Martí et al. 2000); T36, a decline isolate from Florida (Karasev et al. 1995); VT, a stem-pitting isolate from Israel (Mawassi et al. 1996); SY568, a stem-pitting isolate from California (Yang et al. 1999); T385, a mild isolate from Spain (Vives et al. 1999); NuagA, a seeding yellow isolate from Japan (Suastika et al. 2001); T318A, a stem-pitting isolate from Spain (Ruiz-Ruiz et al. 2006); Qaha, a severe isolate from Egypt (GenBank accession no. AY340974); Mexico-ctv, a quick decline and stem-pitting isolate from Mexico (DQ272579). Nucleotide sequence analysis is the most accurate procedure for CTV differentiation and estimation of molecular and genetic variation. Results from recent study suggested that aphid transmission and host passage may significantly alter the composition of CTV populations, and may play an important role in their evolution (Albiach-Martí et al. 2000; Sentandreu et al. 2006). Recent studies on CTV isolates from wild type plants have been reported in China using molecular analysis (Yi et al. 2007). However, study on genetic diversity and difference between the CTV isolates from wild type and cultivated citrus has not been reported. In this study, the differences were estimated among the CTV isolates from both wild type citrus and cultivated citrus by sequence analysis, and theoretical data for analyzing relationship between genetic variation and evolution of CTV isolates is provided.
MATERIALS AND METHODS
YI Long et al.
C. medica in Mengla, Yunnan; CT-W6 and CT-W7 from wild C. ichangensis in Linshui, Sichuan; CT-W8, CTW9 from wild C. ichangensis and C. aurantium respectively in Yizhang, Hunan; CT-W10 from wild C. reticulata in Chongyi, Jiangxi; CT-W11 from wild C. reticulata in Hezhou, Guangxi (Yi et al. 2007). The severe isolate CT3 and the mild isolate CT9, from cultivated pummelo (Zhao et al. 2002), together with the severe isolate CT4 (Zhao et al. 2002) and the mild isolate CT21 (Zhou et al. 2007), from cultivated sweet orange, were kindly provided by Citrus Research Institute, Chinese Academy of Agricultural Sciences as representatives of CTV isolates found in China. CTV isolates were collected from field trees, and were maintained in sweet orange [Citrus sinensis (L.) Osb.] plants in insect-proof greenhouses.
Nucleic acid extraction and RT-PCR Total nucleic acids of the samples, 11 wild CTV isolates and 4 orchard CTV isolates from China, were extracted according to the method proposed by Zhou et al. (2001). The primer T36CP was used as an internal positive control for amplifying the CP gene (672 bp) (Gillings et al. 1993), the sense primer was CP1: 5´-ATGGACGACGA- AACAAAG-3´ , and the anti-sense primer was CP3: 5´ -TCAACGTGTGTTGAATTT-3´. 5 μL cDNA of CTV was amplified and performed in a 25-μL reaction volume containing a 1 × concentration reaction buffer (Promega, Madison, USA), 0.2 mmol L-1 dNTPs, 1.5 mmol L-1 MgCl2, 0.5 μmol L-1 of each primer and 0.625 U of Taq DNA polymerase (TaKaRa, Dalian, China). Amplification profiles were as follows: 94°C for 2 min; 30 cycles and each cycle was 94°C for 30 s, 52°C for 30 s and 72°C for 45 s; an extension step at 72°C for 7 min. PCR products were separated by electrophoresis in a 1.2% agarose gel and detected by ethidium bromide staining.
Sequence alignment and statistical analysis Virus isolates The wild CTV isolates CT-W1, CT-W2 and CT-W3 were collected from wild Poncirus polyandra in Fumin, Yunnan; CT-W4 from wild Citrus medica in Yangbi, Yunnan; CT-W5 from wild hybridism of C. grandis and
The PCR products of approximately 672 bp for the complete CP gene were gel purified and the nucleotide sequences were determined in both directions with an ABI Prism DNA sequencer 377 (Sangon Biological Engineering & Technology and Service, Shanghai, China). © 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Genetic Evolution Analysis on Wild Isolates of Citrus Tristeza Virus Originated in China Based on Coat Protein Genes Sequences
Other CTV nucleotide sequences used in our analysis were obtained from the GenBank entries: American isolates T36, T30, SY568, and T3 (DQ355053); Israel isolate VT; Spain isolates T385 and T318A; Japanese isolate NuagA; Egypt isolate Qaha; Mexico isolate Mexic-ctv, Portuguese isolates 13C (AF184113) and 28C(AF184118); Australia isolates PB61 (AJ297702); Korea isolates Cheju-1 (AF249279) and Cheju-2 (AF339088); New Zealand isolates NZRB-3 (AY896563), NZRB-5 (AY896561), NZRB-6 (AY896568), and NZRB3 (AJ297702); Iran isolates M2 (AY190048); and Indian isolates Bangalore (AF501867) and Pune (AF501869). The nucleotide sequences (636 bp), except the sequences of primers, were aligned with CLUSTAL W (ver. 1.4) (Thompson et al. 1994). The MEGA ver. 4.0 (Kumar et al. 2008) was used to estimate the base content, the transition and transversion, and the Ts/Tv ratio. The rates of non-synonymous (dN) and synonymous (dS) substitutions, and their ratios were determined by the Nei & Gojobori methods (Kumar et al. 2008) , and the differentiation statistic was done using Z-test (P < 0.05) (Kumar et al. 2008). Based on the maximum parsimony method (MP) and neighbor-joining method (NJ), phylogenetic relationships were inferred, taking the sequences of coat protein gene of beet yellow virus (BYV) (NC-001598) as the out-group, and reliability of the clusters was evaluated by bootstrap analysis based on 1000 repetitions, using MEGA 4.0.
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deposited in the GenBank database under accession numbers CT3 (FJ998187), CT4 (FJ998188), CT9 (FJ998189), CT21 (FJ998190), and CT-W1-11 (FJ998191-FJ998201).
RESULTS Sequence identity analysis The nucleotide sequences and amino acid sequences of the CP genes from the 11 wild CTV isolates were found similarity ranging from 92.1 to 99.5% and 94.8 to 100% respectively. The identity of nucleotide sequence ranged from 91.3 to 99.5% and 91.3 to 99.6% when compared with 4 Chinese CTV isolates of cultivated citrus and 21 exotic CTV isolates of cultivated citrus, whereas the identity of amino acid sequences ranged from 95.2 to 100% and 94.3 to 100%, respectively (Table 1). Based on the sequence alignments of all the analyzed CTV isolates it was found that the identity between nucleotide sequences and amino acid sequences of the CTV CP gene was over 90%. The CP gene sequences of wild type citrus plants were generally well conserved, and the CP gene was very important for preserving the characters of the CTV virus during the development. No significant difference was shown between the CTV CP gene sequences of wild type citrus plants and cultivated citrus, either in the nucleotide sequences or amino acid sequences.
Nucleotide sequence accession numbers
Base content analysis
The nucleotide sequence data reported in this paper were
In this investigation, GC% was less than AT%, and base
Table 1 Sequence identity of the CP genes of the wild CTV isolates from different geographical origins Wild CTV isolates CT-W1-11
Comparison CTV isolates (geographical origin) CT-W1-11 (China) CT3, T9, CT4, CT21 (China) T36, T30, SY568, T3 (USA) NuagA (Japan) T385, T318A (Spain) VT (Israel) Qaha (Egypt) Mexico-ctv (Mexico) PB61 (Australia) M2 (Iran) Bangalore, Pune (India) 13C, 28C (Portugal) Cheju-1, Cheju-2 (Korea) NZRB-3, NZRB-5, NZRB-6 (New Zealand)
Identity of nucleotide sequences (%) 92.1-99.5 91.3-99.5 91.6-99.0 92.9-98.8 92.1-98.5 92.4-97.3 91.6-95.2 91.6-95.2 92.2-98.4 92.9-98.7 91.6-99.5 92.6-99.3 91.3-98.8 92.4-99.6
Identity of amino acid sequences (%) 94.8-100 95.2-100 94.3-100 96.2-100 94.8-99.0 95.2-97.1 94.3-96.6 94.3-96.6 95.7-99.0 96.2-99.5 96.2-99.5 95.2-99.0 94.3-98.1 96.2-99.5
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A content was the highest among the four bases in the CP genes of the 11 CTV isolates from wild type citrus, and also in CTV isolates from cultivated citrus (Table 2).
Transition and transversion, non-synonymous and synonymous substitutions The percentage of variable sites of nucleotide sequences and amino acid sequences in the CP genes of the 36 analyzed CTV isolates were 25.2 and 17.5%, respectively. More transitions than transversions were found in the CP genes, with the transition/transversion ratio ranging from 6.3 to 7.0 among species. The substitution frequency was the highest at the third codon, followed by the first and second codon (Table 3). The significant difference was found among the rates of non-synonymous mutations (d N) and synonymous mutations (d S) in the CP genes (P = 0.000 < 0.05). The dN/d S was far lower than 1, suggesting that the CP gene was purely selected during the evolution.
Phylogenetic analysis Taking beet yellow virus (BYV) as the out-group, the
phylogenetic trees of nucleotide sequences of the CP genes were constructed using maximum parsimony method (MP) and neighbor-joining method (NJ). Both methods produced consistent phyogenetic trees with equivalent topology, indicating that the same CTV isolate shared the same grouping in the phylogenetic trees (Fig.). Further analysis indicated that the 11 CTV isolates from Chinese wild type citrus were located in different phylogenetic clusters regardless of their geographical origins. For example, CT-W1, CT-W2, CTW3, CT-W4, and CT-W5 were from Yunnan Province in China, but were located into different clusters. The similar case was observed with the isolates CT-W6 and CT-W7 from Sichuan Province and isolates CT-W8 and CT-W9 from Hunan Province. The investigation revealed that the 11 CTV isolates in Chinese wild type citrus shared a higher homology and closer relationships with foreign CTV isolates, and the CTV isolates classified into one cluster shared a higher homology than those classified into different clusters in phylogenetic trees. The isolates were divided into six distinct groups according to the phylogenetic analysis. Interestingly, there was a trend that the isolates with similar biological characteristics were usually dropped into the same
Table 2 Percentage of the base composition in the CP genes of 3 CTV source Source 1) T
C
A
G
T1
C1
27.4 27.7 27.7
18.8 18.4 18.4
27.9 27.9 27.7
25.9 26.0 26.2
20.9 21.7 21.1
15.4 14.6 15.0
Percentage of the base composition (%) A1 G1 T2 C2 A2
G2
T3
C3
A3
G3
28.3 28.3 28.5
17.7 17.7 17.7
34.4 34.6 35.0
20.9 20.3 20.2
19.9 20.2 19.4
24.7 25.0 25.4
35.4 35.4 35.4
26.9 26.9 26.9
20.0 20.2 20.1
35.4 35.3 35.3
11 CTV isolates from wild type citrus; , 4 CTV isolates from Chinese cultivated citrus; , 21 exotic CTV isolates of cultivated citrus. 1, the base at the first location of codon; 2, the base at the second location of codon; 3, the base at the third location of codon. The same as below.
Table 3 Transition and tranversion, non-synonymous, and synonymous substitutions in the CP genes of 3 CTV sources Source and location of codon1) P1 P2 P3 P1 P2 P3 P1 P2 P3 1)
Ts
Tv
R
dN
dS
dN /dS
P
29 4 0 25 40 5 0 34 33 5 1 26
5 1 1 3 6 2 0 4 5 1 1 3
6.3 3.5 0.3 8.9 6.3 3.2 1.0 8.1 7.0 5.0 1.6 9.0
0.012 ± 0.003
0.153 ± 0.016
0.078
0.000
0.016 ± 0.005
0.215 ± 0.024
0.074
0.000
0.019 ± 0.004
0.160 ± 0.016
0.119
0.000
P1, P2 and P3 indicate the first, second, and third codon. Ts, transition; Tv, transversion; R, Ts/Tv; dN, the rates of non-synonymous substitutions; d S, the rates of synonymous substitutions.
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Genetic Evolution Analysis on Wild Isolates of Citrus Tristeza Virus Originated in China Based on Coat Protein Genes Sequences
A
93 86 92 75
75
96 99
67
62 79
69 99 63
86
78
62 81 99
CT-W10 NuagA CT21 CT-W1 13C CT-W3 I group Cheju-1 CT-W5 CT-W11 SY568 T318A M2 NZRB-5 II group VT 28C T30 T385 III group CT-W4 CT9 NZRB-3 CT4 Pune T3 IV group CT-W8 CT-W6 Bangalore NZRB-6 Cheju-2 CT-W2 V group CT-W9 CT-W7 PB61 CT3 Qaha VI group T36 Mexico-ctv BYV Outgroup
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CT-W3 Cheju-1 CT-W5 95 CT-W11 CT-W1 91 13C I group NuagA CT21 SY568 T318A 82 75 M2 52 CT-W10 71 0.01 VT 0.01 II group 98 0.01 28C NZRB-5 56 0.01 T30 56 0.03 86 T385 III group 96 CT-W4 0.05 CT9 NZRB-3 0.03 CT-W8 0.01 96 Bangalore 58 NZRB-6 IV group CT-W6 T3 56 CT4 Pune 68 0.02 Cheju-2 0.02 0.01 PB61 75 0.01 CT-W7 V group CT-W2 88 CT-W9 CT3 Qaha VI group 72 0.01 T36 90 Mexico-ctv 0.97 BYV Outgroup
B
0.01 change
Fig. Phyogenetic tree of CP gene nucleotide sequences of the 36 CTV isolates. A, the strict consensus tree of 49 maximum parsimony (MP) trees (taxon separation = 21 pixels, tree width = 400 pixels). The figures associated with branches are bootstrap percentages above 50%. B, the neighbor-joining (NJ) tree (Taxon separation = 21 pixels, branch length = 752 pixels/0.1, tree width = 7293 pixels). Numbers above branches indicates the mean distance above 0.01; numbers below branches indicates bootstrap values above 50%.
group, such as severe isolates NuagA, SY568 and T318A cluster into group I, the Qaha, T36, Mexicoctv, and CT3 isolates cluster into group VI, and mild isolates T30, T385 and CT9 cluster into group III (the phylogenetic tree with 70% or greater boot-strap support), and the mild isolate CT9 isolated from Chinese pummelo may be different from all other isolates analyzed because of the far distance (0.05) in the NJ tree.
DISCUSSION Comparative analysis of the nucleotide and deduced amino acid sequences of the CP genes showed a high degree of homology among different strains of CTV. A correlation was found between the biological characteristics and the CP genes sequences (Pappu et al. 1993),
which were conserved at 3´ terminal of the CTV isolates genomes with a nucleotide sequence identity of 90% (Hilf et al. 2005) . In pairwise comparisons between the CP genes of the 11 CTV isolates of wild citrus plants from China, 4 Chinese isolates from cultivated oranges and pummelos and the 21 exotic isolates from cultivated citrus in this study, the identity of nucleotide sequences and deduced amino acid sequences of the CP genes of the CTV isolates in the wild type citrus were over 90%, indicating the CP genes may be very conserved in the CTV genome and functionally important to keep the fundamentality in process of the CTV virus particle assembly, and some of the variants produced by mutation may be eliminated by negative selection. In this study, the GC% was less than AT% in the CP genes of the wild CTV isolates. The similar result was found in those of Chinese CTV isolates and exotic CTV
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isolates from cultivated citrus where lower content of bases GC indicated less variability of sequences. The ratio of transition/transversion was 6.3 in the CTV isolates from wild type citrus, 6.3 was found in the CTV isolates from Chinese cultivated citrus and no difference was found between the CTV isolates of wild type citrus and Chinese cultivated citrus. However, the ratio of 7.0 was found in the exotic CTV isolates from cultivated citrus, with more transversions and this showed they have more mutations than the Chinese CTV isolates. The rates of non-synonymous mutations (dN) and synonymous mutations (dS) in the CTV from wild type citrus was 0.078, those from Chinese cultivated citrus was 0.074, but 0.119 was in the exotic CTV isolates of cultivated citrus. This result suggested that the CP genes of exotic CTV isolates have more non-synonymous mutations than those of Chinese CTV isolates from the wild type citrus and cultivated citrus. When one gene is important to the organism, the nonsynonymous mutations can easily be eliminated by natural selection (Nei et al. 2000; Yang et al. 2000; Hanada et al. 2006). The results suggested that the CP genes of the CTV isolates from the China were more conserved than those of exotic CTV isolates. Phylogenetic analysis showed that 11 CTV isolates in Chinese wild type citrus were located in different phylogenetic clusters, regardless of their geographical origins. They were CT-W1, CT-W2, CT-W3, CT-W4, and CT-W5 from Yunnan which was much lower than that between those and the CTV isolates from different geographical origins. The similar results were found in the Chinese CTV isolates and exotic CTV isolates from cultivated citrus, which indicated complicated genetic relationships among the CTV isolates. Phylogenetic relationship and statistical analysis indicated that the homology among the 11 wild CTV isolates from the same area was much lower than that among other CTV isolates of cultivated citrus from different geographical origins. In addition, the CTV isolates with similar biological characteristics usually located into the same clusters, suggesting that pathogenicity was critical to the evolution and origin of CTV.
Acknowledgements This work was supported by the National Natural Science Foundation of China (30900977), the Key Project
YI Long et al.
210111 of Chinese Ministry of Education and Natural Science Foundation Project of CQ CSTC (2009BB1310).
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Genetic Evolution Analysis on Wild Isolates of Citrus Tristeza Virus Originated in China Based on Coat Protein Genes Sequences
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