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Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China
Journal of Integrative Agriculture 2014, 13(12): 2669-2677
December 2014
RESEARCH ARTICLE
Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China YI Long1, 2 and ZHOU Chang-yong1 1 2
Citrus Research Institute, Chinese Academy of Agricultural Sciences, Chongqing 400712, P.R.China National Navel Orange Engineering Technology Research Center, Ganzhou 341000, P.R.China
Abstract The genetic variation and phylogenetic relationships of Citrus tristeza virus (CTV) isolates collected from Chinese wild type citrus were analyzed by comparing the sequences of nine genomic regions (p23, p20, p13, p18, p25, p27, POL, HEL and k17) with the CTV isolates of cultivated citrus from different countries. The results showed that the divergence pattern of genomic RNA of the CTV isolates from wild type citrus was similar to that of other isolates from cultivated citrus, the 3´ proximal region was relatively conserved, and the 5´ proximal region had greater variability. The nine genomic regions of CTV isolates analyzed were found to have been under purifying selection in the evolution process. Phylogenetic analysis showed that the eleven Chinese wild CTV isolates were located at different clades and did not reflect their geographical origins, suggesting genetic diversity among the Chinese wild CTV populations. These results will aid in the understanding of molecular evolution of the Chinese CTV populations. Key words: Citrus tristeza virus, wild type citrus, genetic diversity, phylogenetic analysis
INTRODUCTION Citrus tristeza virus (CTV), a member of genus Closterovirus within the family Closteroviridae, is distributed worldwide and also the causal agent of one of the most economically important citrus diseases. The virus is dispersed to new areas by propagation of infected buds and locally transmitted by aphids (Bar-Joseph et al. 1989). CTV virions are filamentous flexuous particles about 2 000 nm long with two capsid proteins of 25 and 27 kDa coating about 95 and 5% of the virion length, respectively (Febres et al. 1996). Which have a single-stranded,
positive-sense genomic RNA (gRNA) approximately 20 kb that is organized in 12 open reading frames (ORFs), potentially encoding at least 19 proteins (Karasev et al. 1995). The two 5´ proximal ORFs, encoding replication-related proteins, are directly translated from gRNA. ORF1a encodes a 349-kDa polyprotein that containing papain-protease, helicase-like, and methyltransferase-like domains. ORF1b encodes a protein with RNA-dependent RNA polymerase (RdRp) domains. The remaining 10 ORFs, which are expressed via 3´-coterminal subgenomic RNAs, encode (from 5´ to 3´) proteins p33, p6, p65, p61, p27, p25, p18, p13, p20, and p23 (Hilf et al. 1995; Karasev et al. 1995). Protein p6 may have transmembrane functions; p65 is an HSP70 homologue, which together with p61 and the two coat proteins p27
Received 22 October, 2013 Accepted 24 January, 2014 YI Long, Tel: +86-797-8393068, E-mail: [email protected]; Correspondence ZHOU Chang-yong, Tel: +86-23-68349037, Fax: +86-23-68349592, E-mail: zhoucy@ cric.cn
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60730-3
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and p25 are involved in virion formation (Satyanarayana et al. 2000); p20 accumulates in amorphous inclusion bodies (Gowda et al. 2000); and p23 is an RNA-binding protein (López et al. 2000) that regulates asymmetrical accumulation of positive and negative strands during RNA replication (Satyanarayana et al. 2002) and is involved in symptom expression (Ghorbel et al. 2001; Fagoaga et al. 2005). Proteins p25, p20 and p23 have been shown to act as RNA silencing suppressors (Lu et al. 2004). The roles of the p33, p18 and p13 are presently unknown, but Tatineni et al. (2008) reported that they may be involved in the ability of CTV to infect citrus trees systemically. Nucleotide sequence analysis is the most accurate procedure for CTV isolates differentiation and estimation of molecular or genetic variation (Rubio et al. 2001). Recent study results suggest that aphid transmission and host passage may significantly alter the composition of populations of CTV isolates and may be an important factor in their evolution (Albiach-Martí et al. 2000; Sentandreu et al. 2006). Negative selection, gene flow, sequence recombination and virulence may be important factors driving CTV evolution (Martín et al. 2009). In a previous work, we collected 79 wild type citrus samples from Yunnan, Guangxi, Sichuan, Hunan and Jiangxi provinces (or autonomous region) of China and found that eleven samples were CTV-positive. We characterized the wild CTV isolates by restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP) analysis of their coat protein (CP) genes and found the CP genes of wild CTV isolates were generally well conserved (Yi et al. 2007, 2010). To gain further insight into the molecular characteristics of CTV isolates evolution from wild type citrus, the genetic variation and phylogenetic relationships in nine gRNA regions of these isolates and the CTV isolates from cultivated citrus were further analyzed.
ResUlTs sequence identity, number of variable sites and nucleotide genetic distance Comparing the sequence identity of nine genomic regions of CTV isolates of wild citrus and cultivated
YI Long et al.
citrus, the nucleotide sequences and amino acid sequences of p23, p20, p13, p18, p25 and p27 genomic regions of eleven wild CTV isolates were found to have identity ranging 88.1-100% and 88.2-100%, respectively. Their nucleotide and amino acid sequences identity ranged 87.7-100% and 85.7-100%, with those of group II, respectively. Ranged 88.0-99.7% and 87.4-100% with those of group III, respectively (Table 1). The results of sequence comparisons showed that the nucleotide sequences and amino acid sequences identities around 90% in six genomic regions (p23, p20, p13, p18, p25, p27) at 3´-terminal region of the genome, but only close to 70% in the k17 regions at 5´-terminal region of the genome of wild CTV isolates (Table 1). The number of variable sites of nucleotide sequences and deduced amino acid sequences was also found ranging 13.1-18.9% and 5.7-19.4% in the p23, p20, p13, p18, p25 and p27 genomic regions among three CTV groups, respectively, with 24.5-30.9% and 23.9-31.7% in POL genomic region, 25.1-30.0% and 13.6-20.7% in HEL genomic region, and 35.9-43.3% and 37.5-49.3% in k17 genomic region, respectively (Table 2). When nucleotide genetic distances of nine genomic regions were compared in three groups, we found that the nucleotide distance of k17 (Table 2) was significantly higher than those of regions p23, p20, p13, p18, p25, p27, POL and HEL. The highest nucleotide genetic distance occurred at the k17 region followed by the distance at the HEL and POL regions. In addition, we found that there was a gradual increase in genetic distance towards the 5´ proximal region of the CTV genome.
Base content analysis, transition and transversion, non-synonymous and synonymous substitutions Base content analysis showed that the wild CTV isolates were similar to those of other isolates sourced from cultivated citrus. The GC content of nine genomic regions was less than AU content. Base A content was the highest among the four bases in the p25, p27 and p23 genomic regions, while base U content was the highest in the p20, p13, p18, POL, HEL and k17 genomic regions. Base C content was the lowest in the above nine genomic regions (Table 2). © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China
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Table 1 Identity of nucleotide and amino acid sequences (in parenthesis) of three groups of analyzed CTV isolates Genomic regions p23
p20
p13
p18
p25
p27
POL
HEL
k17
Groups Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ
Group I (%) 88.1-99.8 (88.2-100)
Group II (%) 87.7-97.9 (87.2-97.9) 88.6-98.4 (87.2-98.4)
90.3-99.6 (91.8-100)
88.3-99.6 (90.7-100) 89.2-92.3 (91.2-93.9)
91.1-100 (89.9-100)
88.8-98.6 (85.7-98.3) 90.5-97.2 (89.0-97.4)
90.7-99.1 (93.5-100)
89.8-98.7 (89.6-99.3) 90.0-95.4 (92.8-98.0)
92.1-99.5 (94.8-100)
91.3-99.5 (95.2-100) 91.3-94.3 (96.2-98.1)
89.4-100 (94.2-100)
88.1-100 (94.7-100) 87.7-93.7 (96.0-97.7)
83.3-98.6 (82.1-97.9)
81.9-99.2 (82.6-99.4) 83.0-97.1 (84.6-96.9)
77.0-99.7 (86.3-100)
77.2-99.4 (86.0-100) 77.4-96.2 (86.6-98.5)
65.5-99.7 (63.2-100)
66.0-97.3 (62.5-99.2) 67.2-96.3 (64.7-98.5)
Group III (%) 88.0-99.3 (87.7-100) 88.0-98.8 (86.8-100) 89.1-100 (86.8-100) 88.2-99.0 (87.4-100) 88.5-97.9 (88.5-99.4) 87.6-100 (87.4-100) 89.7-99.7 (88.2-99.1) 88.3-96.6 (85.7-98.3) 89.4-100 (87.3-100) 90.7-98.9 (89.6-99.3) 89.0-97.4 (88.3-99.3) 90.7-100 (90.2-100) 91.6-99.0 (94.3-100) 91.1-99.2 (94.3-100) 92.2-100 (93.8-100) 88.1-99.1 (93.3-99.1) 86.3-96.6 (93.3-98.6) 88.1-100 (93.3-100) 80.6-97.8 (81.1-97.9) 80.6-95.1 (81.6-94.3) 80.7-99.5 (82.1-99.4) 75.6-99.6 (83.5-100) 75.8-99.2 (83.2-100) 74.7-99.4 (81.5-100) 64.0-98.2 (56.6-100) 65.2-97.7 (58.8-98.5) 64.3-100 (54.4-100)
Table 2 Base contents, variable site ratios of nucleotide and amino acid sequences, and average nucleotide distance in nine genomic regions of three groups of analyzed CTV isolates Genomic regions p23
p20
p13
p18
p25
p27
POL
HEL
k17
Groups Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ
Base contents (%) U C A G 24.7 19.4 30.2 25.6 24.9 19.5 29.8 25.8 24.9 19.3 30.0 25.9 29.2 21.0 24.7 25.2 29.4 20.5 24.4 25.7 29.3 20.7 24.4 25.6 31.9 18.9 26.2 22.9 32.3 18.7 26.2 22.8 32.1 19.0 25.7 23.2 35.5 17.2 25.4 21.9 34.8 17.6 25.8 21.7 35.5 17.3 25.4 21.8 27.4 18.8 27.9 25.9 27.7 18.4 27.9 26.0 27.7 18.4 27.9 25.9 27.6 19.1 30.7 22.6 27.9 19.0 30.6 22.5 27.5 19.6 30.5 22.4 32.8 18.6 25.5 23.2 32.4 18.8 25.6 23.2 32.8 18.9 25.5 22.9 30.0 18.4 26.6 24.9 30.5 18.0 26.2 25.3 29.8 18.7 26.5 25.1 35.6 17.7 23.1 23.6 36.0 17.3 22.3 24.3 35.8 18.0 22.3 23.8
V-NN (%)1)
V-AA (%)2)
Average nucleotide distance±SE3)
18.9 14.7 17.5 13.9 16.1 18.7 15.3 13.1 17.8 17.9 13.8 15.1 14.8 13.4 13.7 17.9 17.7 17.1 30.9 24.5 28.4 30.0 25.1 27.8 43.3 35.9 41.6
19.4 14.2 16.8 8.8 6.6 19.0 13.4 12.6 16.8 14.3 9.1 13.0 6.1 5.7 9.9 8.8 4.8 10.6 31.7 23.9 27.3 15.9 13.6 20.7 41.9 37.5 49.3
0.0792±0.0077 0.0923±0.0100 0.0800±0.0082 0.0553±0.0066 0.0976±0.0099 0.0794±0.0080 0.0586±0.0080 0.0751±0.0103 0.0796±0.0095 0.0563±0.0066 0.0798±0.0096 0.0614±0.0076 0.0558±0.0058 0.0775±0.0084 0.0608±0.0067 0.0765±0.0070 0.1063±0.0094 0.0458±0.0056 0.1252±0.0092* 0.1589±0.0132* 0.1425±0.0112* 0.1480±0.0101* 0.1920±0.0127* 0.1619±0.0107* 0.2685±0.0242** 0.3172±0.0311** 0.2692±0.0231**
1)
V-NN, variable site ratio of nucleotide sequences. V-AA, variable site ratio of amino acid sequences. 3) SE, standard errors. * and **, statistically different to the rest of values at P<0.05 and P<0.01, respectively. 2)
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More transitions were found in nine genomic regions than transversions and the transitions/transversions ratio of HEL and k17 genomic regions ranged from 2.1 to 2.6 among three groups-analyzed (Table 3). The number of transitions of HEL and k17 genomic regions was more than that of the rest regions, suggesting they were the most variable regions among the analyzed nine genomic regions. A significant difference was found among the rates of non-synonymous mutations (dN) and of synonymous mutations (dS) in the p25, p27, p23, p20, p13, p18, POL and k17 genomic regions in three groups whereas there was no significant difference in the HEL region (P<0.05) (Table 3). According to Martín et al. (2009), the dN/dS values can be placed into four selection categories: i) strong negative selection (dN/dS<0.1); ii) moderate negative (0.11). Moderate negative selection was detected in most regions, i.e., p23, p20, p13, p18, p25, p27, POL and HEL regions of group III; and weak negative selection occurred in k17 region, HEL region of both group I and group II. In general, dN values were much lower than dS values (dN /dS<1),
suggesting that nine genomic regions have been under purifying selection in the evolutionary process.
Phylogenetic relationships between CTV isolates Most CTV isolates shared the same grouping in the phylogenetic trees obtained from nine genomic regions, regardless of their geographical origins. The eleven CTV isolates in Chinese wild citrus were located in different phylogenetic clusters, such as CT-W1, CT-W2, CT-W3, CT-W4 and CT-W5 from Yunnan Province in China, which dropped into different clades and as do the isolates CT-W6 and CT-W7 from Sichuan Province and the isolates CT-W8 and CT-W9 from Hunan Province. At least three clades were observed in most regions by phylogenetic analysis. One clade (clade I) comprising the isolates CT-W3, CT-W5, CT-W10 (except in p27), CT-W11, T318A, NUagA, and SY568 (except in POL and p27) was supported by bootstrap values exceed 50%. A second clade (clade II) comprising of isolates T36, Qaha, Mexico-ctv, and CT3 (except in p25, p13,
Table 3 The numbers of transition (Ts) and transversion (Tv), the rates of non-synonymous (dN) and synonymous (dS) substitutions and their ratios (R) in the nine genomic regions of three groups of analyzed CTV isolates Genomic regions p23
p20
p13
p18
p25
p27
POL
HEL
k17
*
Groups
Ts
Tv
R (Ts/Tv)
dN±SE
dS±SE
dN /dS
Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ
36 42 34 25 42 33 17 23 21 22 30 23 29 40 12 41 60 41 53 64 57 94 115 96 58 67 56
7 8 9 3 7 8 2 2 6 3 4 3 5 6 2 7 6 8 13 12 14 38 54 46 22 27 26
4.9 5.1 3.7 7.5 6.0 4.4 7.1 14.0 3.8 7.5 8.0 7.1 6.3 6.3 8.2 5.8 9.9 5.2 4.2 5.2 4.0 2.5 2.1 2.1 2.6 2.5 2.1
0.040±0.007 0.051±0.010 0.041±0.007 0.017±0.004 0.021±0.006 0.036±0.007 0.031±0.008 0.037±0.009 0.039±0.010 0.018±0.004 0.025±0.007 0.026±0.005 0.012±0.003 0.016±0.005 0.019±0.004 0.016±0.004 0.017±0.006 0.020±0.004 0.062±0.008 0.070±0.011 0.070±0.010 0.046±0.006 0.064±0.009 0.148±0.012* 0.149±0.020 0.174±0.025 0.161±0.020
0.174±0.021 0.190±0.026 0.179±0.021 0.145±0.020 0.291±0.033 0.190±0.023 0.122±0.020 0.156±0.027 0.182±0.027 0.139±0.018 0.192±0.028 0.135±0.021 0.153±0.016 0.215±0.024 0.148±0.017 0.224±0.023 0.318±0.028 0.221±0.022 0.286±0.026 0.355±0.037 0.332±0.035 0.516±0.042 0.698±0.071 0.166±0.020* 0.634±0.090 0.754±0.120 0.621±0.096
0.230 0.268 0.229 0.117 0.072 0.189 0.254 0.237 0.214 0.129 0.130 0.193 0.078 0.074 0.128 0.071 0.053 0.090 0.217 0.197 0.211 0.089 0.092 0.892 0.235 0.231 0.259
, statistical difference of the dN and dS values of the genomic region in the analyzed group at P<0.05.
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Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China
and p23) was supported by bootstrap values greater than 81% within nine genomic regions. The isolates T30 and T385 were clustered into clade III. Their phylogenetic trees have 56% or greater boot-strap support. While isolates defining clades clustered together regardless of the genomic region, other isolates showed incongruent phylogenetic relationships (Fig.-B). The virulent isolate VT was genetically closer to isolates of clade I, the wild CTV isolates CT-W1, CT-W2, CTW4, CT-W6, CT-W7, and CT-W9 were clustered into different phylogenetic clades, separated from the above clades, with incongruent phylogenetic relationships in the nine genomic regions. The severe isolate CT4 of Chinese cultivated citrus was closely related to isolates of clade I in the HEL, POL, p27 and p20 regions. But it was more closely related to clade III in the k17 region. Similarly, inconsistencies in the phylogenetic relationships of different genomic regions were also observed in mild isolates CT9 and CT21 sourced from Chinese cultivated citrus and cluster into clade III without the mild isolates T30 (Albiach-Martí et al. 2000) and T385 (Vives et al. 1999).
DIsCUssION The nucleotide and deduced amino acid sequences of nine genomic regions in eleven wild CTV isolates from China showed that sequence identity exceeded 87% in the 3´ half of the genome, which was higher than that in the 5´ half of the genome. Genomic RNA within the 3´ half was relatively conserved; whereas the sequences of the HEL and k17 regions in the 5´ proximal region of the genome were more dissimilar with higher diversity. The accumulation of small RNAs of CTV mapping preferentially at the 3´-terminal 2 500-nt of the RNA genome (Ruiz-Ruiz et al. 2011), which most likely results from reduction sequence divergence in the 3´-termianl region of the CTV genomic RNA. The genetic distance of the nine genomic regions were compared with each other and there was a gradual increase in genetic distance from 3´ to the 5´ terminus of the CTV genome. Although no complete gRNA sequence of wild CTV isolate is currently available, our findings suggest the 3´ terminus was relatively conserved and sequence differences were greater in the 5´ terminus, which indicate the genomic RNA of wild CTV isolates have the
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similar patterns of nucleotide variability as those derived from cultivated citrus (Mawassi et al. 1996; López et al. 1998; Vives et al. 1999; Yang et al. 1999; Albiach-Martí et al. 2000; Suastika et al. 2001; Ruiz-Ruiz et al. 2006). GC content is an explanatory variable in the sense that it allows one to predict a certain portion of the variability of each divergence measure (Hardison et al. 2003). The divergence decreases with GC content for low-GC DNA and increases with GC content for higher-GC DNA, consistent with important differences in the patterns of evolution in these two classes of genomic DNA (Bernardi 1995; Fullerton et al. 2001; Castresana 2002). In this study, the GC content was also found to be less than the AU content within nine genomic regions, the dN values were much lower than dS values (dN /dS<1). Analysis of selective pressure acting on different regions suggests that all regions examined are subjected to purifying selection. This pressure was moderate in p23, p20, p13, p18, p25 and p27 regions, but weak in k17 in the 5´ half of the genome. While 3´ half of the genome was more conserved than the 5´ half, some of the variants produced by mutation should be more easily eliminated in the 3´ half of the CTV genome by purifying selection in the evolutionary process. In this study, the wild type citrus samples were collected from high altitudes (>1 100 m), and which were estimated to have been living for over 50 years (some for 200 years or more). From the phylogenetic analysis, eleven CTV isolates from wild type citrus were located in different clades regardless of their geographical origin. Some isolates showed incongruent phylogenetic relationships in different genomic regions, which provide further support to previous inference that the temporal separation of CTV isolates might have occurred several hundred years in the past (Albiach-Martí et al. 2000). CTV isolate sequences could be assigned to three types: T36 genotype, T30 genotype, and VT genotype (López et al. 1998; Hilf et al. 1999; Ayllón et al. 2001), and the same genotype cluster into one clade (Hilf et al. 2005). The genotypes of eleven wild CTV isolates were not clear; however, their segregating into different phylogenetic clusters indicated that they had different genotypes. This demonstrated genetic diversity among the CTV populations from wild citrus plants was similar to those from cultivated citrus (Rubio et al. 2001; Sentandreu et al. 2006; Melzer et al. 2010; Roy and Brlansky 2010). © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
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A
PRO PRO
k17
1b HEL
2
3
p33
4
5
p65
6
7
p27
8
9
p18
10 11 p20
CTV 5´
3´ POL
p6
p16
p25
p13
p23
B
Fig. A, schematic of the genomic structure of the Citrus tristeza virus (CTV) isolate T36, open reading frames (ORFs) are represented by open rectangles with indication of the encoded protein and are numbered in ascending order from the 5´ to the 3´ terminus in the manner of Karasev et al. (1995). Layout of the CTV genome with the regions analyzed indicated as black boxes. B, unrooted nucleotide maximum parsimony phylogenetic trees of CTV genomic regions k17, HEL, POL, p27, p25, p18, p13, p20 and p23. Scale bars indicate number of changes per position for unit branch length. Bootstrap values for significance of nodes are indicated by asterisks (***, 90-100%; **, 70-89%; *, 50-69%).
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Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China
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CONClUsION
sequence alignment and statistical analysis
In summary, analyses of sequence identity, genetic variation and phylogenetic relationships of the CTV isolates from wild and cultivated citrus indicated that the genome of wild CTV isolates have the same sequence divergence as that of cultivated citrus, and the genetic diversity among the CTV populations from wild type citrus in China, which will aid in the understanding of molecular evolution of the Chinese CTV populations.
Nucleotide sequences were translated to proteins using BioEdit 7.0.1 (Hall 1999) and the multiple alignments were performed with the program CLUSTALW (Thompson et al. 1994). The MEGA ver. 4.0 program was used to estimate the base content and calculate the average nucleotide genetic distances of CTV isolates by Kimura two-parameter model. After testing homogeneity of pattern substitution among lineages, the numbers of transitions (Ts) and transversions (Tv) and the Ts/Tv ratio, the rate of non-synonymous (dN), and synonymous (dS) substitutions were determined by the Nei & Gojobori methods (Tamura et al. 2007; Kumar et al. 2008). Phylogenetic relationships were inferred based on the maximum parsimony method, and reliability of the clades was evaluated by bootstrap analysis based on 1 000 repetitions using the MEGA 4.0 program.
MATeRIAls AND MeThODs Nucleotide sequences of CTV isolates The wild CTV isolates CT-W1, CT-W2 and CT-W3 were collected from wild Poncirus polyandra in Fumin County, CT-W4 from wild Citrus medica in Yangbi County, CT-W5 from wild hybridism of C. grandis and C. medica in Mengla County, Yunnan Province; CT-W6 and CT-W7 from wild C. ichangensis in Linshui County, Sichuan Province; CT-W8, CT-W9 from wild C. ichangensis and C. aurantium, respectively in Yizhang County, Hunan Province; CT-W10 from wild C. reticulata in Chongyi County, Jiangxi Province; CTW11 from wild C. reticulata in Hezhou County, Guangxi Autonomous Region of China (Yi et al. 2007). The severe isolate CT3 and the mild isolate CT9 from cultivated pummelos, and the severe isolate CT4 and the mild isolate CT21 from cultivated sweet oranges were kindly provided by the Citrus Research Institute, Chinese Academy of Agricultural Sciences. Their nucleotide sequences data of nine genomic regions (p23, p20, p13, p18, p25, p27, POL, HEL and k17) (Fig.-A) are available in the GenBank database with accession numbers FJ998187-FJ998201 and GQ338540-GQ338659. Other nine CTV nucleotide sequences used in our analyses were obtained from the GenBank entries: American isolates T36 (U16304), T30 (AF260651), and SY568 (AF001623); Israel isolate VT (U56902), Spain isolates T385 (Y18420) and T318A (DQ151548); Japanese isolate NuagA (AB046398), Egypt isolate Qaha (AY340974), and Mexico isolate Mexic-ctv (DQ272579). The CTV-analyzed isolates were compared in three groups. Group I was comprised of eleven CTV isolates (CT-W1-CT-W11) from wild type citrus. Group II and group III constitute four Chinese CTV isolates (CT3, CT4, CT9, CT21) and nine CTV isolates (T36, T30, SY568, VT, T385, T318A, NuagA, Qaha, Mexic-ctv) from cultivated citrus, respectively.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (30900977), the Program for Changjiang Scholars and Innovative Research Team in Universities, China (PCSIRT, IRT0976), the Key Project 210111 of Ministry of Education of China, and the Young Scientist Cultivation Program of Jiangxi, China (2010DQ02300).
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expression of the p23 gene of Citrus tristeza virus are citrus specific and do not correlate with the pathogenicity of the virus strain. Molecular Plant-Microbe Interactions, 18, 435-445. Febres V J, Ashoulin L, Mawasi M, Frank A, Bar-Joseph M, Manjunath K L, Lee R F, Niblett C L. 1996. The p27 protein is present at one end of Citrus tristeza virus particles. Phytopathology, 86, 1331-1335. Fullerton S M, Carvalho A B, Clark A G. 2001. Local rates of recombination are positively correlated with GC content in the human genome. Molecular Biology and Evolution, 18, 1139-1142. Ghorbel R, López C, Fagoaga C, Moreno P, Navarro L, Flores R, Peña L. 2001. Transgenic citrus plants expressing the Citrus tristeza virus p23 protein exhibit viral-like symptoms. Molecular Plant Pathology, 2, 27-36. Gowda S, Satyanarayana T, Davis C L, Navas-Castillo J, Albiach-Martí M R, Mawassi M, Valkov N, Bar-Joseph M, Moreno P, Dawson W O. 2000. The p20 gene product of Citrus tristeza virus accumulates in the amorphous inclusion bodies. Virology, 274, 246-254. Hall T A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symposium Series, 41, 95-98. Hardison R C, Roskin K M, Yang S, Diekhans M, Kent W J, Weber R, Elnitski L, Li J, O’Connor M, Kolbe D, Schwartz S, Furey T S, Whelan S, Goldman N, Smit A, Miller W, Chiaromonte F, Haussler D. 2003. Co-variation in frequencies of substitution, deletion, transposition and recombination during eutherian evolution. Genome Resarch, 13, 13-26. Hilf M E, Karasev A V, Albiach-Marti M R, Dawson W O, Garnsey S M. 1999. Two paths of sequence divergence in the Citrus tristeza virus complex. Phytopathology, 89, 336-342. Hilf M E, Karasev A V, Pappu H R, Gumpf D J, Niblett C L, Garnsey S M. 1995. Characterization of Citrus tristeza virus subgenomic RNAs in infected tissue. Virology, 208, 576-582. Hilf M E, Mavrodieva V A, Garnsey S M. 2005. Genetic marker analysis of a global collection of isolates of citrus tristeza virus: Characterization and distribution of CTV genotypes and association with symptoms. Phytopathology, 95, 909-917. Karasev A V, Boyko V P, Gowda S, Nikolaeva O, Hilf M E, Koonin E V, Niblett C L, Cline K, Gumpf D J, Lee R F, Lewandowski D J, Dawson W O. 1995. Complete sequence of the Citrus tristeza virus RNA genome. Virology, 208, 511-520. Kumar S, Nei M, Dudley J, Tamura K. 2008. MEGA: A biologist-centric software for evolutionary analysis of DNA and protein sequences. Briefings in Bioinformatics, 9, 299-306. López C, Ayllón M A, Navas-Castillo J, Guerri J, Moreno P, Flores R. 1998. Molecular variability of the 5´- and 3´-terminal regions of Citrus tristeza virus RNA.
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Phytopathology, 88, 685-691. López C, Navas-Castillo J, Gowda S, Moreno P, Flores R. 2000. The 23-kDa protein coded by the 3´-terminal gene of Citrus tristeza virus is an RNA-binding protein. Virology, 269, 462-470. Lu R, Folimonov A, Shintaku M, Li W X, Falk B W, Dawson W O, Ding S W. 2004. Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome. Proceedings of the National Academy of Sciences of the United States of America, 101, 15742-15747. Martín S, Sambade A, Rubio L, Vives M C, Moya P, Guerri J, Elena S F, Moreno P. 2009. Contribution of recombination and selection to molecular evolution of Citrus tristeza virus. Journal of General Virology, 90, 1527-1538. Mawassi M, Mietkiewska E, Gofman R, Yang G, Bar-Joseph M. 1996. Unusual sequence relationships between two isolates of Citrus tristeza virus. Journal of General Virology, 77, 2359-2364. Melzer M J, Borth W B, Sether D M, Ferreira S, Gonsalves D, Hu J S. 2010. Genetic diversity and evidence for recent modular recombination in Hawaiian Citrus tristeza virus. Virus Genes, 40, 111-118. Roy A, Brlansky R H. 2010. Genome analysis of an orange stem pitting Citrus tristeza virus isolate reveals a novel recombinant genotype. Virus Research, 151, 118-130. Rubio L, Ayllón M A, Kong P, Fernández A, Polek M, Guerri J, Moreno P, Falk B W. 2001. Genetic variation of Citrus tristeza virus isolates from California and Spain: evidence for mixed infections and recombination. Journal of Virology, 75, 8054-8062. Ruiz-Ruiz S, Moreno P, Guerri J, Ambrós S. 2006. The complete nucleotide sequence of a severe stem pitting isolate of Citrus tristeza virus from Spain: Comparison with isolates from different origins. Archives of Virology, 151, 387-398. Ruiz-Ruiz S, Navarro B, Gisel A, Peña L, Navarro L, Moreno P, Serio F D, Flores R. 2011. Citrus tristeza virus infection induces the accumulation of viral small RNAs (21-24-nt) mapping preferentially at the 3´-terminal region of the genomic RNA and affects the host small RNA profile. Plant Molecular Biology, 75, 607-619. Satyanarayana T, Gowda S, Ayllón M A, Albiach-Martí M R, Rabindran S, Dawson W O. 2002. The p23 protein of Citrus tristeza virus controls asymmetrical RNA accumulation. Journal of Virology, 76, 473-483. Satyanarayana T, Gowda S, Mawassi M, Albiach-Martí M R, Ayllón M A, Robertson C, Garnsey S M, Dawson W O. 2000. Closterovirus encoded HSP70 homolog and p61 in addition to both coat proteins function in efficient virion assembly. Virology, 278, 253-265. Sentandreu V, Castro J A, Ayllon M A, Rubio L, Guerri J, Gonzalez-Candelas F, Moreno P, Moya A. 2006. Evolutionary analysis of genetic variation observed in Citrus tristeza virus (CTV) after host passage. Archives of Virology, 151, 875-894. Suastika C, Natsuake T, Hirotsugu T, Kano T, Ieki H, Okuda S.
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Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China
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December 2014
RESEARCH ARTICLE
Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China YI Long1, 2 and ZHOU Chang-yong1 1 2
Citrus Research Institute, Chinese Academy of Agricultural Sciences, Chongqing 400712, P.R.China National Navel Orange Engineering Technology Research Center, Ganzhou 341000, P.R.China
Abstract The genetic variation and phylogenetic relationships of Citrus tristeza virus (CTV) isolates collected from Chinese wild type citrus were analyzed by comparing the sequences of nine genomic regions (p23, p20, p13, p18, p25, p27, POL, HEL and k17) with the CTV isolates of cultivated citrus from different countries. The results showed that the divergence pattern of genomic RNA of the CTV isolates from wild type citrus was similar to that of other isolates from cultivated citrus, the 3´ proximal region was relatively conserved, and the 5´ proximal region had greater variability. The nine genomic regions of CTV isolates analyzed were found to have been under purifying selection in the evolution process. Phylogenetic analysis showed that the eleven Chinese wild CTV isolates were located at different clades and did not reflect their geographical origins, suggesting genetic diversity among the Chinese wild CTV populations. These results will aid in the understanding of molecular evolution of the Chinese CTV populations. Key words: Citrus tristeza virus, wild type citrus, genetic diversity, phylogenetic analysis
INTRODUCTION Citrus tristeza virus (CTV), a member of genus Closterovirus within the family Closteroviridae, is distributed worldwide and also the causal agent of one of the most economically important citrus diseases. The virus is dispersed to new areas by propagation of infected buds and locally transmitted by aphids (Bar-Joseph et al. 1989). CTV virions are filamentous flexuous particles about 2 000 nm long with two capsid proteins of 25 and 27 kDa coating about 95 and 5% of the virion length, respectively (Febres et al. 1996). Which have a single-stranded,
positive-sense genomic RNA (gRNA) approximately 20 kb that is organized in 12 open reading frames (ORFs), potentially encoding at least 19 proteins (Karasev et al. 1995). The two 5´ proximal ORFs, encoding replication-related proteins, are directly translated from gRNA. ORF1a encodes a 349-kDa polyprotein that containing papain-protease, helicase-like, and methyltransferase-like domains. ORF1b encodes a protein with RNA-dependent RNA polymerase (RdRp) domains. The remaining 10 ORFs, which are expressed via 3´-coterminal subgenomic RNAs, encode (from 5´ to 3´) proteins p33, p6, p65, p61, p27, p25, p18, p13, p20, and p23 (Hilf et al. 1995; Karasev et al. 1995). Protein p6 may have transmembrane functions; p65 is an HSP70 homologue, which together with p61 and the two coat proteins p27
Received 22 October, 2013 Accepted 24 January, 2014 YI Long, Tel: +86-797-8393068, E-mail: [email protected]; Correspondence ZHOU Chang-yong, Tel: +86-23-68349037, Fax: +86-23-68349592, E-mail: zhoucy@ cric.cn
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60730-3
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and p25 are involved in virion formation (Satyanarayana et al. 2000); p20 accumulates in amorphous inclusion bodies (Gowda et al. 2000); and p23 is an RNA-binding protein (López et al. 2000) that regulates asymmetrical accumulation of positive and negative strands during RNA replication (Satyanarayana et al. 2002) and is involved in symptom expression (Ghorbel et al. 2001; Fagoaga et al. 2005). Proteins p25, p20 and p23 have been shown to act as RNA silencing suppressors (Lu et al. 2004). The roles of the p33, p18 and p13 are presently unknown, but Tatineni et al. (2008) reported that they may be involved in the ability of CTV to infect citrus trees systemically. Nucleotide sequence analysis is the most accurate procedure for CTV isolates differentiation and estimation of molecular or genetic variation (Rubio et al. 2001). Recent study results suggest that aphid transmission and host passage may significantly alter the composition of populations of CTV isolates and may be an important factor in their evolution (Albiach-Martí et al. 2000; Sentandreu et al. 2006). Negative selection, gene flow, sequence recombination and virulence may be important factors driving CTV evolution (Martín et al. 2009). In a previous work, we collected 79 wild type citrus samples from Yunnan, Guangxi, Sichuan, Hunan and Jiangxi provinces (or autonomous region) of China and found that eleven samples were CTV-positive. We characterized the wild CTV isolates by restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP) analysis of their coat protein (CP) genes and found the CP genes of wild CTV isolates were generally well conserved (Yi et al. 2007, 2010). To gain further insight into the molecular characteristics of CTV isolates evolution from wild type citrus, the genetic variation and phylogenetic relationships in nine gRNA regions of these isolates and the CTV isolates from cultivated citrus were further analyzed.
ResUlTs sequence identity, number of variable sites and nucleotide genetic distance Comparing the sequence identity of nine genomic regions of CTV isolates of wild citrus and cultivated
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citrus, the nucleotide sequences and amino acid sequences of p23, p20, p13, p18, p25 and p27 genomic regions of eleven wild CTV isolates were found to have identity ranging 88.1-100% and 88.2-100%, respectively. Their nucleotide and amino acid sequences identity ranged 87.7-100% and 85.7-100%, with those of group II, respectively. Ranged 88.0-99.7% and 87.4-100% with those of group III, respectively (Table 1). The results of sequence comparisons showed that the nucleotide sequences and amino acid sequences identities around 90% in six genomic regions (p23, p20, p13, p18, p25, p27) at 3´-terminal region of the genome, but only close to 70% in the k17 regions at 5´-terminal region of the genome of wild CTV isolates (Table 1). The number of variable sites of nucleotide sequences and deduced amino acid sequences was also found ranging 13.1-18.9% and 5.7-19.4% in the p23, p20, p13, p18, p25 and p27 genomic regions among three CTV groups, respectively, with 24.5-30.9% and 23.9-31.7% in POL genomic region, 25.1-30.0% and 13.6-20.7% in HEL genomic region, and 35.9-43.3% and 37.5-49.3% in k17 genomic region, respectively (Table 2). When nucleotide genetic distances of nine genomic regions were compared in three groups, we found that the nucleotide distance of k17 (Table 2) was significantly higher than those of regions p23, p20, p13, p18, p25, p27, POL and HEL. The highest nucleotide genetic distance occurred at the k17 region followed by the distance at the HEL and POL regions. In addition, we found that there was a gradual increase in genetic distance towards the 5´ proximal region of the CTV genome.
Base content analysis, transition and transversion, non-synonymous and synonymous substitutions Base content analysis showed that the wild CTV isolates were similar to those of other isolates sourced from cultivated citrus. The GC content of nine genomic regions was less than AU content. Base A content was the highest among the four bases in the p25, p27 and p23 genomic regions, while base U content was the highest in the p20, p13, p18, POL, HEL and k17 genomic regions. Base C content was the lowest in the above nine genomic regions (Table 2). © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
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Table 1 Identity of nucleotide and amino acid sequences (in parenthesis) of three groups of analyzed CTV isolates Genomic regions p23
p20
p13
p18
p25
p27
POL
HEL
k17
Groups Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ
Group I (%) 88.1-99.8 (88.2-100)
Group II (%) 87.7-97.9 (87.2-97.9) 88.6-98.4 (87.2-98.4)
90.3-99.6 (91.8-100)
88.3-99.6 (90.7-100) 89.2-92.3 (91.2-93.9)
91.1-100 (89.9-100)
88.8-98.6 (85.7-98.3) 90.5-97.2 (89.0-97.4)
90.7-99.1 (93.5-100)
89.8-98.7 (89.6-99.3) 90.0-95.4 (92.8-98.0)
92.1-99.5 (94.8-100)
91.3-99.5 (95.2-100) 91.3-94.3 (96.2-98.1)
89.4-100 (94.2-100)
88.1-100 (94.7-100) 87.7-93.7 (96.0-97.7)
83.3-98.6 (82.1-97.9)
81.9-99.2 (82.6-99.4) 83.0-97.1 (84.6-96.9)
77.0-99.7 (86.3-100)
77.2-99.4 (86.0-100) 77.4-96.2 (86.6-98.5)
65.5-99.7 (63.2-100)
66.0-97.3 (62.5-99.2) 67.2-96.3 (64.7-98.5)
Group III (%) 88.0-99.3 (87.7-100) 88.0-98.8 (86.8-100) 89.1-100 (86.8-100) 88.2-99.0 (87.4-100) 88.5-97.9 (88.5-99.4) 87.6-100 (87.4-100) 89.7-99.7 (88.2-99.1) 88.3-96.6 (85.7-98.3) 89.4-100 (87.3-100) 90.7-98.9 (89.6-99.3) 89.0-97.4 (88.3-99.3) 90.7-100 (90.2-100) 91.6-99.0 (94.3-100) 91.1-99.2 (94.3-100) 92.2-100 (93.8-100) 88.1-99.1 (93.3-99.1) 86.3-96.6 (93.3-98.6) 88.1-100 (93.3-100) 80.6-97.8 (81.1-97.9) 80.6-95.1 (81.6-94.3) 80.7-99.5 (82.1-99.4) 75.6-99.6 (83.5-100) 75.8-99.2 (83.2-100) 74.7-99.4 (81.5-100) 64.0-98.2 (56.6-100) 65.2-97.7 (58.8-98.5) 64.3-100 (54.4-100)
Table 2 Base contents, variable site ratios of nucleotide and amino acid sequences, and average nucleotide distance in nine genomic regions of three groups of analyzed CTV isolates Genomic regions p23
p20
p13
p18
p25
p27
POL
HEL
k17
Groups Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ
Base contents (%) U C A G 24.7 19.4 30.2 25.6 24.9 19.5 29.8 25.8 24.9 19.3 30.0 25.9 29.2 21.0 24.7 25.2 29.4 20.5 24.4 25.7 29.3 20.7 24.4 25.6 31.9 18.9 26.2 22.9 32.3 18.7 26.2 22.8 32.1 19.0 25.7 23.2 35.5 17.2 25.4 21.9 34.8 17.6 25.8 21.7 35.5 17.3 25.4 21.8 27.4 18.8 27.9 25.9 27.7 18.4 27.9 26.0 27.7 18.4 27.9 25.9 27.6 19.1 30.7 22.6 27.9 19.0 30.6 22.5 27.5 19.6 30.5 22.4 32.8 18.6 25.5 23.2 32.4 18.8 25.6 23.2 32.8 18.9 25.5 22.9 30.0 18.4 26.6 24.9 30.5 18.0 26.2 25.3 29.8 18.7 26.5 25.1 35.6 17.7 23.1 23.6 36.0 17.3 22.3 24.3 35.8 18.0 22.3 23.8
V-NN (%)1)
V-AA (%)2)
Average nucleotide distance±SE3)
18.9 14.7 17.5 13.9 16.1 18.7 15.3 13.1 17.8 17.9 13.8 15.1 14.8 13.4 13.7 17.9 17.7 17.1 30.9 24.5 28.4 30.0 25.1 27.8 43.3 35.9 41.6
19.4 14.2 16.8 8.8 6.6 19.0 13.4 12.6 16.8 14.3 9.1 13.0 6.1 5.7 9.9 8.8 4.8 10.6 31.7 23.9 27.3 15.9 13.6 20.7 41.9 37.5 49.3
0.0792±0.0077 0.0923±0.0100 0.0800±0.0082 0.0553±0.0066 0.0976±0.0099 0.0794±0.0080 0.0586±0.0080 0.0751±0.0103 0.0796±0.0095 0.0563±0.0066 0.0798±0.0096 0.0614±0.0076 0.0558±0.0058 0.0775±0.0084 0.0608±0.0067 0.0765±0.0070 0.1063±0.0094 0.0458±0.0056 0.1252±0.0092* 0.1589±0.0132* 0.1425±0.0112* 0.1480±0.0101* 0.1920±0.0127* 0.1619±0.0107* 0.2685±0.0242** 0.3172±0.0311** 0.2692±0.0231**
1)
V-NN, variable site ratio of nucleotide sequences. V-AA, variable site ratio of amino acid sequences. 3) SE, standard errors. * and **, statistically different to the rest of values at P<0.05 and P<0.01, respectively. 2)
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More transitions were found in nine genomic regions than transversions and the transitions/transversions ratio of HEL and k17 genomic regions ranged from 2.1 to 2.6 among three groups-analyzed (Table 3). The number of transitions of HEL and k17 genomic regions was more than that of the rest regions, suggesting they were the most variable regions among the analyzed nine genomic regions. A significant difference was found among the rates of non-synonymous mutations (dN) and of synonymous mutations (dS) in the p25, p27, p23, p20, p13, p18, POL and k17 genomic regions in three groups whereas there was no significant difference in the HEL region (P<0.05) (Table 3). According to Martín et al. (2009), the dN/dS values can be placed into four selection categories: i) strong negative selection (dN/dS<0.1); ii) moderate negative (0.1
suggesting that nine genomic regions have been under purifying selection in the evolutionary process.
Phylogenetic relationships between CTV isolates Most CTV isolates shared the same grouping in the phylogenetic trees obtained from nine genomic regions, regardless of their geographical origins. The eleven CTV isolates in Chinese wild citrus were located in different phylogenetic clusters, such as CT-W1, CT-W2, CT-W3, CT-W4 and CT-W5 from Yunnan Province in China, which dropped into different clades and as do the isolates CT-W6 and CT-W7 from Sichuan Province and the isolates CT-W8 and CT-W9 from Hunan Province. At least three clades were observed in most regions by phylogenetic analysis. One clade (clade I) comprising the isolates CT-W3, CT-W5, CT-W10 (except in p27), CT-W11, T318A, NUagA, and SY568 (except in POL and p27) was supported by bootstrap values exceed 50%. A second clade (clade II) comprising of isolates T36, Qaha, Mexico-ctv, and CT3 (except in p25, p13,
Table 3 The numbers of transition (Ts) and transversion (Tv), the rates of non-synonymous (dN) and synonymous (dS) substitutions and their ratios (R) in the nine genomic regions of three groups of analyzed CTV isolates Genomic regions p23
p20
p13
p18
p25
p27
POL
HEL
k17
*
Groups
Ts
Tv
R (Ts/Tv)
dN±SE
dS±SE
dN /dS
Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ
36 42 34 25 42 33 17 23 21 22 30 23 29 40 12 41 60 41 53 64 57 94 115 96 58 67 56
7 8 9 3 7 8 2 2 6 3 4 3 5 6 2 7 6 8 13 12 14 38 54 46 22 27 26
4.9 5.1 3.7 7.5 6.0 4.4 7.1 14.0 3.8 7.5 8.0 7.1 6.3 6.3 8.2 5.8 9.9 5.2 4.2 5.2 4.0 2.5 2.1 2.1 2.6 2.5 2.1
0.040±0.007 0.051±0.010 0.041±0.007 0.017±0.004 0.021±0.006 0.036±0.007 0.031±0.008 0.037±0.009 0.039±0.010 0.018±0.004 0.025±0.007 0.026±0.005 0.012±0.003 0.016±0.005 0.019±0.004 0.016±0.004 0.017±0.006 0.020±0.004 0.062±0.008 0.070±0.011 0.070±0.010 0.046±0.006 0.064±0.009 0.148±0.012* 0.149±0.020 0.174±0.025 0.161±0.020
0.174±0.021 0.190±0.026 0.179±0.021 0.145±0.020 0.291±0.033 0.190±0.023 0.122±0.020 0.156±0.027 0.182±0.027 0.139±0.018 0.192±0.028 0.135±0.021 0.153±0.016 0.215±0.024 0.148±0.017 0.224±0.023 0.318±0.028 0.221±0.022 0.286±0.026 0.355±0.037 0.332±0.035 0.516±0.042 0.698±0.071 0.166±0.020* 0.634±0.090 0.754±0.120 0.621±0.096
0.230 0.268 0.229 0.117 0.072 0.189 0.254 0.237 0.214 0.129 0.130 0.193 0.078 0.074 0.128 0.071 0.053 0.090 0.217 0.197 0.211 0.089 0.092 0.892 0.235 0.231 0.259
, statistical difference of the dN and dS values of the genomic region in the analyzed group at P<0.05.
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Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China
and p23) was supported by bootstrap values greater than 81% within nine genomic regions. The isolates T30 and T385 were clustered into clade III. Their phylogenetic trees have 56% or greater boot-strap support. While isolates defining clades clustered together regardless of the genomic region, other isolates showed incongruent phylogenetic relationships (Fig.-B). The virulent isolate VT was genetically closer to isolates of clade I, the wild CTV isolates CT-W1, CT-W2, CTW4, CT-W6, CT-W7, and CT-W9 were clustered into different phylogenetic clades, separated from the above clades, with incongruent phylogenetic relationships in the nine genomic regions. The severe isolate CT4 of Chinese cultivated citrus was closely related to isolates of clade I in the HEL, POL, p27 and p20 regions. But it was more closely related to clade III in the k17 region. Similarly, inconsistencies in the phylogenetic relationships of different genomic regions were also observed in mild isolates CT9 and CT21 sourced from Chinese cultivated citrus and cluster into clade III without the mild isolates T30 (Albiach-Martí et al. 2000) and T385 (Vives et al. 1999).
DIsCUssION The nucleotide and deduced amino acid sequences of nine genomic regions in eleven wild CTV isolates from China showed that sequence identity exceeded 87% in the 3´ half of the genome, which was higher than that in the 5´ half of the genome. Genomic RNA within the 3´ half was relatively conserved; whereas the sequences of the HEL and k17 regions in the 5´ proximal region of the genome were more dissimilar with higher diversity. The accumulation of small RNAs of CTV mapping preferentially at the 3´-terminal 2 500-nt of the RNA genome (Ruiz-Ruiz et al. 2011), which most likely results from reduction sequence divergence in the 3´-termianl region of the CTV genomic RNA. The genetic distance of the nine genomic regions were compared with each other and there was a gradual increase in genetic distance from 3´ to the 5´ terminus of the CTV genome. Although no complete gRNA sequence of wild CTV isolate is currently available, our findings suggest the 3´ terminus was relatively conserved and sequence differences were greater in the 5´ terminus, which indicate the genomic RNA of wild CTV isolates have the
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similar patterns of nucleotide variability as those derived from cultivated citrus (Mawassi et al. 1996; López et al. 1998; Vives et al. 1999; Yang et al. 1999; Albiach-Martí et al. 2000; Suastika et al. 2001; Ruiz-Ruiz et al. 2006). GC content is an explanatory variable in the sense that it allows one to predict a certain portion of the variability of each divergence measure (Hardison et al. 2003). The divergence decreases with GC content for low-GC DNA and increases with GC content for higher-GC DNA, consistent with important differences in the patterns of evolution in these two classes of genomic DNA (Bernardi 1995; Fullerton et al. 2001; Castresana 2002). In this study, the GC content was also found to be less than the AU content within nine genomic regions, the dN values were much lower than dS values (dN /dS<1). Analysis of selective pressure acting on different regions suggests that all regions examined are subjected to purifying selection. This pressure was moderate in p23, p20, p13, p18, p25 and p27 regions, but weak in k17 in the 5´ half of the genome. While 3´ half of the genome was more conserved than the 5´ half, some of the variants produced by mutation should be more easily eliminated in the 3´ half of the CTV genome by purifying selection in the evolutionary process. In this study, the wild type citrus samples were collected from high altitudes (>1 100 m), and which were estimated to have been living for over 50 years (some for 200 years or more). From the phylogenetic analysis, eleven CTV isolates from wild type citrus were located in different clades regardless of their geographical origin. Some isolates showed incongruent phylogenetic relationships in different genomic regions, which provide further support to previous inference that the temporal separation of CTV isolates might have occurred several hundred years in the past (Albiach-Martí et al. 2000). CTV isolate sequences could be assigned to three types: T36 genotype, T30 genotype, and VT genotype (López et al. 1998; Hilf et al. 1999; Ayllón et al. 2001), and the same genotype cluster into one clade (Hilf et al. 2005). The genotypes of eleven wild CTV isolates were not clear; however, their segregating into different phylogenetic clusters indicated that they had different genotypes. This demonstrated genetic diversity among the CTV populations from wild citrus plants was similar to those from cultivated citrus (Rubio et al. 2001; Sentandreu et al. 2006; Melzer et al. 2010; Roy and Brlansky 2010). © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
YI Long et al.
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A
PRO PRO
k17
1b HEL
2
3
p33
4
5
p65
6
7
p27
8
9
p18
10 11 p20
CTV 5´
3´ POL
p6
p16
p25
p13
p23
B
Fig. A, schematic of the genomic structure of the Citrus tristeza virus (CTV) isolate T36, open reading frames (ORFs) are represented by open rectangles with indication of the encoded protein and are numbered in ascending order from the 5´ to the 3´ terminus in the manner of Karasev et al. (1995). Layout of the CTV genome with the regions analyzed indicated as black boxes. B, unrooted nucleotide maximum parsimony phylogenetic trees of CTV genomic regions k17, HEL, POL, p27, p25, p18, p13, p20 and p23. Scale bars indicate number of changes per position for unit branch length. Bootstrap values for significance of nodes are indicated by asterisks (***, 90-100%; **, 70-89%; *, 50-69%).
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China
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CONClUsION
sequence alignment and statistical analysis
In summary, analyses of sequence identity, genetic variation and phylogenetic relationships of the CTV isolates from wild and cultivated citrus indicated that the genome of wild CTV isolates have the same sequence divergence as that of cultivated citrus, and the genetic diversity among the CTV populations from wild type citrus in China, which will aid in the understanding of molecular evolution of the Chinese CTV populations.
Nucleotide sequences were translated to proteins using BioEdit 7.0.1 (Hall 1999) and the multiple alignments were performed with the program CLUSTALW (Thompson et al. 1994). The MEGA ver. 4.0 program was used to estimate the base content and calculate the average nucleotide genetic distances of CTV isolates by Kimura two-parameter model. After testing homogeneity of pattern substitution among lineages, the numbers of transitions (Ts) and transversions (Tv) and the Ts/Tv ratio, the rate of non-synonymous (dN), and synonymous (dS) substitutions were determined by the Nei & Gojobori methods (Tamura et al. 2007; Kumar et al. 2008). Phylogenetic relationships were inferred based on the maximum parsimony method, and reliability of the clades was evaluated by bootstrap analysis based on 1 000 repetitions using the MEGA 4.0 program.
MATeRIAls AND MeThODs Nucleotide sequences of CTV isolates The wild CTV isolates CT-W1, CT-W2 and CT-W3 were collected from wild Poncirus polyandra in Fumin County, CT-W4 from wild Citrus medica in Yangbi County, CT-W5 from wild hybridism of C. grandis and C. medica in Mengla County, Yunnan Province; CT-W6 and CT-W7 from wild C. ichangensis in Linshui County, Sichuan Province; CT-W8, CT-W9 from wild C. ichangensis and C. aurantium, respectively in Yizhang County, Hunan Province; CT-W10 from wild C. reticulata in Chongyi County, Jiangxi Province; CTW11 from wild C. reticulata in Hezhou County, Guangxi Autonomous Region of China (Yi et al. 2007). The severe isolate CT3 and the mild isolate CT9 from cultivated pummelos, and the severe isolate CT4 and the mild isolate CT21 from cultivated sweet oranges were kindly provided by the Citrus Research Institute, Chinese Academy of Agricultural Sciences. Their nucleotide sequences data of nine genomic regions (p23, p20, p13, p18, p25, p27, POL, HEL and k17) (Fig.-A) are available in the GenBank database with accession numbers FJ998187-FJ998201 and GQ338540-GQ338659. Other nine CTV nucleotide sequences used in our analyses were obtained from the GenBank entries: American isolates T36 (U16304), T30 (AF260651), and SY568 (AF001623); Israel isolate VT (U56902), Spain isolates T385 (Y18420) and T318A (DQ151548); Japanese isolate NuagA (AB046398), Egypt isolate Qaha (AY340974), and Mexico isolate Mexic-ctv (DQ272579). The CTV-analyzed isolates were compared in three groups. Group I was comprised of eleven CTV isolates (CT-W1-CT-W11) from wild type citrus. Group II and group III constitute four Chinese CTV isolates (CT3, CT4, CT9, CT21) and nine CTV isolates (T36, T30, SY568, VT, T385, T318A, NuagA, Qaha, Mexic-ctv) from cultivated citrus, respectively.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (30900977), the Program for Changjiang Scholars and Innovative Research Team in Universities, China (PCSIRT, IRT0976), the Key Project 210111 of Ministry of Education of China, and the Young Scientist Cultivation Program of Jiangxi, China (2010DQ02300).
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