Genetic characterization between the Citrus tristeza virus isolates in wild and cultivated citrus on the basis of p23 gene sequences

YI Long;ZHOU Changyong;College of Life and Environmental Sciences, Gannan Normal University;Citrus Research Institute, Chinese Academy of Agricultural Sciences/Southwest University;

Wild citrus;;Cultivated citrus;;Citrus tristeza virus;;p23 gene;;Genetic characterization

【Objective】This study focused on comparative analysis of the genetic evolution characteristics of the Citrus tristeza virus(CTV) isolates between wild citrus and cultivated citrus, which provided the theoretical basis for further study on the genetic evolution of CTV.【Methods】A total of 11 CTV isolates in this study were collected from naturally wild citrus in Yunnan(isolates CT-W1, CT-W2, CT-W3, CTW4, CT-W5), Sichuan(isolates CT-W6, CT-W7), Hunan(isolates CT-W8, CT-W9), Jiangxi(isolate CTW10), Guangxi(isolate CT-W11) provinces of China. The severe isolate CT3 and the mild isolate CT9 from cultivated pummelos, together with 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 as being representative of CTV isolates from cultivated citrus in China. The nucleic acids of the samples with 11 CTV isolates from wild citrus and 4 CTV isolates from cultivated citrus in China were each extracted. The primer pairs of p23 f and p23 r were used as a specific primer for amplifying the p23 gene, the sense primer was p23f:5'-ATGGATAATACTAGCGGACA-3',and the anti-sense primer was p23r:5'-TCAGATGAAGTGGTGTTCAC-3'. The c DNA of CTV was amplified and performed in a 25 μLreaction volume. PCR products were separated by electrophoresis in a 1.2% agarose gel and detected by ethidium bromide staining, and the amplified products of approximately 630 bp for the complete p23 gene were gel purified and the nucleotide sequences were determined in both directions by means of an ABI Prism DNA sequencer 377(Sangon Biological Engineering & Technology and Service, Shanghai, China).Other CTV nucleotide sequences used in this study were obtained from the indicated Gen Bank entries:American isolates T36, T30 and SY568, Israel isolate VT, Spain isolates T385 and T318 A, Japanese isolate Nuag A, Egypt isolate Qaha, Mexico isolate Mexic-ctv. Multiple alignments of the nucleotide sequences were realized using the Bio Edit program. The version 6.0 of the MEGA program was used to estimate the base content, the transition and transversion, and their ratio(Ts/Tv), the rates of non-synonymous(dN)and synonymous(dS) substitutions and their ratios were determined by the Nei & Gojobori methods, the statistic for differentiation using the Z-test(P<0.05). Based on the neighbor-joining method, phylogenetic relationships were inferred taking the homologous sequences of Beet yellow virus(BYV) as the outgroup, and the reliability of the clusters was evaluated by using the bootstrap analysis base on 1 000 repetitions.【Results】The sequences of p23 genes of the 11 wild CTV isolates were found having a similarity ranging from88.1 to 99.8%, the identity of the sequence ranged from 87.7 to 97.9% and 88.0 to 99.3% when compared with 4 Chinese CTV isolates of cultivated citrus and 9 exotic cultivated citrus, respectively. In this investigation, the GC base contents of the p23 gene were less than the AT base contents, the base A content was the highest and the base C was the lowest among the four bases in the p23 genes in the 11 CTV isolates from wild type citrus, which was the same as that from cultivated citrus. More transitions were found in the p23 genes than transversions with the transitions/transversions ratio ranging from 3.4 to 5.3 among the species; the substitution frequency was the highest at the third codon, followed by the first and the second codon. The significant difference was found among the rates of non-synonymous mutations(dN) and of synonymous mutations(dS) in the p23 genes(P=0.000<0.05). The ratio of non-synonymous(dN) to synonymous(dS)substitutions is far lower than 1, suggesting that the p23 gene might have experienced a purifying selection in the CTV evolution process. Phylogenetic analysis revealed that the 11 CTV isolates from wild citrus belonged to different phylogenetic clusters regardless of their geographic origins, and shared higher homology and lower genetic distance with other CTV isolates of cultivated citrus from different geographic origins, for example, isolates CT-W1, CT-W2, CT-W3, CT-W4, CT-W5 were from Yunnan province in China, but dropped into different clusters, the same as the isolates CT-W6, CT-W7 from Sichuan province and the isolates CT-W8, CT-W9 from Hunan province.【Conclusion】Comparative analysis found that the genetic characteristics of the base content, transition and tranversion, non-synonymous and synonymous substitutions of CTV isolates from wild citrus were similar to those of CTV isolates from cultivated citrus. Phylogenetic analysis showed that there was no significant correlation between the geographical origins of Citrus tristeza virus isolates from wild citrus and its clustering relationship of p23 gene sequences.