Population characteristics analysis of citrus tristeza virus isolates from wild citrus in southern Hunan and Jiangxi based on two conserved genes

Abstract:【Objective】The experiment aimed to clarify the population characteristics of Citrus tristeza virus (CTV) in wild citrus, and to provide an important basis for analyzing the genetic evolution of CTV population.【Methods】With 10 positive CTV samples from four different geographic regions of Chongyi (Jiangxi), Daoxian (Hunan), Mangshan (Hunan) and Jiangyong (Hunan), the CP gene and p23 gene amplification were carried out. The samples were named Chongyi 1-10 (C-1 to C-10), Daoxian 1-10 (D-1 to D-10), Mangshan 1-10 (M-1 to M-10), and Jiangyong 1-10 (Y-1 to Y-10). The samples were preserved at - 40 ℃ for later assay. After purification and recovery, the PCR products were sent to Shanghai Bioengineering Company for paired and sequencing. MEGA-X was used to calculate the con-servative site, abbreviated information site, mutation site, descent locus and the ratio of the base transi- tion rate Ts to the transversion rate Tv. The software Arlequin 3.1 was used for the analysis of molecular variance (AMOVA), and GenAIEx 6.5 was used for gene flow Nm calculation. DnaSP was used to per-form three neutrality tests and mismatch analysis to infer population expansion and genetic evolution. RDP 5 was used to perform recombination event analysis. In order to clarify the genetic relationship  among the CTV isolates of wild citrus from different regions of the same gene, this study combined the 40 CP gene sequences and p23 gene sequences obtained by sequencing and the CP gene sequences and p23 genes of eight CTV isolates downloaded from GenBank and built phylogenetic trees. In the pro-cess, it used maximum likelihood (ML) and the Kimura 2- parameter model respectively, and each branch node confidence in the phylogenetic tree underwent repetitive verification for 1000 times based on bootstrap. The isolates data downloaded from GenBank included those of US isolates T36 (U16034) and T30 (AF260651), Spanish isolates T318A (DQ151548) and T385 (Y18420), Israeli isolates VT (U56902), Egyptian isolates Qaha (AY340974), the Mexican isolate Mexico-ctv (DQ272579) and the Japanese isolate NuagA (AB046398). Through Network 10.0 and the Median-joining method, we con-structed the haplotype network based on CP gene and p23 gene respectively, and analyzed the evolution-ary relationship among those isolates.【Results】The base composition analysis showed that the CP genes base A, base T, base C, and base G nucleotide base contents of CTV isolates from wild citrus in four regions were 28.32%, 26.99%, 18.49% and 26.20%, respectively. The ratio of transition to transver- sion was 5.049, with 555 conservative sites, 80 abbreviated information sites, 117 mutation sites, and 37 descent loci. The nucleotide content of base A, base T, base C, and base G in p23 gene were 30.48%,24.74%, 19.60%, and 25.18%, respectively. The ratio of transition to transversion was 4.415, of which the transition was higher than the transversion, with 485 conservative sites, 97 abbreviated information sites, 145 mutation sites, 48 descent loci, and AT base content was higher than that of GC. According to the AMOVA results of the CP gene and p23 gene, it showed that the molecular variation within the pop-ulation accounted for 85.8% and 82.38%, and the molecular variation between the populations account-ed for 14.20% and 17.62%, respectively, which indicated that the genetic variation mainly came within the population. The gene flow Nm was 6.749 and 1.475, respectively, indicating that there was a high level of gene exchange among the four regions. The result of the neutrality test showed that the CTV population may remain in a stable state or experienced population expansion, but the P value was not significant. Through base mismatches, it was further found that the two genes both formed the multi-modal distribution curve, indicating that the CTV population from four regions remained stable. Through recombination analysis, it showed that there was no recombination event detected for both genes. The phylogenetic trees constructed based on the two genes showed that most of the CTV popula-tion of the same geographic origin were aggregated in the same cluster, and a few were scattered in dif-ferent clusters. For example, in the phylogenetic tree constructed based on p23 gene, the first group in-cluded 8 Chongyi isolates; the second group included 5 Daoxian isolates, 8 Jiangyong isolates and 2 Mangshan isolates; the third group included 2 Jiangyong isolates, 5 Daoxian isolates, 7 Mangshan iso-lates, 2 Chongyi isolates and VT, NuagA, T318A, T385 isolates; the fourth group included 1 Mangshan isolate and Qaha, T36, Mexico-ctv isolates. It indicated that the genetic relationship among CTV popula-tions on wild citrus from different geographic sources was related to geographic distance, and the haplo-type network structure yielded similar analyzing results.【Conclusion】The genetic variation among CTV populations on wild citrus in the four regions mainly came within the population. Although gene exchanges were frequent among regions, genetic differences were small, and CTV populations re-mained stable. Moreover, phylogenetic analysis and haplotype network structure showed that the genet-ic relationship among CTV populations on wild citrus from different geographical sources was correlat-ed with geographical distance.