Genetic and evolutionary analysis of Citrus chlorotic dwarf-associated virus infecting Ruby green pomelo in Guangxi

【Objective】To clarify the pathogen of the Ruby green pomelo showing symptoms of leaf deformation, curling, shrinking, chlorotic and dwarfing, and provide a theoretical basis for effective prevention and treatment of the disease.【Method】Several leaf samples were collected from suspected Ruby green pomelo in some cities of Guangxi, and two pairs of specific primers of Citrus chlorotic dwarf-associated virus(CCDaV) were designed and used for detected the suspected samples, the full-length of genome sequence were obtained by segmented amplification and gene cloning. RDP5 and MEGA 7.0 softwares were applied for analyze the recombination and the genetic diversity of the full length genome.【Result】PCR with expected size of 642 bp were obtained from all of the collected Ruby green pomelo samples, which confirmed that all of the samples were infected by CCDaV. The full-length genome sequences of seven locations representing isolates LA-1, LA-2, WM-1, GG-1, GG-2, WZ-1 and WZ-2 were obtained by using segmented amplification and gene cloning, and the full-length sequences of the seven isolates were 3641, 3642, 3642, 3640, 3642, 3644 and 3644 bp, respectively. The nucleotide sequence identities between the seven isolates were over 98%, and the identities against other CCDaV isolates available in GenBank were over 99%, this results showed that the seven isolates obtained in this research were CCDaV isolates. Recombination analysis showed that no recombination occurred in the seven isolates. Phylogenetic analysis showed that two CCDaV Guangxi isolates(WZ-1 and WZ-2) clustered with Tha1-19 and Tha30 in a sub-clade, indicating their close relation. The other isolates of Guangxi were distributed in different sub-clade, indicating that there were evolutionary diversity among the CCDaV Guangxi isolates.【Conclusion】CCDaV is the pathogen that infects the Ruby green pomeloshowing symptoms of V shape, shrinking, curling, chlorotic and dwarfing, and there is regional diversity of the isolates among different regions.