Detection Of Viruses In Cherry And Analysis Of Genome Sequences Of Cherry Virus A

Cherry is one of the important fruit trees in China. Cherry virus diseases are important factors that affect the quantity and quality of cherry fruits. Among cherry viruses, infection rate of Cherry virus A(CVA) in China and other countries was found to be high; however, researches of CVA is currently only limited to detection and detection methods, and few studies on CVA population genetics were reported. Here, firstly, 12 cherry viruses were investigated and detected in four main cultivated areas of cherry in China, so as to provide more comprehensive basis for distribution and identification investigations of pathogens. Secondly, we determined the complete nucleotide sequences of CVA Chinese isolates and analyzed the population genetics of CVA to study the genetic variation mechanism of CVA Chinese isolates and provided theoretical basis for preventing prevalence of CVA.1. Virus diseases were investigated in cultivated areas in Beijing, Dalian in Liaoning province, Tai’an and Yantai in Shandong province. Symptoms mainly included roll leaf, mosaic, yellow and green mottle, plant dwarf etc. 60 leaf samples were collected from these areas. RT-PCR and sequence analysis for the detection of 12 viruses infecting cherry showed that infection rates of CVA, Prunus necrotic ringspot virus(PNRSV), Cherry green ring mottle virus(CGRMV), Apple chlorotic leaf spot virus(ACLSV), Little cherry virus-1(LChV-1), Prune dwarf virus(PDV), Plum bark necrosis stem pitting-associated virus(PBNSPaV) were 70%, 53.3%, 45%, 21.7%, 35%, 5%, 3.3%, respectively. This is the first report of LChV-1 in cherry in China.2. We determined complete nucleotide sequences of two CVA isolates(named ChTA11 and ChTA12) from Tai’an, Shandong province using high fidelity PCR enzymes and specific primers for amplifying long fragments in RT-PCR and RACE. Full length sequences of ChTA11 and ChTA12 isolates are both 7382 nts, excluding the poly(A) tail, encode two open reading frames(ORFs) and have similar genome structures with the other two isolates from Germany and India in GenBank. The complete nucleotide sequence of ChTA11 is 98.2% and 81.2% nt identity to German and Indian isolates, respectively, and the ChTA12 isolate is 98.2% and 81.0% similar with the two isolates. Nucleotide and amino acid sequence analysis showed that the domain of unknown function(DUF1717) is more variable compared with other domains. Phylogenetic analysis based on complete genomic sequences and partial CP gene sequences of CVA showed that the two CVA isolates in this study are more closely related to the German isolate than the Indian isolate. This is the first report of complete nucleotide sequences of CVA isolates infecting sweet cherry in China.3. Genetics parameters based on CP, RdRp and MP of thirty-one CVA Chinese isolates were analyzed using analysis methods of population genetics. Our results showed that: 1) Putative recombination events were found in the CP, RdRp and MP genes of CVA. We found four recombinants; 2) Phylogenetic analysis based on CP and RdRp genes revealed that CVA isolates were divided into three phylo-groups, suggesting that the genetic evolutionary history of CP and MP genes is similar; however, phylogenetic analysis based on MP gene showed that CVA isolates were divided into six phylo-groups. The difference may be due to different evolution levels of different ORFs in the genome structure in which CP and RdRp were encoded by ORF1 and MP was encoded by ORF2. Most of CVA Chinese isolates were clustered into group I with German isolates; 3) Selection pressure analysis of CVA based on the ratio of non-synonymous(dN) to synonymous(dS) revealed that all groups in its CP, RdRp and MP genes were identified to be under negative selection; 4) Analyses of nucleotide / haplotype diversities and neutrality test showed that CVA Chinese isolates had high genetic diversity and the CVA Chinese population in the group I was in a expansion; 5) Gene flow and genetic differentiation analysis showed that gene flow between different groups was less and there were some genetic differences between them. However, gene flow between different geographic groups was frequent.