Population Composition And Molecular Diversity Of Pear Viruses Revealed By High Throughput Sequencing Technology

Pear(Pyrus spp.)has a long cultivation history in China.Several viruses infecting pear trees have been reported in China.Apple stem grooving virus(ASGV),apple stem pitting virus(ASPV)and apple chlorotic leaf spot virus(ACLSV)are the most common viruses infecting pear trees.These viruses often mixed-infect pear trees and cause growth decline of some susceptible pear trees.These three viruses are members in the family Betaflexiviridae.Previously,the molecular variation characteristics of these viruses have been analyzed basing on single gene or genome sequences amplified by RT-PCR.However,the population compositions of these viruses in single pear tree are rarely known.In this study,the variation characteristics of these three virus populations were revealed by using high-throughput sequencing combined with informatics analysis.The results enriched the molecular information of these viruses and provided useful reference for understanding their pathogenic mechanism.The main results are as follows:1.Sixteen pear samples were analyzed by RNA-seq.Blast N search against NCBI databases using contig sequences derived from RNA-seq samples identified seven plant viruses,including ASGV,ASPV,ACLSV,apple rubbery wood virus 2(ARWV2),citrus virus A(Ci VA),pear chlorotic leaf spot-associated virus(PCLSa V)and Pyrus pyrifolia cryptic virus(Pp CV).ASPV was detected in all RNA-seq samples,and 14 samples were co-infected with two to five viruses,with a mixed infection rate of 87.5%.Further analyzes showed that ASPV had was relatively complicated population constitution,with relatively more contigs matched to the viral genome and multiple haplotypes in single sample.The contigs matched to ASGV and ACLSV had relatively simple population structures with were relatively less contigs matched to their genomes compared with ASPV.The infections of the three viruses in RNA-seq samples,including 16 RNA-seq samples and 16 independent samples composed of mixed RNA-seq samples,confirmed by RT-PCR using specific primers was detected in totally,26,27 and 14 samples were positive for ASGV,ASPV and ACLSV,respectively.2.By using primers designed based on RNA-seq sequences,10,3 and 2 genomic sequences of ASGV,ASPV and ACLSV were amplified from high-throughput sequencing samples,respectively.Phylogenetic analysis for 19,32 and 6 genome sequences of the three viruses obtained by RNA-seq and RT-PCR amplification showed that the genome sequences of each virus obtained from the same pear sample had long genetic distances and distributed in different phylogenetic branches.Nucleotide diversity(Pi)analysis revealed relatively high variation occurring in variable region V1 of ASGV,at sites2000-2100 nt and 2700-2800 nt in ORF1 and coding sequence for CP C-terminus of ASPV,and near 2000 nt of ACLSV ORF1.Analyses of genetic parameters showed that two ASPV populations from hosts pear and apple had significant genetic differentiation and frequent gene drift,and the evolution of ASPV was mainly regulated by negative selection pressure.