Identification Of Novel Genotype Of Citrus Tristeza Virus Based On Deep Sequencing

Tristeza caused by Citrus tristeza virus(CTV)is an economically important citrus disease,which has caused the death of at least 100 million citrus plants.In the field,CTV mainly transmitted by grafting and aphid causes,huge economic losses due to rapid death and stem-pitting resulting in small fruits and low yield.CTV has complex characteristics of strains differentiation and genetic diversity.Traditional detection methods are limited in genomic characteristics of different CTV isolates and make it difficult to obtain the complete genome sequence.Transcriptome sequencing technology overcomes these obstacles and can be used for detection and identification of new CTV strains and other unknown viruses.In this study,identification and verification of Citrus trifoliata isolates was carried out by high-throughput sequencing and RT-PCR,respectively.Complete sequences,genotypes and genetic evolution relationship of the CTV isolates from C.trifoliata were expected to obtain by bioinformatically analyzing the available data.This may lay a foundation for the further study on the basis of CTV complete genome sequences.The results are as follows:1.Transcriptome sequencing results: The reads(>200 nt)obtained by transcriptome sequencing were aligned and assembled after removing the reads from the host.The results showed that the contigs related to virus had higher similarity with two CTV isolates only.The similarity between contig14 and SY568(AY001623)was 87%,and that between contig15 and T36(EU937521)was 89% by BLAXTx,suggesting that the trifoliate plant was co-infected by two CTV isolates.2.PCR verification: The whole genome sequences of the two CTV isolates(CN-L1-ZT1 and CN-M1-ZT1)were obtained by RT-PCR and RACE-PCR.The full genome length of CN-L1-ZT1 and CN-M1-ZT1 was 19244 nt and 19265 nt,respectively.3.Similarity comparison: The similarity of full-lentgh genome between CN-L1-ZT1 and CN-M1-ZT1 was 81.8%.Sequence comparison showed that the similarity between CN-L1-ZT1 and SY568(AF001623)was 87.3%,the highest at the whole genome level,and similarity between CN-L1-ZT1 and other CTV genotypes ranged from 79.0% to 86.9%.The similarity between CN-M1-ZT1 and Florida T36 genotype was 89.1%,the highest,and similarity between CN-M1-ZT1 and other CTV genotypes wasranged from 79.3% and 88.9%.4.Phylogenetic analysis: The phylogenetic analysis of complete genomes of available CTV isolates by MP and NN showed that CN-L1-ZT1 and CN-M1-ZT1 were distinct from the other CTV genotypes and existed alone.MP analysis showed that CN-L1-ZT1 was clustered into a cluster,which was different from the extant CTV genotypes and was close to T30 and A18.CN-M1-ZT1 was a single sub-cluster near T36 genotype.NN analysis showed that CN-L1-ZT1 and CN-M1-ZT1 were significantly different from other sequence groups,not clustered with reported CTV genotypes,but a sequence alone.The phylogenetic tree of CP and RdRp isolates with available CTV genotypes was constructed by NJ.The results showed that in the phylogenetic tree of CP gene,CN-L1-ZT1 and CN-M1-ZT1 did not cluster into other reported CTV genotypes,but a new cluster,suggesting they werenew genotypes.In the evolutionary tree of RdRp nucleotide sequences,CN-L1-ZT1 was clustered into one cluster,which was different from the other CTV genotypes,close to T3 and S1,and CN-M1-ZT1 was a sub-cluster close to T36 genotype,which was similar to the outcome by the whole genome phylogenetic analysis.5.Recombination analysis: 24 significant recombination events were detected by genome-wide recombination analysis of 2 CTV isolates in this study and 60 CTV isolates reported in NCBI.CN-L1-ZT1 had a recombination sequencebetween 10700 and 19165 nt from VT(EU937519)and an unknown sequence as the major and minor parents,respectively,which all the algorithms supported.The recombinant site of CN-M1-ZT1 was 10793-19238 nt from FS674-T36(KC517485)and another unknown sequence as major and minor parents,respectively,which all the algorithms supported.6.Genotype classification: According to the definition of CTV genotype classification,when the similarity of whole genome is less than 92.5%,the similarity of ORF1 a nucleotide and amino acid sequence is less than 92%,and recombination between different genotypes occurs,it can be considered as a novel genotype.The two CTV isolates obtained in this study met the above classification criteria.Therefore,CN-L1-ZT1 and CN-M1-ZT1 may be two novel CTV genotypes named L1 and M1 genotypes,respectively.7.Establishment of detection system: According to the specific nucleotides sequences in P33 region,primers were designed to identify L1 and M1 genotypes.The detection system of these two novel CTV genotypes was established by RT-PCR.8.Field occurrence: Through field investigation,citrus samples from Chongqing,Sichuan,Jiangxi,Guangdong,Guangxi,Hunan,Hubei,Zhejiang and Guizhou were found to have high CTV infection rate(85.1%),but the detection rate varied from different areas.Population analysis of positive samples showed that almost all of the samples were infected by a mixture of several CTV genotypes.These CTV isolates contained all reported CTV genotypes,which indicated that the Chinese isolates had complex population structure.The L1 and M1 genotypes in this study were detected only in 16 isolates from Chongqing.The reason may result from less amount of samples taken in other areas or the range of aphid activity.