Advance In Small RNA Deep Sequencing Technology For Identification Virus Disease From Hollyhock

Hollyhock[Althaea rosea（Linn.）]is a flower planted in the world widely with good ornamental value,its resistibility is strong but hollyhock virus diseases cause a serious impact on the growth of hollyhock.Identification of viral pathogens species and mutation evolution is the basis work of prevention and cure hollyhock virus disease.The high throughput sequencing technology detects plant virus and breaks through the traditional methods,it enables to detection the known and unknown viruses.In this study,base on small RNA high-throughput sequencing technology identify the virus disease of hollyhock leaves showed the symptoms of mosaic and vein cleaning.The contigs were obtained by the splicing of small RNAs.Three kinds of viruses were identified by comparison analysis:Mala vein cleaning virus（MVCV）,Watermelon mosaic virus（WMV）and a novel RNA virus tentatively named Althaea rosea virus 1（ArV1）.MVCV is one of the members of the genus potyviridae.The virus has been discovered for a long time,but there is no full-sequence report yet.Using 7 pairs of primers and RACE technology to obtain a full genome of 9530 nt.It has a 205nt length 5’-UTR and a 290nt length 3’-UTR,encoding a single open reading frame（ORF）of 3010 amino acid（aa）.The nucleotide homology analysis showed that high similarity between MVCV and Plum pox virus was 61.1%,the low similarity between MVCV and Sweetpotato mild mottle virus was 41.2%.Nucleotide phylogenetic analysis revealed that MVCV is clustered with Plum pox virus,Sweet potato virus G,and Sweet potato virus 2,kinship nearly.The amino acid homology analysis showed that the consistency of MVCV and Plum pox virus was as high as 60.4%,the low consistency between MVCV and Sweetpotato mild mottle virus was 26.3%.Amino acid evolution analysis showed that MVCV clustered with Euphorbia ringspot virus,Tobacco vein mottling virus,Yam mosaic virus,Pokeweed mosaic virus and Potato virus A,the more nearly kinship is Euphorbia ringspot virus.Comparing the entire MVCV genome with 2651 contigs,a total of 97 contigs were found to be relatively evenly distributed on the MVCV genome.By analyzing the polarity of the results of MVCV-derived vsiRNAs,the sense chain was 51%,and the negative sense chain was 49%,the proportion was nearly close to 1:1.The 5’ base preference A and U are greater than G and C.The hotspots of the siRNAs appear at 1500 nt-1800 nt and 5100 nt-5400 nt,especially in the5100 nt-5400 region,the total number of siRNAs reaches 50000,and most of them are from the sense strand.ArV1 genome of 15516nt is a single-stranded RNA virus,it has genomic characteristics of the genus Closteroviridae,containing 9 open reading frames（ORFs）,which are encoded to encode 1a,1b,p7,HSP70h,CPh,CPm,CP,p20,and p21,respectively.The nucleotide homology analysis showed that the highest similarity to Tobacco virus 1 was 69.1%,followed by Mint virus 1 similarity 61.8%.Systematic evolution was clustered with Tobacco virus 1 and Mint virus 1.The HSP70h amino acid sequence homology analysis between members of the Closteroviridae family and ArV1 virus showed that the lowest similarity was 11.3%for Grapevine leafroll-associated virus 4,and the highest was 80.4%for Tobacco virus 1.Which was associated with the strain of Closterovirus,the homology was 32.1%-80.4%;the HSP70h amino acid phylogenetic analysis yielded the same results as the nucleotides phylogenetic.The ArV1 whole genome was compared with 2651 contigs and a total of 195 contigs were found to match the ArV1 genome.The sense chain of sRNA accounted for 59.28%and the negative sense chain accounted for 40.72%.The proportion of siRNAs derived from the positive and negative strands approaches 1:1,indicating that the dsRNA intermediates generated during ArV1 replication may be the major source of small RNA production.The base bias of small RNAs of 18-24 nt in length is different,and the 5’-terminal base of siRNA is mainly "A".The hotspots are mainly concentrated in three regions.especially 14500 nt-14800 nt,producing13 5,00 small RNAs.Watermelon mosaic virus is also a member of the genus potyviridae.It was amplified from hollyhock to a total length of 10026 nt.Nucleotide homology analysis showed 80.3%-90.2%homology with the WMV-Tg and other WMV genomes at the nucleotide level,and the highest similarity to the Korean ginseng isolate WMV-Pg was 90.2%.The lowest similarity of WMV-Buan2₂014 with Korean watermelon was 83.3%.It can be seen from the phylogenetic relationship that WMV-Tg forms a separate branch with WMV-Pg.The consistency of amino acids was between 80.3%and 95.8%,and the identity with Korean ginseng isolate WMV-Pg was up to 95.8%.The phylogenetic relationship was closest to WMV-Pg.The 87 contigs of the 2561 contigs can be matched with the WMV-Tg full sequence.The 18-24 nt visRNA numbers were 1584,2345,7491,76619,43437,1876 and 167 respectively.The proportion of polar distribution of WMV-Tg-derived sRNA is closer to 1:1.The small RNA 5’ base preference has a characteristic U/A ratio that is highest.This is consistent with the preference of miRNA 5’ bases in plants,and they all show a certain preference for A/U base.The small RNA-specific sequences of WMV accumulate particularly in the range of 7000-9000nt,but few in other regions.