Exploring The Viral Diversity Associated With Wild Rice Plants By High-Throughput Next-Generation Sequencing

The core objectives of this research work have been to use Next-generation sequencing and bio informatics tools to comprehensively analyze the viral diversity associated with wild rice plants.Rice is one of the most widely produced crops worldwide.The production of rice is ranked second after corn,mainly in Asian countries,most of South and Central America,and in some African countries.The hot and humid climate of South,East,and Southeast Asian areas favors the rice production.Additionally,this specific climate also favor the viruses and the vectors that infect rice plants.According to the literature around 30 rice viruses diseases have been reported till the date.Over the last few years,next-generation sequencing(NGS)has revolutionized the viral research and remain the most fascinating approach to study viral diversity.Viruses are ubiquitous in nature and can infect almost all living entity.The diversity of viruses associated with plants are explored tremendously by using NGS.Both known and novel viruses are identified and characterized in different plant samples.The viral diseases of plants are usually devastating and cause enormous loss to plants growth and yields.Majority of viral diagnostics techniques employed for pathogen detection are antigen/antibody-dependent and require prior knowledge of the disease-causing agents.Additionally,these techniques can't detect novel pathogens present in the sample.Because of these serious concerns and drawback of traditional viral diagnostics techniques,NGS has emerged an alternative and attractive tool that require no prior knowledge of viral pathogens and can detect both known and novel viruses more rapidly and more accurately.In the current study,we adopted a metagenomics next-generation sequencing approach to analyze and characterize the viral diversity associated with wild rice plants.Using the Illumina platform,we performed next-generation sequencing(NGS)of transcriptome and small RNAs from one hundred rice plants.The bioinformatics pipeline developed here has combined the assembly of RNA-seq and small RNA data to deeply analyze the viral diversity associated with rice plants.The assembly result of transcriptomic data combined with sRNA assembled contigs has identified and characterized a total of 210 assembled viral contigs.These assembled viral contigs were further classified as complete,near complete,and fragmented sequences based on their coverage.Among the total identified assembled contigs,61 belongs to the complete viral sequences,8 belongs to the near complete,and 141 represented fragmented contigs of viral sequences of both know and novel viruses.The length of the assembled viral contigs ranges from a few hundred bp to over 15000bp.These assembled contigs were then classified into different viral families.The detailed analysis revealed that GC content of these assembled viral contigs ranges from 30-55%.Among the rice infecting viruses,we have identified and assembled the genomes of five rice viruses that are well known devastating pathogens to rice crops.These include rice grassy stunt virus(RGSV),rice ragged stunt virus(RRSV),rice gall dwarf virus(RGDV),southern rice black-streaked dwarf virus(SRBSDV),and Oryza sativa alphaendornavirus.Additionally,we have characterized the 21-24 nt viral derived small RNA for these assembled viral genomes to confirm their circulation in the plant samples.Additionally,five novel polerovirus genomes sequences were also assembled during de novo assembly of RNA-seq data.These viruses were assigned a tentative name of Rice associated Poleroviruses(RaPV).All of the five assembled viruses were confirmed by reverse transcription polymerase chain reaction(RT-PCR)assays and Sanger sequencing.The maximum nucleotide sequence identities observed for RaPV1 was 75%,RaPV2 76%,RaPV374%,RaPV4 75%and RaPV5 73%respectively.However,the amino acid sequence identities range from 46 to 62%.Phylogenetic analysis of these novel isolates showed a separate single cluster close to Sugarcane yellow leaf virus and Wheat leaf yellowing virus in the family Luteoviridae.Here in this study,we have shown the superiority of next-generation sequencing technique over other traditional viral diagnostic techniques to effectively identify and characterize viral diversity associated with wild rice plants.Our findings clearly demonstrate that using the combination of RNA-seq and small RNA data for exploring the viral diversity of rice plants is a valid and ideal approach which can be extended to further epidemiological studies.