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Identification of viruses infecting cigar and flue-cured tobacco using next generation sequencing and establishment of multiplex RT-PCR


 Backgrounds: Infection of plant viruses cause extensive damage to plants and reduce crop yield. Early, precise and simultaneous identification of the plant viruses is of great significance on preventing the spread of the viruses as well as reducing losses on agricultural yield.Methods: Identification of plant viruses from symptomatic samples collected from cigar tobacco planting area in Deyang and flue-cured tobacco planting area in Luzhou city of Sichuan Province China was conducted by the deep sequencing of small RNAs (sRNAs) through an Illumina sequencing platform and plant virus specific contigs were generated based on the virus derived siRNA sequences. Additionally, sequence alignment and phylogenetic analysis was performed to determine the species or strains of these viruses. Subsequently, specific primers were designed for simultaneous detection of five RNA viruses infecting tobacco.Results: A total of 27930450, 21537662 and 28194021 clean reads were generated from three pooled samples and a total of 105 contigs that can be mapped to the closest plant viruses with the length range from 34~1720 nt. The results indicated that the major viruses were potato virus Y (PVY), Chilli veinal mottle virus (ChiVMV), tobacco vein banding mosaic virus (TVBMV), tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). A fast and sensitive multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for the simultaneous detection of the most frequent RNA viruses infecting cigar and flue-cured tobacco in Sichuan.Conclusion: These results provide theoretical basis and convenient methods for rapid detection and control of viruses on cigar and flue-cured tobacco.