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Deep sequencing of total RNAs in papaya for genome characterization of Papaya ringspot virus Bangladesh strain

John Hu: Department of Plant & Environmental Protection Sciences, University of Hawaii at Manoa

<div>Papaya (<em>Carica papaya </em>L.) is an economically important horticultural crop in Bangladesh. During a survey conducted on papaya viruses in different papaya growing regions of Bangladesh in December 2016 to January 2017, nearly 100% of papaya plants in some locations were observed to have typical symptoms of <em>Papaya ring spot virus </em>(PRSV) infection. Using a PRSV-specific single-tube nested PCR assay, the presence of PRSV was confirmed in several symptomatic plants. PRSV positive samples were then subjected to deep sequencing analyses on an Illumina<sup>®</sup> NextSeq 500 platform. A nearly complete genome (10282bp) of PRSV-Bangladesh strain (PRSV-BD) was generated. To validate the results of the deep sequencing analysis, 8 pairs of PRSV-specific primers were designed and overlapping DNA fragments were amplified by RT-PCR from an isolate (G52158) collected from Gazipur district. The amplified DNA fragments were cloned and sequenced by Sanger sequencing method and the resulting sequences were found to be 100% identical at the nucleotide level to the deep sequencing-derived sequence of PRSV-BD. Further analysis of genome using BLASTn showed that PRSV-BD was the most closely related to PRSV-Aus-53C (GenBank accession no. KX655866), a PRSV-W strain reported from cucurbits in Australia.</div>