Transcriptome sequencing reveals novel Citrus bark cracking viroid (CBCVd) variants from citrus and their molecular characterization.

Yafei Wang,Fangyun Yang,Xuefeng Wang,Sagheer Atta,Changyong Zhou,Mengji Cao,Rajesh Mehrotra

doi:10.1371/journal.pone.0198022

Yafei Wang, Fangyun Yang + Show 5 more

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https://doi.org/10.1371/journal.pone.0198022

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Abstract

Citrus bark cracking viroid (CBCVd), previously called Citrus viroid IV, belongs to the genus Cocadviroid within the family Pospiviroidae. CBCVd has been identified as an important causative agent in citrus and hops. In this study, we obtained the full-length genomes of different variants of all detected citrus viroids from Pakistan through transcriptome sequencing. Different CBCVd variants were first found in Pakistan. These newly discovered Pakistani CBCVd variants were provisionally called “CBCVd-LSS” for their low sequence similarity (80.9%–88.9%) with the CBCVd RefSeq sequence (NC_003539). The two most predominant CBCVd sequences from Pakistan had the closest identity, 90.6% and 87.9%, with two CBCVd sequences isolated from hops. Identification and molecular characterization of CBCVd from citrus in Pakistan and China were also reported. The length of CBCVd from China ranged from 282 to 286 nucleotides, while that of the one from Pakistan ranged from 273 to 277 nucleotides. Based on genetic diversity and phylogenetic analysis, two main CBCVd clades were identified. CBCVd sequences from Pakistan, China, and other countries were further divided into six sub-clades. Sequence alignment revealed some nucleotide changes between these sub-clades, and analysis indicated that several mutations could significantly affect the primary and secondary structure of the viroid. Our results indicated that the CBCVd sequences from Pakistan and China were significantly different with respect to genome and secondary structure and Pakistan might be one of the independent geographical origins of CBCVd worldwide.

Highlights

Viroids, the known smallest pathogens, are closed-loop, single-stranded RNA molecules of 246–401 nucleotides [1, 2]
The full genomes of six viroids including Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), CDVd, Citrus bark cracking viroid (CBCVd), and Citrus viroid V (CVd-V) were obtained from PCV-I and PCV-II
Previous studies have shown that the rRNA-depleted total RNA method is generally more effective than the sRNA method when reassembling sequencing reads [25]

Summary

Introduction

The known smallest pathogens, are closed-loop, single-stranded RNA molecules of 246–401 nucleotides (nt) [1, 2]. Eight viroid species of the family Pospiviroidae: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus dwarfing viroid. Novel CBCVd variants infecting citrus from Pakistan. Funds for the Central Universities (XDJK2018AA002, XDJK2016B021, SWU116012), Chongqing Research Program of Basic Research and Frontier Technology (cstc2017jcyjBX0016). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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