Next-Generation Sequencing Combined With Conventional Sanger Sequencing Reveals High Molecular Diversity in Actinidia Virus 1 Populations From Kiwifruit Grown in China.

Shaohua Wen,Yanxiang Wang,Ni Hong,Qian Lu,Zuokun Yang,Min Rao,Guoping Wang

doi:10.3389/fmicb.2020.602039

Abstract

Kiwifruit (Actinidia spp.) is native to China. Viral disease–like symptoms are common on kiwifruit plants. In this study, six libraries prepared from total RNA of leaf samples from 69 kiwifruit plants were subjected to next-generation sequencing (NGS). Actinidia virus 1 (AcV-1), a tentative species in the family Closteroviridae, was discovered in the six libraries. Two full-length and two near-full genome sequences of AcV-1 variants were determined by Sanger sequencing. The genome structure of these Chinese AcV-1 variants was identical to that of isolate K75 and consisted of 12 open reading frames (ORFs). Analyses of these sequences together with the NGS-derived contig sequences revealed high molecular diversity in AcV-1 populations, with the highest sequence variation occurring at ORF1a, ORF2, and ORF3, and the available variants clustered into three phylogenetic clades. For the first time, our study revealed different domain compositions in the viral ORF1a and molecular recombination events among AcV-1 variants. Specific reverse transcriptase–polymerase chain reaction assays disclosed the presence of AcV-1 in plants of four kiwifruit species and unknown Actinidia spp. in seven provinces and one city.

Highlights

The family Closteroviridae contains many plant viruses causing different diseases of economically important crops (Martelli et al, 2012)
Homologous recombination has been described for coat protein (CP) and P20 genes of citrus tristeza virus (CTV) (Rubio et al, 2001, 2013; Bar-Joseph and Mawassi, 2013), a member in the genus Closterovirus, for CP genes of ampeloviruses grapevine leafrollassociated virus 3 (GLRaV-3) (Turturo et al, 2005; Farooq et al, 2012), GLRaV-1 (Fan et al, 2015), and GLRaV-11 (Boulila, 2010), for HSP70h gene of plum bark necrosis stem pitting-associated virus (PBNSPaV) (Qu et al, 2014), and for ORF1a of GLRaV-4 (Adiputra et al, 2019)
The RNA-seq analyses of two samples individually collected from two kiwifruit plants and four pooled kiwifruit samples revealed the presence of the eight kiwifruitinfecting viruses Actinidia virus 1 (AcV-1), Actinidia chlorotic ringspot-associated emaravirus (AcCRaV), Actinidia seed-borne latent virus (ASbLV), Actinidia virus A (AcVA), Actinidia virus B (AcVB), citrus leaf blotch virus (CLBV), Actinidia emaravirus 2 (AcEV-2), and apple stem grooving virus (ASGV), and three viruses related to the families Bromoviridae, Betaflexiviridae, and Secoviridae

Summary

Introduction

The family Closteroviridae contains many plant viruses causing different diseases of economically important crops (Martelli et al, 2012). Members of the family Closteroviridae share a common genome organization, containing a replication-related module encoded by open reading frame (ORF) 1a and 1b, and a five-gene module encoding a small hydrophobic protein, a homolog of the plant heat shock protein HSP70 (HSP70h), a ∼60 kDa protein, a major coat protein (CP), and a minor coat protein (CPm) (Martelli et al, 2012). A notable exception is that viruses in the genus Ampelovirus have smaller genomes, which do not possess CPm and have unique ORFs. Previous studies showed that viruses in the family Closteroviridae have great genetic variability, and molecular evolution might be necessary for adaptation to new environments (Holland et al, 1982; Kreuze et al, 2002; Cuellar et al, 2008; Bertazzon et al, 2010; García-Arenal and Fraile, 2011; Esteves et al, 2012). Homologous recombination has been described for CP and P20 genes of citrus tristeza virus (CTV) (Rubio et al, 2001, 2013; Bar-Joseph and Mawassi, 2013), a member in the genus Closterovirus, for CP genes of ampeloviruses grapevine leafrollassociated virus 3 (GLRaV-3) (Turturo et al, 2005; Farooq et al, 2012), GLRaV-1 (Fan et al, 2015), and GLRaV-11 (Boulila, 2010), for HSP70h gene of plum bark necrosis stem pitting-associated virus (PBNSPaV) (Qu et al, 2014), and for ORF1a of GLRaV-4 (Adiputra et al, 2019)

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Results

Conclusion