sRNA deep sequencing aided plant RNA virus detection in cultivated raspberries and molecular characterization of Raspberry bushy dwarf virus and Black raspberry necrosis virus from Finland

Abstract

Plant RNA silencing machinery cleaves exogenous nucleic acids into 21, 22 and 24nt siRNAs, which constitute the core of basal defence against RNA/DNA virus infection. sRNA deep sequencing and assembly of 21-24nt size reads from virus-derived siRNAs (vsiRNAs) details plausible information of viral genomes targeted, following sequence homology search in virus databases. Pooled RNA sample marked (HXR-2) from 21 raspberry cultivars subjected to sRNA deep sequencing and vsiRNA read alignment revealed the presence of Raspberry bushy dwarf virus (RBDV) and Black raspberry necrosis virus (BRNV). Countercheck using VirusDetect, a sensitive virus finding software also confirmed these raspberry infecting plant RNA viruses through contig assembly. Read size calculation attested 21nt and 22nt vsiRNA read length as leads in alignment to reference that covered genomic regions in both viruses. Viral genome mapping displayed vsiRNA distribution regions, RBDV genomes distinctly masked from 5’ end, towards coding regions in RNA-1 and RNA-2, scarcely towards 3’ end. The putative stem-loop occurring 3’UTRs were figured as least covered regions. BRNV genomes mapped fractionally at 5’UTRs, and densely at 3’UTRs including a vsiRNA 22nt size hotspot position. RdRp encoding region in RNA-1 and small coat protein (CPs) encoding region in RNA-2 were markedly not spanned by vsiRNAs. RT-PCR for individual samples validated RBDV presence in 5 cultivars and BRNV in 6 cultivars. Molecular characterization following Sanger sequencing distinguished virus isolate diversity, RBDV sequence phylogeny showed limited variability in the CP encoding region among geographical isolates. BRNV CPs encoding region showed 25% nucleotide non-identity among cultivar isolates, tree phylogeny supported the interchange of isolates among cultivars and wild raspberries in Finland. The study highlights novel virus detection tool and significant vsiRNA benchmark profile for discovering RNA viruses. Remarked RNA recombination in BRNV CPs encoding sequence is moreover surmised.