Small RNA deep sequencing for plant virus discovery in cultivated Rubus and Ribes in Finland
Small RNA (sRNA) sequences are precisely 21, 22 and 24 nt (nucleotide) in length. Deep sequencing of sRNAs generates sRNA datasets. This procedure allows for the discovery of novel viruses and the identification of viral genomes for use in reference databases. RNA samples from Rubus and Ribes germplasm in Finland were combined for a large-scale virus diagnosis. The collection included ten red raspberry (Rubus idaeus L.) mother plants and one accession each from blackcurrant (Ribes nigrum L.) and redcurrant (Ribes rubrum L.) cultivars. VirusDetect, a user-friendly sRNA software, identified genomes of Raspberry bushy dwarf virus (RBDV), Black raspberry necrosis virus (BRNV), Blackcurrant reversion virus (BRV), Rubus yellow net virus (RYNV) and Gooseberry vein banding virus (GVBaV) based on homology-dependent contig assembly. Reverse-transcription PCR and direct sequencing of PCR products validated positive results in samples extracted from raspberry leaves. RBDV was detected in five samples (three raspberry cultivars); BRNV in six (three cultivars). Genome wide distribution of the sRNA read profile (21 and 22 nt) in RBDV and BRNV genomes provided novel “hotspot” information. In RBDV genomes, the lowest coverage by sRNAs was observed at 3’UTR (untranslated region). The upstream coat protein (CP) encoding sequence in RBDV was PCR amplified from one cultivar and aligned to geographical isolates previously characterized from red raspberries (96-99% identity). The analysis revealed that RBDV diversity is restricted in red raspberry plants worldwide. The genomic regions encoding RNA dependent RNA polymerase (RdRp) and small coat protein (CPs) in BRNV had the lowest coverage with sRNAs. The CPs nucleotides sequenced from three cultivars aligned poorly (~79%) or highly (99%) with BRNV isolates found in Finland and exhibited diversity (~25%) to BRNV Alyth (UK). In addition, the presence of PstI endonuclease (CTGCA^G) site in CPs region distinguished isolates from Finland. The sRNA data assists unbiased virus detection. In this study, molecular diagnosis of CPs sequences rapidly detected BRNV, a virus at low titres in red raspberry.
Fhilmar Raj Jayaraj-Mallika won the ISHS Young Minds Award for the best poster presentation at the XII International Rubus and Ribes Symposium: Innovative Rubus and Ribes Production for High Quality Berries in Changing Environments in Switzerland in June 2019.
Fhilmar Raj Jayaraj-Mallika, Department of Agricultural Sciences, University of Helsinki, FI-0014 Helsinki, Finland, e-mail: firstname.lastname@example.org
The article is available in Chronica Horticulturae