Small-RNA analysis of pre-basic mother plants and conserved accessions of plant genetic resources for the presence of viruses

Abstract

Pathogen-free stocks of vegetatively propagated plants are crucial in certified plant production. They require regular monitoring of the plant germplasm for pathogens, especially of the stocks maintained in the field. Here we tested pre-basic mother plants of Fragaria , Rubus and Ribes spp., and conserved accessions of the plant genetic resources of Rubus spp. maintained at research stations in Finland, for the presence of viruses using small interfering RNA (siRNA) -based diagnostics (VirusDetect). The advance of the method is that unrelated viruses can be detected simultaneously without resumptions of the viruses present. While no virus was detected in pre-basic mother plants of Fragaria and Ribes species, rubus yellow net virus (RYNV) was detected in pre-basic mother plants of Rubus . Raspberry bushy dwarf virus (RBDV), black raspberry necrosis virus (BRNV), raspberry vein chlorosis virus (RVCV) and RYNV were detected in the Rubus genetic resource collection. The L polymerase encoding sequence characterized from seven RVCV isolates showed considerable genetic variation. The data provide the first molecular biological evidence for the presence of RYNV in Finland. RYNV was not revealed in virus indexing by indicator plants, which suggests that it may be endogenously present in some raspberry cultivars. In addition, a putative new RYNV-like badnavirus was detected in Rubus spp. Blackcurrant reversion virus (BRV) and gooseberry vein banding associated virus (GVBaV) were detected in symptomatic Ribes plants grown in the field. Results were consistent with those obtained using PCR or reverse transcription PCR and suggest that the current virus indexing methods of pre-basic mother plants work as expected. Furthermore, many new viruses were identified in the collections of plant genetic resources not previously tested for viruses. In the future, siRNA-based diagnostics could be a useful supplement for the currently used virus detection methods in certified plant production and thus rationalize and simplify the current testing system.

High-Throughput Sequencing of Small RNAs for the Sanitary Certification of Viruses in Grapevine

“…In the last years, performing RNA-sequencing analysis for determination and discovery of plant viruses in crop plants has become widespread ( Rott et al, 2017 ; Zheng et al, 2017 ; Maliogka et al, 2018 ; Massart et al, 2019 ; Rajamäki et al, 2019 ; Mehetre et al, 2021 ). More specifically, Al Rwahnih et al (2009) pioneered the use of HTS for virus determination in grapevine; and since then, it has proved a valuable tool ( Coetzee et al, 2010 ; Pantaleo et al, 2010 ; Al Rwahnih et al, 2015 ; Maree et al, 2015 ; Jo et al, 2015 ; Czotter et al, 2018 ; Hily et al, 2018 ; Vigne et al, 2018 ).…”

Integration of Rubus yellow net virus in the raspberry genome: A story centuries in the making

“…Another genome (RYNV-BS, KM078034, Diaz-Lara et al, 2015) was sequenced from red raspberry 'Baumforth's Seedling A' using DNA from rolling circle amplification. Since then, several RYNV sequences were published using small RNA (Rajamäki et al, 2019) or whole genome sequencing (MN245240). Diaz-Lara (2016) observed that red raspberry plants, supposedly free of RYNV based on aphid or graft transmission onto R. occidentalis 'Munger' indicator, yielded positive results when indexed by PCRbased assays.…”

Successful Cryopreservation of Dormant Buds of Blackcurrant (Ribes nigrum L.) by Using Greenhouse-Grown Plants and In Vitro Recovery

“…Blackcurrant reversion virus (BRV), transmitted by the gall mite ( Cecidophyopsis ribis ), is the most significant virus in blackcurrants, causing disease symptoms to the leaves and flowers, proliferation, and ultimately yield losses [ 33 , 34 ]. Pest and disease infections can be prevented in certified plant production by maintaining pre-basic mother plants (stock material) in insect-proof greenhouse [ 35 ].…”