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

Minna-Liisa Rajamäki,Anne Lemmetty,Janne Streng,Jari P T Valkonen,Archana Vishwakarma,Satu Latvala,Elina Roininen,Jaana Laamanen,Ulrich Melcher

doi:10.1371/journal.pone.0220621

Minna-Liisa Rajamäki, Anne Lemmetty + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0220621

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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.

Highlights

Virus-free and genetically true-to-type propagation materials are crucial for certified production of small fruits
Raspberry bushy dwarf virus (RBDV) and raspberry yellow net virus (RYNV) were detected in pool GEN18, whereas only rubus yellow net virus (RYNV) was detected in pool GEN19 (Table 1)
Mapping of the virus-derived small interfering RNA (siRNA) reads to the RBDV RNA1 and RNA2 showed high coverage of 72.6% and 80.3%, respectively, and high depth of coverage of 27.9 and 19.6, respectively (Table 1)

Summary

Introduction

Virus-free and genetically true-to-type propagation materials are crucial for certified production of small fruits. Certified production of small fruits has been carried out in Finland since 1977 [1]. Pre-basic mother plants must be virus-free and are tested using recommended indexing procedures [1,2,3,4,5]. Basic methods consist of bioassays such as sap-inoculation and graftinoculation from the tested plant to recommended indicator plants that display symptoms when infected, and serological assays such as enzyme-linked immunosorbent assays (ELISA). Nucleic acid analysis–based methods, such as polymerase chain reaction (PCR) and reverse transcription (RT)-PCR, have become commonplace. Pathogens not permissible in pre-basic mother plants of small fruits are regulated by a decree announced by the European Union (EU) in 2017. The new legislation will change the testing requirements and requires more frequent testing of pests in many plant species

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