Small RNA Sequencing and Multiplex RT-PCR for Diagnostics of Grapevine Viruses and Virus-like Organisms

Vanja Miljanić; Jernej Jakše; Denis Rusjan; Andreja Škvarč; Nataša Štajner

doi:10.3390/v14050921

Small RNA Sequencing and Multiplex RT-PCR for Diagnostics of Grapevine Viruses and Virus-like Organisms

Viruses. 2022 Apr 28;14(5):921. doi: 10.3390/v14050921.

Authors

Vanja Miljanić¹, Jernej Jakše¹, Denis Rusjan¹, Andreja Škvarč², Nataša Štajner¹

Affiliations

¹ Department of Agronomy, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia.
² Chamber of Agriculture and Forestry of Slovenia, Agriculture and Forestry Institute Nova Gorica, 5000 Nova Gorica, Slovenia.

Abstract

Metagenomic approaches used for virus diagnostics allow for rapid and accurate detection of all viral pathogens in the plants. In order to investigate the occurrence of viruses and virus-like organisms infecting grapevine from the Ampelographic collection Kromberk in Slovenia, we used Ion Torrent small RNA sequencing (sRNA-seq) and the VirusDetect pipeline to analyze the sRNA-seq data. The used method revealed the presence of: Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2 (GLRaV-2), Grapevine leafroll-associated virus 3 (GLRaV-3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine fanleaf virus (GFLV) and its satellite RNA (satGFLV), Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV), Grapevine Pinot gris virus (GPGV), Grapevine satellite virus (GV-Sat), Hop stunt viroid (HSVd), and Grapevine yellow speckle viroid 1 (GYSVd-1). Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for validation of sRNA-seq predicted infections, including various combinations of viruses or viroids and satellite RNA. mRT-PCR could further be used for rapid and cost-effective routine molecular diagnosis, including widespread, emerging, and seemingly rare viruses, as well as viroids which testing is usually overlooked.

Keywords: Vitis vinifera L.; mRT-PCR; sRNA-seq; virome.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA Viruses / genetics
Plant Diseases
RNA, Satellite / genetics
RNA, Small Untranslated*
Reverse Transcriptase Polymerase Chain Reaction
Sequence Analysis, RNA
Viroids* / genetics
Viruses, Unclassified* / genetics
Vitis*

Substances

RNA, Satellite
RNA, Small Untranslated

Grants and funding

This research was funded by the Slovenian Research Agency (SRA–ARRS), grant number P4-0077, research program: Genetics and Modern Technologies of Crops, awarded to the research group of the Chair of Genetics, Biotechnology, Statistics and Plant breeding at the University of Ljubljana, Biotechnical Faculty. In addition, a scholarship for nationals of the Western Balkans for postgraduate study in Slovenia (Public Scholarship, Development, Disability and Maintenance Fund of the Republic of Slovenia, 245. Public Announcement) was awarded to V.M.