Development of an Oligoprobe-Based Targeted High-Throughput Sequencing Method for Grapevine Virus Detection

Viral diseases are a major threat to global grapevine production, resulting in substantial economic impacts and trade restrictions. Accurate determination of the phytosanitary status of grapevine planting material is therefore critical for both domestic certification and international movement of grapevine material. This study aimed to develop and evaluate a targeted high-throughput sequencing (HTS) strategy using a custom-designed oligoprobe panel to enhance the detection of grapevine-associated viruses. Ampliseq Custom DNA oligoprobe panels were designed to detect 24 RNA and six DNA viruses infecting grapevines, targeting structural and non-structural viral genes as regions of interest (ROIs). Four HTS-based approaches were compared in this study. Two were oligoprobe-based methods (OM): oligoprobe method total-RNA (OM-TR) and oligoprobe method-double stranded RNA (OM-DR), and two were non-oligoprobe-based methods (NOM): non-oligoprobe method-total-RNA (NOM-TR) and non-oligoprobe method-double stranded RNA (NOM-DR). Among these four approaches, the OM-TR and OM-DR demonstrated enhanced virus detection providing greater viral diversity and improved viral read intensity (RI) values. Virtool v8 application was used for HTS data curation and viral sequence analysis. The OM-DR approach detected seven viruses among the seven virus-infected grapevines followed by OM-TR detecting six viruses. The viruses detected by OM-DR included Grapevine leafroll-associated virus (GLRaV)-1, -2, -3, Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine Pinot gris virus (GPGV), Grapevine virus H (GVH), and Grapevine red blotch virus (GRBV). The oligoprobe-based methods enhanced virus detection compared to the non-oligoprobe based approaches. The OM approach improved the detection of grapevine viruses and could be tested as a sensitive and scalable tool for grapevine virus diagnostics, providing valuable support for quarantine programs and the domestic certification of grapevine propagative material in Canada.