Application of High-Throughput sequencing approach to define the sanitary status of certified authocthonous grapevine cultivars

In Italy, grapevine certification system requires the status of "virus controlled" for multiplication and commercialization. This system entails free plant propagation material from Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine leafroll-associated virus 1, 2, 3 (GLRaV-1, -2, -3), Grapevine fanleaf virus (GFLV), Grapevine fleck virus (GFkV) and Arabis mosaic virus (ArMV)]. Routinely applied bio- and molecular assays for the detection of systemic plant pathogens, such as wood indexing, serological and (RT-) PCR molecular tests, are time-consuming and always require a priori knowledge of the target. On the other hand, the availability of High-Throughput Sequencing (HTS) techniques, which are free from the abovementioned constraints, allows the characterization of the "virome" of the analysed plants. Indeed, data originating from HTS give an unbiased picture of the virus/viroid content of a plant. In this frame, a task of the Apulian Regional Project Re. Ge. Vi. P (Recovery of Apulian grape germplasm) aims to assess the sanitary status of 20 certified autochthonous grapevine clones, table and wine cultivars, by HTS. In 2016, small RNAs (sRNAs) libraries were synthesized from 20 grape accessions and subjected to Illumina sequencing. The downstream data were processed using a custom-developed bioinformatics pipeline including the quality control of the libraries, the de novo assembly of the reads using Velvet assembler and the Blast annotation of the obtained contigs. Annotated contigs confirmed the absence of the above-regulated viruses. Also, this approach showed the presence of two viruses and two viroids not targeted in routinely performed assays, i. e. Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine rupestris vein feathering virus (GRVFV), Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd-1). These results, confirmed by RT-PCR assays, strengthen the potentiality of HTS approach in plant "virome" definition and its application in grapevine certification schemes.