Next generation RNA sequencing analysis of nuclear grapevines to identify viruses and viroids

Abstract

Abstract The use of next generation sequencing technology has allowed for greater elucidation of viruses infecting grapevine, which number more than 85. Many of these viruses can result in disease symptoms and it is therefore vital to the industry that they be supplied with healthy plant material. This is done through grapevine certification schemes. In this study, 97 nuclear grapevine vines subjected to a virus elimination process and maintained in vector-free conditions, were subjected to total RNA sequencing using the Ion-Torrent platform to determine the presence of viruses and viroids. In total, five virus species, four viroid species, and a further putative viroid species were identified in these nuclear grapevines through NGS analysis and confirmed using RT-PCR and Sanger sequencing. Grapevine Rupestris stem pitting-associated virus was identified in six nuclear accessions, while grapevine fleck virus, grapevine Syrah virus, grapevine leafroll-associated virus 4, and grapevine Rupestris vein feathering virus were identified in one nuclear accession each. Viroids were identified in 73 of the 97 nuclear accessions, with hop stunt viroid present in 63 of the 97 samples followed by grapevine yellow speckle viroid 1, present in 52 of the 97 samples, grapevine yellow speckle viroid 2, present in 17 of the 97 samples, Australian grapevine viroid present in three of the 97 samples and grapevine hammerhead viroid-like RNA was identified in two of the 97 samples. These findings suggest that some viruses, especially GRSPaV, and viroids are recalcitrant to currently employed virus elimination techniques, and how advantageous the use of NGS is to grapevine certification schemes.