Phylogenetic Clade Identification of GRBV Isolates

To determine the phylogenetic clade of GRBV isolates in symptomatic, infected grapevines, PCR products corresponding to a fragment of the GRBV replication ORF [22 (link),36 (link),38 (link)] were Sanger sequenced at the Cornell Biotechnology Resource Center in Ithaca, New York. GRBV sequences were assembled and analyzed using DNASTAR Lasergene software suite, version 14.1. Additionally, some PCR products underwent restriction digestion with AleI-v2 (New England Biolabs, Ipswich, MA, USA) to determine the GRBV phylogenetic clade, as previously described [41 (link)]. Digestions were resolved via electrophoresis on agarose gels and visualized using UV illumination post-staining with GelRed (Biotium, Fremont, CA, USA). Restriction digests of approximately 201 and 117 bp in size reflected a GRBV clade 1 isolate, while a single uncut 318 bp fragment indicated a GRBV clade 2 isolate [41 (link)].