Genome Sequences of Two Grapevine Rupestris Stem Pitting-Associated Virus Variants from Vitis vinifera cv. Riesling in Idaho, USA

ABSTRACT We report the genome sequences of two genetic variants of grapevine rupestris stem pitting-associated virus (GRSPaV) from Idaho, USA. The coding-complete, positive-strand RNA genome of 8,700 nucleotides contains six open reading frames characteristic of foveaviruses. The two Idaho genetic variants belong to GRSPaV phylogroup 1.

(Bioo Scientific). After bead-based size selection, the resulting libraries were multiplexed and subjected to high-throughput sequencing (HTS) on a NovaSeq 6000 platform through the University of Idaho Genomics and Bioinformatics Resources Core Facility. A total of 31,500,611 paired-end 250-bp read pairs were produced for the CC04 sample. Raw reads were adapter and quality cleaned using Trimmomatic v0.38 (10) and were mapped against the V. vinifera reference genome using bowtie2 v2.4.4 in local mode (9); unmapped paired-end reads were subjected to assembly using SPAdes v3.15.3 in RNA mode, and analyses using BLASTn and DIAMOND programs were performed with the nonredundant database (11). One of the samples, CC04 from an own-rooted, 38-year-old Riesling vine collected in Nez Perce County, Idaho, yielded a large, 8,700-nucleotide (nt) contig (GRSPaV-ID1) that spanned six ORFs and, based on the BLASTn program (National Center for Biotechnology Information), exhibited 98.5% identity to the GRSPaV isolate 12G412 (GenBank accession number MZ484771) from British Columbia, Canada. The same Riesling sample also yielded three additional contigs of 2,514, 2,893, and 506 nt, which exhibited 98.3%, 98.6%, and 98.8% identity, respectively, to the GRSPaV isolate AMCF clone 3 (GenBank accession number MG938311) from France (12) as determined by BLASTn. Apparently, this sample of Riesling vine contained two distinct genetic variants of GRSPaV. Specific primers (Table 1) FIG 1 Phylogenetic relationships of the genome sequences of 25 GRSPaV strains from the four main phylogroups of GRSPaV identified by Hily et al. (11) and the GRSPaV sequences obtained in this work, i.e., GRSPaV-ID1 and GRSPaV-ID2. Genome sequences for two other foveaviruses, i.e., apple stem pitting virus (ASPV) and peach chlorotic mottle virus (PeCMV), were added as an outgroup. Nucleotide sequences were aligned with the MAFFT program in Geneious, with default settings in G-insi mode. Phylogenies were inferred using IQ-TREE 2 (13), with ModelFinder for best model fit (14), and branch support was estimated with SH-aLRT/UFBoot (1,000 replicates), as implemented in IQ-TREE 2 (15,16). The tree was then edited in FigTree v1. 4.4. were designed to confirm/validate the presence of both GRSPaV variants in the original sample. Using reverse transcription (RT)-PCR as described (9), virus-specific bands were amplified for both GRSPaV genetic variants in the original Riesling vine sample; the specificity of the amplified PCR products was confirmed by Sanger sequencing (9). To extend the genome sequence for the GRSPaV-ID2 variant, additional primers (Table 1) were used in RT-PCR to amplify virus-specific sequences between the three HTS-derived contigs, and the assembly was then verified by remapping the HTS reads to it; the resulting GRSPaV-ID2 contig of 8,481 nt was 92.3% identical to that of GRSPaV-ID1 in a pairwise comparison by BLASTn. The sequenced GRSPaV-ID1 and GRSPaV-ID2 genomes had GC contents of 42.5% and 42.0%, respectively (as determined by Geneious), and were covered by 3,711 and 289 reads, respectively (after assembly with Sanger and HTS contigs). In a phylogenetic analysis (Fig. 1), GRSPaV-ID1 and GRSPaV-ID2 were placed in the same clade 1 designated by Hily et al. (12).
Data availability. The coding-complete genome sequences of GSPaV-ID1 and GSPaV-ID2 are available in GenBank under accession numbers OQ134941 and OQ134942, respectively. The raw sequence data were deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA931750.

ACKNOWLEDGMENTS
The help of Idaho wine grape growers who permitted our access to and sampling in their vineyards is greatly appreciated.
This work was funded, in part, through grants from the Idaho State Department of Agriculture (ISDA) Specialty Crop Block Grant, the Idaho Wine Commission, the Northwest Center for Small Fruits Research, the USDA Agricultural Research Service (grants 5358-21220-002-18G, 2072-21000-057-00D, and 2072-21220-003-00D), the USDA National Institute of Food and Agriculture (Hatch projects IDA01560 and IDA01712), and the Idaho Agricultural Experiment Station. Data collection performed by the Institute for Interdisciplinary Data Sciences (IIDS) Genomics and Bioinformatics Resources Core at the University of Idaho was supported in part by NIH COBRE grant P30GM103324.
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