The genome assemblies of eight grapevines, ‘Catawba’, ‘Concord’, ‘Chambourcin’, ‘Horizon’, MN1264, ‘Norton’, NY84.0101.03 and PN40024, were used in this study. These individuals were included because they were known or implicated via SSRs as carriers for either specific Rpv3 alleles or Rpv27. Among the French-American hybrids, NY84.0101.03 is heterozygous for Rpv3.1, ‘Horizon’ and MN1264 both carry single copies of Rpv3.2, while ‘Chambourcin’ is heterozygous for both Rpv3.1 and Rpv3.2 (Di Gaspero et al., 2012; Clark et al., 2018; Patel et al., 2023; Reisch, 2024). ‘Norton’ is commonly described as being derived from V. aestivalis and is one of the known carriers of Rpv27 (Hammers et al., 2017; Sapkota et al., 2019). ‘Catawba’ and ‘Concord’ are related hybrids between Vitis labrusca and V. vinifera that date back to the early to mid-1800s. Specifically, ‘Catawba’ is an F₁ of V. labrusca and V. vinifera ‘Semillon’, while ‘Concord’ is the product of a ‘Catawba’ backcross to V. labrusca (Vitis International Variety Catalogue: www.vivc.de). While both were found to carry the SSR fragment lengths for Rpv3.3, neither could be tied to a known source through pedigree. Lastly, PN40024 is a highly homozygous V. vinifera, recently shown to originate from the repeated selfing of ‘Helfensteiner’, an F₁ of ‘Pinot noir’ (Velt et al., 2023) and is often used as the primary reference genome for grapevines.

Through the use of haplotype-resolved genome assemblies, the Rpv3 / Rpv27 region of sixteen haploid genome assemblies were characterized. Genome assemblies for ‘Catawba’, ‘Concord’, MN1264, ‘Norton’, and NY84.0101.03 were constructed at Cornell University while ‘Horizon’ was assembled at the University of California–Davis. Genome assembly followed the same methodology described in detail by Zou et al. (2023) for the Vitis x doaniana ‘PI 588149’ genome assembly. Briefly, SMRTbell Template Prep Kit SP v2 (PacBio, CA, USA) libraries were constructed from high molecular weight DNA that was filtered for DNA between 15–20 kbp using BluePippin (Sage Science, MA, USA). The libraries were then sequenced with a PacBio Sequel II using 1 SMRT cell by the DNA Sequencing and Genotyping Center at the University of Delaware. The resulting PacBio reads were then used to construct two phased assemblies using Hifiasm (v0.16.0) (Cheng et al., 2021). ‘Chambourcin’ and PN40024 had preexisting, publicly available genome assemblies that were used in this study. The ‘Chambourcin’ assembly constructed by Patel et al. (2023) was diploid with haplotypes separated into ‘primary’ and ‘haplotig’ assemblies and was accessed via the article’s supplemental data. PN40024, as an inbred accession, was constructed as a haploid genome assembly in its role as reference genome. However, including both high quality versions of PN40024, PN40024.v4 (Velt et al., 2023) and PN_T2T (Shi et al., 2023), allowed for a bioinformatic comparison of these versions in addition to the opportunity to update the physical positions of Rpv3 and Rpv27 in the latest versions of the reference genome. Both versions of PN40024 were downloaded from grapedia.org/genomes.

As SSR markers are intrinsically tied to the allele classification and demarcation of Rpv3 and Rpv27, the primer sequences of associated SSR were used to validate the presence and the physical boundaries of these loci within each diploid assembly (Table 1).

*Abbreviations: VVCS1, VVCS1H077H16R1-1.

Using BBMap (v39.03) (Bushnell, 2024) under default parameters, both forward and reverse primer sequences from six SSR markers were aligned to all sixteen haplotypes from eight assemblies, separately. Based on the predicted fragment lengths from BBMap, each haplotype assembly was then assigned to an Rpv3 allele or Rpv27 based on the combination of UDV305–UDV737 or VVCS1H077H16R1-1–UDV737 for Rpv3 (Di Gaspero et al., 2012) or Rpv27 (Sapkota et al., 2019), respectively. Fragment length combinations for Rpv3 allele assignment, as UDV305–UDV737, were: Rpv3.1, 299–279; Rpv3.2, null–297; and Rpv3.3, null–271 while the fragment length for Rpv27, as VVCS1H077H16R1-1–UDV737, was 99—294. An exact match was not required, however, given that small differences were expected between the in-silico fragment lengths derived through BBMap and the reported PCR amplification products for each allele. Three additional SSRs, UDV730, UDV734, and UDV736, were included as associated markers that span the gap between the diagnostic pairs described above.

In order to focus solely on Rpv3 and Rpv27, the physical positions of SSR markers VVCS1H077H16R1-1 and UDV737, the most physically distant pair, were used to isolate the region from the rest of each haplotype assembly using the ‘seqinr’ package (Charif & Lobry, 2007) to manually isolate the sequence in R (R Core Team, 2023). In most of the haplotype assemblies, the Rpv3 / Rpv27 region was contiguous. Extensions of 5 kb and 100 kb were added to the positions of VVCS1H077H16R1-1 and UDV737, respectively. A larger extension was included beyond UDV737 to include more of the neighboring NLR (Nucleotide-binding Leucine-rich Repeat) gene cluster found to reside nearby. For haplotype assemblies where the SSRs were split across multiple contigs, all-to-all genome alignments were performed using ‘nucmer’ with default parameters from MUMmer4 (v4.0.0) (Marçais et al., 2018). The discontiguous haplotype was aligned to a contiguous haplotype with the same allele. This allowed for the visual selection of contigs via ‘mummerplot’ and the formation of an Rpv3 / Rpv27 region for each haplotype assembly. Once the haplotype assemblies were narrowed down, AUGUSTUS (v3.4.0) (Stanke et al., 2008) was used for gene prediction, followed by functional annotation with diamond (v2.1.8) (Buchfink et al., 2021) and Blast2GO (v1.5.1) (Gotz et al., 2008) each under default parameters. NLR genes were annotated using NLR-Annotator (Steuernagel et al., 2020) with their functionality and completeness predicted using NLRexpress (Martin et al., 2022), again under default parameters. Visualization of the Rpv3 / Rpv27 regions, including positions of mapped SSR and annotated NLR genes was produced in Python (v3.9) using custom script incorporating tools from ‘pyGenomeViz’ (v0.4.4) (Moshi, 2024).

The candidate genes conferring Rpv3.1 downy mildew resistance were first identified by Foria et al. (2020) and later corroborated and expanded upon by Wairich et al (2022). These candidate genes were two TIR-NBS-LRR (TNL), ‘VIT_18s0041g01330’ (TNL1) and ‘VIT_18s0041g01340’ (TNL2), and a leucine-rich repeat kinase (LRRk), ‘VIT18s0041g01350’. Both articles demonstrated the role TNL2 (as paralogs TNL2a and TNL2b in Rpv3.1) played in triggering a defense response through expression analysis. Only Wairich et al. (2022), however, found evidence that TNL1 and LRRk may also play a role. All three gene models were included here for completeness in characterizing the locus. The reported gene model identifiers were based on a previous version of the PN40024 reference genome, however. In order to characterize the presence, functionality, and abundance of these genes, the gene model identifiers were updated to their PN40024.v4 counterparts through the PN40024 reference genome browser provided by grapegenomics.com (hosted by UC-Davis). These translations were: TNL1, ‘Vitvi18g02077’; TNL2, ‘Vitvi18g03133’; and LRRk, ‘Vitvi18g02080’. The genomic sequences for these three genes were aligned to each haplotype assembly using nucleotide BLAST (Camacho et al., 2009), (-max_target_seq 5 -evalue 1e-10) including the diploid Muscadinia rotundifolia ‘Trayshed’ assembly (Minio et al., 2022) as an outgroup. The high sequence similarity between TNL1 and TNL2 made it necessary to identify short, 60 bp sequences of high divergence that could be used to sort through the blast results. These sequences were identified using a combination of R (R Core Team, 2023) and alignment in MEGA11 (Tamura et al., 2021). These short sequences were then blasted back to the extracted alignment results of the initial blastn (-evalue 1e-10). Manual classification of the results to either TNL1 or TNL2 was based, in part, on the presence and quality of the alignment of these short sequences. Alignment quality was assessed based on the combination of percent identity and alignment length. Multiple sequence alignment of the resulting TNL1, TNL2, and LRRk sequences from each haplotype genome was followed by the construction of neighbor-joining trees using MEGA11 and visualized in iTOL (v5) (Letunic & Bork, 2021). In order to compare the sequence similarity among the LRRk from each haplotype, pairwise distances were calculated under default settings in MEGA11.

In order to have high quality representation of each haplotype allele at Rpv3 and Rpv27, assemblies of six new grapevines were constructed. These grapevines, ‘Catawba’, ‘Concord’, ‘Horizon’, MN1264, ‘Norton’, and NY84.0101.03 were included alongside ‘Chambourcin’ and PN40024 to account for alleles Rpv3.1, Rpv3.2, Rpv3.3, and the co-located Rpv27 as assigned based on diagnostic SSR. The primary or haplotype-resolved contigs for the six new diploid assemblies ranged in contig N50 from 4.9–21 Mb with complete BUSCO scores from 95.8–98.7% (Table 2).

*SSR alleles designated as Rpv3.3 were based solely on SSR fragment lengths and were unable to be confirmed via pedigree analysis. ^#Rpv27 fragment lengths (VVCS1H077H16R1-1–UDV737); SSR alleles labeled V. vinifera refer to haplotypes descending from V. vinifera, while NC reflects non-characterized haplotypes. Contig N50 and BUSCO scores were skipped for haplotig assemblies as they do not represent the complete genome.

The combination of SSR markers UDV305 and UDV737, or VVCS1H077H16R1-1 and UDV737 for Rpv27, were used to classify each haploid assembly with an Rpv3 allele according to the fragment lengths described in Di Gaspero et al. (2012). There were two assembled haplotypes of Rpv3.1, Chambourcin Primary and NY84.0101.03 Hap1. Rpv3.2 was assembled three times from Chambourcin Haplotig, Horizon Primary, and MN1264 Hap1. Potential haplotypes of Rpv3.3 based on SSRs were assembled from Catawba Hap1, Concord Hap2, and Horizon Haplotig. Norton Hap 2 was the only instance of Rpv27. An additional conserved haplotype from Di Gaspero et al. (2012) representing a V. vinifera background, UDV305: null–UDV737: 287, from PN40024.v4, PN_T2T, Concord Hap1, and Catawba Hap2. The final three haplotypes, MN1264 Hap2, Norton Hap1, and NY84.0101.03 Hap2 were distinct from each other and have yet to be characterized.

Using the alignment positions of the six SSR markers, the Rpv3 / Rpv27 region was extracted from each haplotype assembly based on the two most physically separated markers, VVCS1H077H16R1-1 and UDV737. Across all haplotypes, this region ranged from 0.8 to 1.7 Mb (Table S1). Both examples of Rpv3.1 were contiguous in their assemblies and covered a physical distance of 1.36 Mb and 1.40 Mb on the Chambourcin Primary and NY84.0101.03 Hap1 assemblies, respectively. For Rpv3.2, both the Chambourcin Haplotig and MN1264 Hap1 assemblies were contiguous and spanned 1.3 Mb, while the Horizon Primary Rpv3.2 was not contiguous. This assembly was thusly aligned to Chambourcin Haplotig, another Rpv3.2, contig ‘vitin_chambo_h_18_02’. The Rpv3.2 from Horizon Primary was scaffolded using two contigs (‘VITHorizon_Primary000139F’ and ‘VITHorizon_Primary000043F’) spaced roughly 3 kb apart for a total length of 1.19 Mb (Figure S1). More variation in length existed among the three representatives of Rpv3.3. Catawba Hap1 and Concord Hap2 were both contiguous and 1.29 and 1.34 Mb in length. The third, Horizon Haplotig, was not contiguous and also lacked successful alignments for both VVCS1H077H16R1-1 and UDV730. Regardless, the Horizon Haplotig assembly was aligned to the Concord Hap 2 contig ‘h2tg000005l’ and scaffolded using four contigs with cumulative added spacing of 125 kb for a total length of 0.8 Mb (Figure S2). The single carrier of Rpv27, Norton Hap2, had a contiguous length of 1.3 Mb. The V. vinifera haplotype alleles were all contiguous and consistent in length, with the two versions of PN40024 at 1.52 Mb and both Concord Hap1 and Catawba Hap2 at 1.53 Mb. Of the remaining three haplotype assemblies, NY84.0101.03 Hap2 and Norton Hap1 were both contiguous yet varied in length at 1.43 Mb and 1.7 Mb, respectively. Lastly, the MN1264 Hap2 haplotype was not contiguous and due to its novelty within the study was aligned to the PN_T2T assembly contig ‘PN18’, resulting in a scaffolded region of 1.27 Mb consisting of two contigs, spaced 12.6 kb apart (Figure S3). The extracted genomic sequences for Rpv3/Rpv27 haplotypes, including those scaffolded for this analysis, are provided in Additional Data 1.

Visualization of the extracted Rpv3/Rpv27 regions of each haplotype, specifically the distribution of the SSR markers and predicted gene structure, allowed for the resolution of existing sequence variation within and among haplotype alleles. While the gene structure between individuals carrying the same Rpv3 allele differed, all haplotypes became more consistent as they approached UDV737, especially within allelic groups (Figure 1).

Figure 1. Rpv3 and Rpv27 region SSR alignments and annotated genes by SSR haplotype.

^a VVCS1: VVCS1H077H16R1-1; *Haplotypes labeled as Rpv3.3 were based solely on SSR fragment lengths and were unable to be confirmed via pedigree analysis. The black boxes on Chambourcin Primary and NY84.0101.03 Hap1 show the within-haplotype Rpv3.1 candidate gene region containing TNL1, TNL2a, TNL2b, and LRRk. Gray arrows within each haplotype are predicted gene feature annotations. The regional alignments are anchored to the position of SSR marker UDV737 (green) to facilitate comparison among haplotype alleles.

Three candidate genes have been found in connection to the resistance conferred by Rpv3.1. The first TNL, TNL2 was identified by Foria et al. (2020) as a pair of paralogs, TNL2a and TNL2b with the second TNL, TNL1, and a nearby LRRk added later by Wairich et al. (2022). The gene model identifiers were translated to the PN40024.v4 reference genome (Velt et al., 2023), functionally annotated, and aligned to the extracted Rpv3/Rpv27 regions. In every analyzed haplotype, the candidate genes clustered closely to UDV737, within 220 kb, with a single copy of the LRRk roughly 25 kb upstream (Figure S2).

Figure 2. Rpv3.1 candidate gene positions and predicted functionality by haplotype allele.

^a *Haplotypes labeled as Rpv3.3 were based solely on SSR fragment lengths and were unable to be confirmed via pedigree analysis. Gray arrows within each haplotype are predicted, non-candidate gene feature annotations. The letters next to some genes serve to distinguish multiple genes of the same type for comparisons between Figure 2 and Figure 3. The distinction between complete, partial, and pseudogene was based on in-silico prediction using NLRexpress. Each region is anchored to the position of SSR marker UDV737 (green bar) to allow for comparison among haplotype alleles.