Long-read, chromosome-scale assembly of Vitis rotundifolia cv. Carlos and its unique resistance to Xylella fastidiosa subsp. fastidiosa
BMC Genomics volume 24, Article number: 409 (2023)
Muscadine grape (Vitis rotundifolia) is resistant to many of the pathogens that negatively impact the production of common grape (V. vinifera), including the bacterial pathogen Xylella fastidiosa subsp. fastidiosa (Xfsf), which causes Pierce’s Disease (PD). Previous studies in common grape have indicated Xfsf delays host immune response with a complex O-chain antigen produced by the wzy gene. Muscadine cultivars range from tolerant to completely resistant to Xfsf, but the mechanism is unknown.
We assembled and annotated a new, long-read genome assembly for ‘Carlos’, a cultivar of muscadine that exhibits tolerance, to build upon the existing genetic resources available for muscadine. We used these resources to construct an initial pan-genome for three cultivars of muscadine and one cultivar of common grape. This pan-genome contains a total of 34,970 synteny-constrained entries containing genes of similar structure. Comparison of resistance gene content between the ‘Carlos’ and common grape genomes indicates an expansion of resistance (R) genes in ‘Carlos.’ We further identified genes involved in Xfsf response by transcriptome sequencing ‘Carlos’ plants inoculated with Xfsf. We observed 234 differentially expressed genes with functions related to lipid catabolism, oxidation-reduction signaling, and abscisic acid (ABA) signaling as well as seven R genes. Leveraging public data from previous experiments of common grape inoculated with Xfsf, we determined that most differentially expressed genes in the muscadine response were not found in common grape, and three of the R genes identified as differentially expressed in muscadine do not have an ortholog in the common grape genome.
Our results support the utility of a pan-genome approach to identify candidate genes for traits of interest, particularly disease resistance to Xfsf, within and between muscadine and common grape.
Muscadine (Vitis rotundifolia Michx., syn. Muscadinia rotundifolia 2n = 2x = 40) is a species of grape native to the southeastern United States (Supplemental Figure S1). It is part of the Muscadinia subgenus of Vitis, which contains only three species (Hickey et al., 2019). In comparison, the subgenus Euvitis (bunch grapes) exhibits greater diversity and includes about 60 species. Compared to the common table grape (V. vinifera; a member of the Euvitis subgenus), muscadine grapes are a small-market crop , but they are also more pest and drought tolerant . Based on phylogenetic evidence, the Vitis genus originated in North America 28 million years ago, and the Muscadinia subgenus diverged from Euvitis approximately 18 million years ago . Native Americans used the muscadine fruit both as a food source and a dye prior to European colonization, and wineries in the Southeast have produced special red and white wines from muscadine berries since the 16th century . Muscadine grapes have increased in commercial interest outside of culinary applications, including medicinal use and nutritional supplements, due to higher antioxidant content and an increased diversity of bioactive compounds relative to other grape species. Several studies demonstrate treatment of cancer cells in vitro with muscadine grape skin extracts (MGSEs) has been found to increase cytotoxicity and trigger apoptosis in breast cancer cells [5, 6] and prostate cancer cells . In the context of grape breeding, muscadines have attracted interest in providing a resource for beneficial traits to be introduced into bunch grape species. However, breeding efforts are complicated by the different number of chromosomes caused by fusion of two muscadine chromosomes in the Vitis vinifera genome [8, 9].
Pierce’s Disease (PD) has become a major limiting factor in the production of wine and table grapes in southern California, the center of winemaking in the continental United States (US). Symptoms include leaf necrosis, leaf scorch, and dieback, leading to vine death within three to five years (Supplemental Figure S2). PD is caused by the xylem-limited bacterial pathogen Xylella fastidiosa subsp. fastidiosa (Xfsf) and is spread by xylem-feeding sharpshooter family (Cicadelladea) insect vectors . Disease spread was limited by short flying distance and low feeding rate of the insect vector, however, the introduction of a more efficient vector in the 1990s, the glassy-winged sharpshooter (Homalodisca vitripennis), exacerbated the impact of PD in California . Xfsf is a gram-negative bacterium that is indigenous to the Gulf Coast region of the US, but it has since spread to other regions of both American continents as an invasive disease . The bacteria clog the xylem vessels of susceptible grape genotypes, leading to water and nutrient stress. PD symptoms usually appear in late summer during high temperatures or when the plants are exposed to drought conditions. In most muscadine grapes, PD is of little concern, with disease symptoms typically limited to occasional necrosis or marginal leaf burn on tolerant cultivars (Supplemental Figure S1).
Previous studies have found clues to the physiology and molecular basis of resistance to PD in both muscadines and common grapes. In comparison to susceptible bunch grapevines, tolerant muscadine cultivars infected with Xfsf produced higher frequencies of tyloses and gums, which occlude xylem vessels to encapsulate and prevent internal spread of the bacteria . In addition, gums and tannins occur more frequently in the PD-resistant muscadine grape cultivar ‘Noble’ after PD inoculation than in the PD-tolerant cultivar ‘Carlos’ [14, 15]. Furthermore, higher amounts of xylem sap β-1,3-glucanase and peroxidase was observed in PD-tolerant muscadine grapes compared to PD-susceptible bunch grapes . In PD, knockout of the wzy gene in Xfsf was observed to result in a tolerant host response . The wzy mutants were unable to produce a complex lipopolysaccharide O-antigen that, in wild-type Xfsf, delays host immune response. Comparison of infected and uninfected grape vines (var. Thompson Seedless) found an up-regulation of antioxidant strategies, cell wall modification enzymes, and pathogenesis-related proteins . Although structural barriers provide some degree of resistance/tolerance against pathogens, little is known about the relationship between muscadine grape anatomy and its tolerance to Xfsf.
At present, two muscadine genomes have been published. The first genome was a haplotype-phased, chromosome-level assembly of the male cultivar ‘Trayshed,’ representing about 86.2% of the estimated 484.3 Mbp genome size . The genome was used to verify the fusion of muscadine chromosome 7 and 20 in common grape as chromosome 7, previously only supported by muscadine linkage group evidence [20, 21]. Compared to the assembly of Cabernet Sauvignon, a disease-susceptible cultivar of bunch grape, the ‘Trayshed’ assembly had an expansion of Toll/Interleukin-1 Receptor-like Nucleotide-Binding Site Leucine-Rich Repeat (TIR-NBS-LRR) proteins, a class of disease resistance genes. A second assembly was recently published for the ‘Noble’ cultivar and represents 81.5% of the estimated total genome size . Additionally this study included a genome-wide association study (GWAS) which identified 52 quantitative trait nucleotides (QTNs) associated with 12 berry-related traits. Due to the similarity between Muscadinia and Euvitis genomes, these QTN markers can be used to study comparative genetics between species. As the dynamic of genomics shifts towards a pan-genome model, which covers genetic content across multiple cultivars of a species, these two assemblies can be used together to provide a more accurate understanding of the relationship between different cultivars [23, 24].
To examine gene-expression changes in response to inoculation with Xfsf, RNA-Seq was performed on the muscadine ‘Carlos,’ a bronze-skinned, commercial cultivar. Unlike most muscadine cultivars with PD resistance, Carlos is considered tolerant and may show symptoms . ‘Carlos’ is the predominant bronze-skinned juice cultivar and is generally selected for its high yield and dry stem scars. A ‘Carlos’ reference genome was constructed to improve the accuracy of the RNA-Seq data analysis and expand existing muscadine genomic resources. To establish a genomic framework for this comparison within and across species, we developed a pan-genome resource incorporating three muscadine genome assemblies and a common grape genome assembly. To further understand the differences in response between muscadine and common grapes, we compared our gene expression data to grapes inoculated with both wild-type Xfsf and wzy knock-out mutants .
Genome Assembly Information and Statistics.
Our ‘Carlos’ chromosome-scale assembly utilized PacBio CLR long-read sequencing to generate DNA scaffolds. The initial assembly consisted of 384 contigs arranged into 355 scaffolds with a total assembly size of 413 Mb and a contig N50 of 2.3 Mb (Supplemental Table S1). Reference-guided scaffolding of the initial ‘Carlos’ assembly to the ‘Trayshed’ haplotype 1 assembly  arranged the contigs into 76 scaffolds, including 20 chromosome-level scaffolds that contained 99% of the sequenced bases. After resolving gaps, the assembly consisted of 112 contigs in 76 scaffolds with a low percentage of uncalled bases (Table 1). The final assembly has a total sequence length of 413 Mb with the 20 assembled chromosomes representing 97.7% of the total length. Assessment of our assembly by benchmarking universal single-copy orthologs (BUSCOs) indicated the sequence was largely complete . This assembly is slightly longer than the 394 Mb for ‘Noble’ and 400 Mb for ‘Trayshed’, but all three are notably lower than the flow cytometry estimate of 483 Mb, suggesting the flow cytometry is likely an overestimate [19, 22].
Gene Annotation Report.
Using a combination of de novo repeat calling and known plant repeats, 51.12% of the ‘Carlos’ assembly was identified as repetitive (Supplemental Table S2). Long-terminal repeats (LTRs) were the most common repeats (15.96%), primarily consisting of Gypsy/DIRS1 (8.06%) and Ty1-Copia (7.33%) retrotransposon families. A total of 27,923 genes were identified in the ‘Carlos’ assembly, of which 27,747 (99.4%) of these genes were located on the 20 assembled chromosomes. The inclusion of RNA-Seq alignment data allowed GeMoMa to identify 40,219 gene isoforms , and tRNAscan identified 611 tRNAs across all 20 chromosomes . RNAmmer identified two nucleolus organizing regions (NORs) with 18 S-5.8 S-26 S (35 S) rDNA arrays on chromosomes 15 and 17, as well as two distinct 5 S rDNA regions on chromosome 17 (Fig. 1) .
Comparison of assembly to existing cultivars and common grape genome.
Overall chromosome size was consistent among all three assemblies, though chromosome 18 was notably longer in the ‘Trayshed’ assembly than in the other cultivar assemblies. Alignment of the ‘Carlos’ assembly to the existing ‘Trayshed’ and ‘Noble’ assemblies demonstrated an overall similar structure among cultivars (Fig. 1) [19, 22]. Most regions in all three genomes were syntenic, with 83.8% of the ‘Carlos’ genome syntenic with the ‘Trayshed’ assembly and 73.4% syntenic with the ‘Noble’ assembly (Table 2). In these syntenic comparisons with ‘Carlos’, ‘Trayshed’ had a higher number of sites with translocations compared to ‘Noble’. In contrast, the ‘Noble’ genome assembly had a larger number of inversions despite containing more syntenic regions with ‘Carlos’. Chromosome 8 in the ‘Carlos’ assembly had a gap in synteny in its alignments to both ‘Trayshed’ and ‘Noble’ at approximately 5 Mb.
Alignment of the ‘Carlos’ cultivar to the common grape genome, V. vinifera version 2.1 , showed strong collinearity between the two assemblies, and chromosome 7 in the common grape assembly was shown to be split between chromosomes 7 and 20 in the ‘Carlos’ assembly, consistent with previous muscadine cultivar assemblies and linkage maps (Fig. 2) [19, 21, 22, 30].
Comparison of the three muscadine and the common grape genome annotation sets with GENESPACE, version 0.9.4, produced a synteny-based pan-genome (Table 3, Supplemental Table S3) . GENESPACE predicted a total of 34,970 synteny-constrained orthogroups. Of these, 17,457 orthogroups contain at least one gene from each genome, representing the core Vitis gene set. Of these genes, 10,750 match exactly one gene from each genome, representing single-copy orthologs. The remaining 17,513 accessory orthogroups are missing gene(s) from at least one genome. 4,919 accessory orthogroups are represented by only genes from one genome, or “single-individual”. Of these, 4,321 have a single gene from a single genome, hereafter referred to as singleton genes, and 598 contained more than one gene from a single organism. Of the singletons, 426 were observed in ‘Carlos’, 607 were observed in ‘Noble’, 455 were observed in ‘Trayshed’, and 2,833 were observed in common grape. Annotation of the ‘Carlos’ singletons indicated the presence of RPV1 and RUN1, two muscadine disease-resistance genes , among other plant defense proteins (Supplemental Table S4), and annotation of the non-singleton, ‘Carlos’-only Orthogroups identified lignin-degrading proteins and germin-like proteins (Supplemental Table S4). The GENESPACE riparian synteny plot is consistent with the synteny plots produced by SyRi and RIdeogram (Supplemental Figure S3).
We ran RNAmmer, version 1.2 , on the assemblies for ‘Trayshed’, ‘Noble’, and common grape to compare predicted rDNA regions between other muscadine and grape assemblies (Fig. 1, Supplemental Tables S6-9). In the ‘Carlos’ assembly, recall from above there were two nucleolus organizing regions (NORs) found on chromosomes 15 and 17, and two 5S arrays were found on chromosome 17. In ‘Trayshed’, an NOR was only detectable on chromosome 17. In ‘Noble’, a single NOR on chromosome 15 was detected. We did not detect any complete NORs in the Version 2.1 assembly of the common grape genome (Fig. 2). Two 5S arrays along chromosome 17 were detected in all four assemblies.
We identified and classified predicted plant disease resistance-related genes (R-genes) in ‘Carlos’, ‘Trayshed’, ‘Noble’, and common grape to compare between tolerant, resistant, and susceptible individuals/species (Fig. 3). We first identified genes with R-gene relevant domains, including coiled-coil (CC), kinase, leucine rich repeat (LRR), nucleotide binding site (NBS), Toll/interleukin-1 receptor (TIR), and transmembrane helix domains. ‘Carlos’ contained a total of 3,522 genes containing domains associated with R-genes, a comparable number to ‘Trayshed’ (3,274), but higher than the number observed in ‘Noble’ (2,394) and common grape (2,332). These sets of genes were further categorized by domain combinations into common R-gene classes: receptor-like kinases (RLK), receptor-like proteins (RLP), TIR-NBS-LRRs (TNL), CC-NBS-LRRs (CNLs), and other NBS-LRRs (NLs). ‘Carlos’ and ‘Trayshed’ have similar gene counts in most classes, and are consistently higher than in ‘Noble’, except the TNL class. Common grape has the lowest number in all R-gene categories except NLs, where it has one gene more than ‘Noble’, yet still less than the other two muscadine cultivars. This reveals an overall trend of expansion of R-genes in muscadine grape species that is not present in common grape species. All four gene sets had a higher number of RLK and RLP class genes than other NBS-related classes (Supplemental Table S10).
We then identified orthogroups from GENESPACE that contained one or more R-gene candidates from ‘Carlos’. The 3,522 candidate R-genes were placed into 2,061 synteny-constrained orthogroups. Of these orthogroups, 250 were predicted to function as RLKs, 303 as RLPs, 84 as TNLs, 132 as CNLs, and 89 as other NLs. In comparing the number of genes in these orthogroups from ‘Carlos’ to those of common grape, we found that 518 of these orthogroups contain genes novel to ‘Carlos’, and 392 contain more genes in ‘Carlos’ compared to grape. The orthogroups unique to ‘Carlos’ contained 64 genes predicted to function as RLKs, 109 as RLPs, 50 as TNLs, 54 as CNLs, and 37 as other NLs. Of the 392 orthogroups with more ‘Carlos’ genes, 65 are predicted to function as RLKs, 166 as RLPs, 36 as TNLs, 87 as CNLs, and 57 as other NLs. For the remaining orthogroups, 244 contained more genes in grape than in ‘Carlos’, and 907 contained an equal number of genes between ‘Carlos’ and grape. These numbers support that a number of R-genes have either undergone expansions in muscadine or are unique to muscadine.
Differential expression in response to Pierce’s Disease.
Four weeks after stem inoculation of Xfsf in ‘Carlos’ cultivar plants, RNA sequencing of leaf tissue revealed 234 differentially expressed (DE) genes compared to mock-inoculated control plants (Table 4, Supplemental Table S11). Of DE genes, 89 were up-regulated in infected ‘Carlos’ vines, and 145 were down-regulated. Annotation of these DE genes indicated a down-regulation of genes associated with lipid catabolism (mostly consisting of GDSL lipases), both up and down regulation of genes associated with oxidation-reduction, and an up-regulation of genes associated with ubiquitin, a proteolysis mediator associated with plant defense . Genes predicted to function as lignin-degrading laccases were down-regulated, possibly indicating a strengthening of the cell wall through lignin fortification . Genes associated with abscisic acid (ABA) biosynthesis, a pathway involved in drought resistance and stress response in plants , were mostly up-regulated, though some genes activated by ABA were down-regulated. Sixteen of the DE genes were found to have R-gene domains, 5 of which were assigned to the Kinase R-gene class, indicating a potential R-gene mediated plant defense response after inoculation with Xfsf (Tables 5, Supplemental Table S13). Of these candidate R-genes, 9 were down-regulated and 7 were up-regulated. VITMroCarlos_v1.3.g17552, an up-regulated gene with RLP domains, is predicted to function as Eix2, a receptor driving disease resistance in plants . GhostKOALA, version 2.2 , analysis of the differentially expressed genes indicated an enrichment of pathways associated with metabolism, cell signaling, and genetic processing. Up-regulated genes were associated with the Toll and immune deficiency (IMD) signaling pathways, ubiquitin-mediated proteolysis, and terpenoid biosynthesis. The Toll and IMD pathways are consistent with up-regulation of R-genes containing Toll domains, and plant terpenoids have well-characterized roles in plant defense . Down-regulated genes were associated with biosynthesis of phenylpropanoids and other plant secondary metabolites, plant-pathogen interactions, and cell cycle processes.
In common grape, tolerance can be induced in otherwise susceptible cultivars by knockout of the wzy gene in Xfsf, which results in truncation of the complex, O-chain antigen that delays PAMP-triggered immunity (PTI) [17, 39]. It is currently unclear how muscadine grapes, which have greater tolerance to wild type Xfsf, are able to bypass the O-chain antigen, or if it has an alternative method of tolerance. To gain insight into the possible differences or overlaps of these two grape species to Xsfs, we examined the overlap of the gene orthogroups between our dataset and data from Rapicavoli et al., 2018. Rapicavoli et al. 2018 reported an RNASeq experiment with inoculation of ‘Cabernet Sauvignon’ grapes with wild type WZY Xfsf, mutant wzy Xfsf, or control buffer. RNA sequencing in tissue both local and systemic to the site of inoculation spanned collection times ranging from hours to 4 weeks after inoculation . We selected data that mirrored our experimental design, i.e. systemic tissue collected four weeks after inoculation, for comparison.
Genes were compared between species by orthogroups, with each orthogroup representing a common ancestral grape gene for all orthogroup members. The 25,556 orthogroups identified by OrthoFinder analysis were filtered for orthogroups containing the genes of interest - including orthogroups containing two or more differentially expressed genes - and an overlap between all four conditions was determined (Fig. 4). Most orthogroups containing muscadine DE genes were unique to the muscadine response, with any grape gene members not reported as differentially expressed. Only 1 orthogroup, OG0000266, shared genes that were differentially expressed (all up-regulated) in all four comparisons (Table 6). Functional annotation of the muscadine DE gene defined it as a Dirigent protein, a plant defense gene associated with lignin biosynthesis [40, 41]. Four orthogroups contained genes that were differentially expressed in the three comparisons associated with greater tolerance to PD. Functional annotation of these orthogroups of interest identified more genes associated with plant pathogen response, including Snakin-1 and an auxin-binding protein (Table 6). Half of these genes were up-regulated in muscadine, and the other half were down-regulated. A full list of orthogroups, including those containing DE muscadine genes, is available in Supplemental Table S12.
The 16 differentially expressed R-gene candidates from the ‘Carlos’ DE gene set were placed into 13 orthogroups in this analysis. While most of these orthogroups contained genes from the common grape assembly, only the muscadine genes were differentially expressed in response to infection with Xfsf (Table 5, Supplemental Table S13). One orthogroup contained more genes in common grapes compared to ‘Carlos’, and 5 contained an equal number of genes between the two species. The remaining orthogroups contained more genes from ‘Carlos’ than in common grapes. Of these, 2 orthogroups - OG0013253 and OG0015829 - contained no genes from the common grape annotation, indicating proteins novel to the muscadine annotation (Table 5, bold; Supplemental Table S13, bold). The R-genes in OG0013253 are located on Chromosome 16, and the gene in OG0015829 is located on Chromosome 10. These R-genes are predicted to function as kinases - from both the annotation results and the R-gene analysis - with the R-genes of OG0013253 being down-regulated in response to Xfsf and the R-gene of OG0015829 being up-regulated. Another orthogroup, OG0005036, contained VITMroCarlos_v1.3.g17552, previously established as a predicted plant defense gene. Orthogroup analysis indicates an expansion in this family in ‘Carlos’ compared to common grapes, supporting an R-gene expansion.
Pierce’s Disease, caused by the gram-negative bacteria Xylella fastidiosa subsp. fastidiosa, represents a limiting factor in the production of Californian common grapes, yet is inconsequential to wild and commercial muscadine grapes grown as a native food crop in the Southeast. While current understanding of muscadine’s mechanisms of tolerance and resistance is limited, current research suggests that muscadine grapes produce novel proteins that improve host tolerance  or produce tyloses to prevent spread of the bacteria . A better understanding of genetic and molecular resistance mechanisms will allow researchers to improve tolerance in common grape varieties through either gene introgression or selection of traits through breeding. To that end, we have worked to expand our existing knowledge of the muscadine genome and PD tolerance by providing a genome assembly and Xfsf response transcriptome for the tolerant cultivar ‘Carlos’.
The ‘Carlos’ muscadine genome assembly is the third public reference genome from muscadine, following the ‘Trayshed’ and ‘Noble’ reference genomes [19, 22]. Using the ‘Trayshed’ assembly as a guide, our assembly anchored over 99% of sequenced bases into 20 chromosome-level scaffolds. By default, this method resulted in chromosome-level scaffolds, but still containing a number of gap regions. We were able to resolve a number of the gaps using raw PacBio reads, resulting in an assembly with a low percentage of uncalled bases. Structural comparison of the ‘Carlos’ assembly to the ‘Trayshed’ assembly showed that, because of the high-quality contig assembly, we were able to identify structural variants between the two assemblies . Comparison of ‘Carlos’ with the ‘Noble’ assembly is consistent with the collinear analysis from the ‘Noble’ paper , finding most structural variants previously observed between ‘Noble’ and ‘Trayshed’ were also identified when comparing ‘Carlos’ and ‘Noble’. Due to the ‘Noble’ assembly utilizing a combination Illumina and PacBio CLR reads - as opposed to our PacBio CLR-only approach -it is likely that some of these structural variations may be artifacts of a lower contiguity assembly in ‘Noble’. Supporting this, the contig N50 of the ‘Noble’ assembly is 105.8 KB, while the contig N50 for the ‘Carlos’ assembly is 11.3 MB. Recent developments in long-read sequencing, in particular the PacBio HiFi technology, have resulted in more accurate assemblies using long-reads alone than either hybrid- or short-read assemblies only [43, 44]. As accurate long-read sequencing becomes more common, future genome assemblies will provide more accurate insights into true structural variation, as genome assembly quality is a key factor in these pan-genome type comparisons .
Ribosomal gene arrays (rDNA arrays) are fundamental parts of genome architecture with high tandem copy numbers. In plants, the two array types − 5 and 35 S (18 S-5.8 S-26 S) - can occur in one or a few locations in the genome. In Vitis species, rDNA arrays have been used to profile chromosome architecture and to infer phylogenies, and results of the available assemblies indicate the number and length of both array types varies within V. vinifera cultivars and across Vitis species [46, 47]. These arrays represent a challenge to most assembly algorithms, as their total length may exceed even long read sequencing reads. We compared placement of rDNA arrays along the ‘Carlos’ assembly to those of existing muscadine and grape cultivars. The two 5 S rDNA locations on ‘Carlos’ chromosome 17 were shared with the ‘Trayshed’ and ‘Noble’ muscadine assemblies, but each of the existing genomes missed one of the two 35 S arrays we identified in ‘Carlos’. Often, rDNA array presence was associated with large structural variations (Fig. 1). For example, the NOR along Chromosome 15 in ‘Carlos’ is associated with a large inversion between the ‘Carlos’ and ‘Noble’ assemblies. These differences in rDNA array locations between otherwise similar genomes highlight the difficulty in capturing these repetitive regions in genome assemblies. Similarly, we found that NORs could not be reliably found in the common grapevine version 2.1 genome. Further cytogenetic work in these specific cultivars is needed to confirm the actual 5S and 35S loci locations, confirm nearby structural variants (or indicate errors in assembly), and tie the reference genomes back to the chromosomal karyotypes.
There are over 100 cultivars of muscadine available in and beyond its native range . Recent studies have found substantial genetic diversity within muscadine cultivars [49, 50]. Attempting to use a single one of the three available genomes to characterize every cultivar will result in gaps in knowledge due to the phenotypic and genotypic differences between each cultivar [23, 51]. The solution is to further develop resources for these three cultivars - as well as additional cultivars - to develop a pan-genome. Pan-genomes cover the complete gene content of multiple cultivars of a single species, or multiple species of a closely-related genus. The pan-genome approach has been applied with great success to common crops such as maize, tomato, and rice [52,53,54,55]. In the context of Vitis species, pan-genome data can be used to better understand the mechanisms driving disease resistance, drought tolerance, and other traits of interest. For this study, we produced a foundational muscadine pan-genome consisting of the three muscadine cultivar annotations, as well as the common grape annotation. Of the 34,970 synteny-constrained orthogroups, almost half contain at least one gene from all four annotations, representing the “core” gene set across all four genomes. The other orthogroups represent “accessory” genes, including 4,919 orthogroups containing only genes from a single genome. As more grape, muscadine, and other Vitis genomes continue to be produced, we will add new Vitis genomes to our pan-genome, and our understanding of what genes are currently considered “core” and “accessory” to evolve.
In common grapes, pattern-associated molecular pattern (PAMP)-triggered immunity (PTI) is delayed by a long O-chain antigen associated with lipopolysaccharides (LPSs) in Xfsf. Knock-out of the wzy gene results in grapevines mounting a faster immune response and having greater tolerance to infection . By contrast, muscadine cultivars range from tolerant to resistant against the bacteria, indicating that it is successfully detecting the pathogen by overcoming the O-chain antigen or utilizing another mechanism of detection. Plant resistance (R) genes are of particular importance to pathogen detection and response signaling , and the immunity observed in muscadine cultivars could be attributed to an expanded suite of R-genes compared to the R-genes of common grapes . The ‘Trayshed’ assembly paper reported an expansion of R genes in muscadine grapes compared to common grapes . Analysis of R-genes in the ‘Carlos’ assembly supports this expansion, with more R-genes identified in the ‘Carlos’ assembly than in ‘Trayshed’. The Trayshed genome paper described an expansion of Toll/Interleukin-1 Receptor-like Nucleotide-Binding Site Leucine-Rich Repeat (TIR-NBS-LRR) genes compared to the common grape. R-gene analysis of our gene annotation supported this expansion and indicated a nearly two-fold expansion of TIR-NBS-LRR genes in ‘Carlos’ compared to ‘Trayshed’. The ‘Noble’ assembly appears to have a contraction in most R-genes compared to other muscadine cultivars, though this may be the result of a different annotation strategy and deserves further exploration. Despite the overall lower number of R-genes compared to the other cultivars, ‘Noble’ had more proteins containing TIR-NBS-LRR domains compared to the ‘Trayshed’ assembly. The disparity in R-genes identified in Noble may be attributed to a lower quality assembly. An enriched set of R-genes in muscadine grape genomes supports the notion of improved disease resistance mechanisms in muscadine compared to the common grape. A recent GWAS (genome wide association study) in V. arizonica identified eight genomic regions associated with PD resistance, many containing R genes . The authors attempted to extend their results to an additional six wild Vitis species using a k-mer bioinformatics approach. They identified a greater overlap of resistance-associated k-mers between closely-related species than between more distant species, suggesting that the resistance trait evolved independently and more than once . Further work will be needed to tease out candidates in V. rotundifolia associated with improved response to infection with Xfsf.
The results of our gene expression analysis in ‘Carlos’, reveal that abiotic stress response, particularly drought tolerance, were up-regulated in ‘Carlos’ inoculated with Xfsf, with a few up-regulated genes associated with defense. Of these defense genes, we observed a candidate gene, VITMroCarlos_v1.3.g17552, that showed similarity to EIX2, a receptor-like protein that confers resistance to a fungal pathogen of tomato after detecting an elicitor protein . While LPSs are well-characterized elicitors in common grapes, they are not the only PAMP that can induce PTI . Orthogroup placement of this gene indicates an expansion in this family compared to grape, which could point to an expanded suite of PAMP response elements in muscadine compared to common grape (Table 5). It is important to note that, instead of samples collected from the site of inoculation, our sequencing data measured systemic responses to Xfsf inoculation. These results, therefore, offer no conclusions on parenchymal cell response in the xylem, where initial detection of Xfsf occurs. Further research will be needed to fully characterize the muscadine response to inoculation with Xfsf. In particular, it will need to be determined if the tolerance observed in ‘Carlos’, as opposed to resistance in other cultivars, can be attributed to a different immune response, or if it is the result of greater drought tolerance compared to common grapes.
To contextualize how these observed genes overlap with the common grape response to Xfsf, we compared the gene expression of ‘Carlos’ muscadines inoculated with Xfsf to grapes inoculated with two strains: a wild-type strain and a wzy-knockout strain . Rather than attempting to map reads from one organism to the genome of another, we utilized OrthoFinder to identify genes with a common ancestor of both species. Of the 208 orthogroups containing differentially expressed genes in muscadine, 197 were unique to the muscadine response to PD. Of note, 19 of these orthogroups contained no genes from the common grape annotation, suggesting them as interesting candidates for follow up study. For the remaining orthogroups with both muscadine and common grape members, unique changes in gene sequence or expression patterns may have emerged after the organisms diverged evolutionarily to drive a unique host response to PD.
Despite the variation in host susceptibility, some overlaps in DE genes between muscadine and common grape were found. One orthogroup contained genes that were differentially expressed across all four conditions, and four contained genes that were differentially expressed among all PD-tolerant responses (Fig. 4). The single orthogroup representing all four conditions, OG0000266, was annotated as a Dirigent (DIR) protein. Among other important functions, plant (DIR) proteins play a role in plant defense response by modulating cell wall metabolism through lignin accumulation . In muscadine, this gene was up-regulated in response to inoculation with Xfsf. The four orthogroups associated with PD-tolerant responses were also functionally annotated as genes associated with plant pathogen responses [59,60,61,62]. Among these, the muscadine gene coding for a GDSL lipase was down-regulated, which is consistent with the observed DEGs. While GDSL lipases can play a role in plant defenses , some plant systems see improved host resistance when GDSL lipases are silenced . While there is overlap in genes differentially expressed in muscadines inoculated with X. fastidiosa subsp. fastidiosa, our main conclusion is that most differentially expressed genes were uniquely expressed in muscadine, supporting a unique host response not found in common grapes. While hybrids between common grapes and muscadine grapes have been produced, these efforts are made complicated by a chromosome-fusion event in common grapes. By identifying specific genes with high association to PD-resistance in muscadine, researchers will be better able to focus their efforts on candidate resistance genes.
Our efforts in this study have provided three major outcomes to advance the understanding of the muscadine grape. First, our ‘Carlos’ cultivar assembly adds to the existing genome reference sequences and gene annotation sets available for muscadine grapes. Second, our Vitis pan-genome provides a new resource for comparative genomics between muscadine cultivars and those of the common grape, while allowing room for expansion. Third, we provide a comparison of differentially expressed genes that are informative in comparing the immune response between muscadine and common grapes. There is more work to be done to fully characterize the muscadine response to Pierce’s disease, from an expanded time course at different times and locations after infection to host and pathogen knockout experiments. Our results have provided new resources to expand this understanding and provided a framework for future sequencing efforts.
Materials and methods
Plant sampling - DNA sequencing.
The muscadine grape clone ‘US19-33’, a self-pollinated accession of V. rotundifolia Michx., (2n = 2x = 40) cultivar ‘Carlos’ , maintained at the USDA ARS Southern Horticultural Research Laboratory in Poplarville, MS was selected for genomic DNA (gDNA) extraction (30.8402° N, 89.5342° W). Young leaves were collected from a single plant of clone ‘US19-33’ and used for gDNA extraction using a modified hexadecyltrimethylammonium bromide (CTAB) protocol (Nishiyama et al. 2021). Isolated gDNA was quantified with a Nanodrop 2000 spectrophotometer and a Qubit dsDNA HS assay kit (ThermoFisher Scientific, Waltham, MA), and gDNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Tech, Santa Clara, CA, USA). Ultimately, gDNA libraries for Illumina short-read sequencing were prepared, and paired-end sequencing (2 × 150 bp, 30× coverage) was performed on the Illumina NovaSeq 6000 sequencing platform (Illumina, San Diego, CA, USA).
To isolate high molecular weight (HMW) DNA suitable for PacBio long-read sequencing, young expanding leaves were dark-treated for 48 h prior to harvest and flash frozen in liquid nitrogen prior to gDNA extraction. Nuclei were isolated using the Bionano Prep Plant Tissue DNA Isolation kit (Bionano Genomics, SanDiego, CA, USA). Subsequently, HMW DNA was extracted from the nuclei using the Circulomics Nanobind Plant Nuclei Big DNA kit (Pacific Biosciences, Menlo Park, CA, USA). HMW DNA was used to construct libraries following the manufacturer’s protocol and sequenced using the continuous long read (CLR) protocol on the Pacific Biosciences (PacBio) sequencing platform (Pacific Bioscience, Menlo Park, CA, USA).
Plant sampling - RNA sequencing.
A total of 8 plantlets from tissue culture propagated muscadine grape cultivar ‘Carlos’ were transferred from PlantMedia Magenta boxes (PlantMedia, Dublin, OH, USA) into 1-gallon plastic pots containing a mixture of pine bark mulch and sand (1:1, v/v) with a pH of 5.6. Stems were needle-inoculated approximately 7 cm above the media surface with 10 µl of succinate-citrate buffer containing 106 cells of X. fastidiosa subsp. fastidiosa (Xfsf) strain NOB1 . Control plants were mock inoculated with 10 µl of succinate-citrate buffer. Plants were stored inside an incubator maintained at the following conditions: 25° C temperatures during 16 h photoperiods of 154 µmol m− 2s− 1 photon flux density, 22° C temperatures during 8 h skotoperiods, and 65% consistent relative humidity.
Approximately 0.1 g of leaf tissues were collected 28 days post-inoculation and used for DNA and RNA extraction. Total genomic DNA was extracted using a Cetyltrimethyl ammonium bromide (CTAB) method and used for the detection of Xfsf using RST31/RST33 primer pairs as described by Minsavage et al. 1994 . Total RNA was isolated from leaf tissues collected from control and PD-inoculated plants using the Spectrum plant total RNA kit protocol (Sigma-Aldrich, MO, USA). RNA quantity and purity were assessed with a Nanodrop 2000 spectrophotometer and a Qubit 2.0 Fluorometer (Thermofisher, Waltham, MA, USA), respectively, and RNA quality was evaluated using an Agilent 2100 Bioanalyzer (Agilent Tech, Santa Clara, CA, USA). RNA sequencing libraries were constructed using the NEBNextTM II Directional RNA library prep kit, and sequencing was performed on the Illumina NovoSeq 6000 platform (Illumina, San Diego, CA, USA).
Genome scaffolding and assembly.
CLR reads were initially assembled into contigs using MECAT2 version 2019.03.04 , which implemented CANU version 1.9 . Resulting contigs underwent one round of polishing with Arrow (Pacific BioSciences SMRT Tools Reference Guide, 2019), followed by additional polishing with Pilon . Circular contigs, which were labeled in the output of CANU v. 1.9, were removed from the assembly. We labeled this initial assembly version 1.1.
Following initial contig-joining, RagTag version 1.0.1  was used to align the ‘Carlos’ contigs to the previously published ‘Trayshed’ assembly (focusing on haplotype 1). This produced a chromosome-level assembly with gap regions, labeled assembly version 1.2. These gaps were resolved with raw PacBio reads by running TGS-GapCloser, version 1.1.1 , producing our final assembly, labeled version 1.3. Benchmarking Universal Single-Copy Orthologs (BUSCO), version 5.2.2, was run to assess the completeness of the ‘Carlos’ cultivar assembly . Statistical analysis of the scaffolds was performed using the BBtools “stats” command . Repetitive regions were identified using RepeatModeler  and softmasked using RepeatMasker . RNA-associated repetitive sequences were left unmasked.
Gene Model Maker (GeMoMa) version 1.8 was used to predict the ‘Carlos’ gene annotation using the existing annotation for the ‘Trayshed’ haplotype 1 genome assembly . GeMoMa includes an option to enhance the lift-over by incorporating RNASeq read alignment files, which we used to include the PD-inoculation transcriptome data in this analysis. Thereafter, input RNASeq reads were trimmed with Skewer version 0.2.2 , and trimmed reads were aligned to the ‘Carlos’ assembly with STAR version 2.7.9a . All alignment files were merged into a single file using the Samtools version 1.10 “merge” command . In addition to including the alignment data, GeMoMa was run with options to include untranslated regions (UTRs) and other features from the ‘Trayshed’ annotation. Identified genes were renamed, and gFACs version 1.1.2 was run without any filtering parameters to produce updated coding sequence (CDS) and amino acid (AA) FASTA files . Non-coding RNA features such as rRNA and tRNA were annotated using RNAmmer version 1.2  and tRNAScan version 2.0.9 [27, 78].
Comparative genomics of ‘Carlos’ to other genomes.
The ‘Carlos’ chromosomes were aligned to the ‘Trayshed’ haplotype 1 chromosomes and the ‘Noble’ chromosomes using MiniMap2 version 2.24 with default settings . The resulting alignment files were run through synteny and rearrangement identifier (SyRI), version 1.6.3, to identify any structural rearrangements . The resulting output was placed into SyRI’s sister program PlotSR, version 0.5.4, to visualize all structural rearrangements between ‘Carlos’ and the aforementioned ‘Noble’ and ‘Trayshed’ genomes . In-depth gene annotation comparison and initial pan-genome construction was performed using GENESPACE, version 0.9.4 . The common grape (Vitis vinifera), version 2.1, annotation was included in the GENESPACE analysis . A custom script was developed to obtain statistics from the GENESPACE results . Plant disease resistance-related genes (R-genes) were identified in these four genomes using the Disease Resistance Analysis and Gene Orthology (DRAGO2) pipeline .
Due to grape and muscadine having a different total number of chromosomes, SyRI could not be run to compare assembly structure. Instead, Orthofinder version 2.3.12 was used to identify orthologs between ‘Carlos’ muscadine and common grape . Protein sets from a total of 7 other species, as well as the ‘Trayshed’ haplotype 1 annotation, were included as outgroups to enhance OrthoFinder phylogenetic results based on the suggested best practices in the OrthoFinder documentation . Multiple sequence alignment (MSA) mode was selected using MAFFT version 7.467 to obtain MSA files . Orthogroups with a single gene member from both ‘Carlos’ and common grape were selected [87, 88], and the related orthologous links were plotted using RIdeogram version 0.2.2 to visualize synteny between the genomes .
Differential expression analysis.
The ‘Carlos’ RNASeq data was trimmed using Skewer, version 0.2.2, with identical parameters to those used in the gene annotation Sect. . STAR, version 2.7.9a , was rerun for the muscadine samples against the ‘Carlos’ genome with gene annotation included. Following alignment, gene features were associated with read counts using HTSeq-Count version 0.13.5 . Differential expression was determined using DESeq2 version 1.26.0 analysis of the read count data . Expression was compared between the control samples and the inoculated samples. Significantly differentially expressed genes were called with an adjusted p-value of 0.05. DRAGO2 was rerun on the differentially expressed genes to identify candidate R-genes.
Common grapevine RNASeq data were obtained from a previous study that confirmed the effect of wzy knockout in Xfsf, strain Temecula1, on host response (NCBI accession: PRJNA345471) . Due to the difference in strains, we compared the existing annotations of both strains to confirm that no significant structural variation occurred in Wzy between the strains. To match the conditions of our ‘Carlos’ RNASeq data, samples were only included if collected 4 weeks after inoculation from petioles. A total of 9 samples were analyzed: 3 representing control, 3 representing infection with wild-type Temecula1 strain Xfsf, and 3 representing infection with mutant wzy knockout Temecula1 strain Xfsf. For both sets of samples, Skewer, version 0.2.2, was run using identical parameters to those used in the gene annotation section. The same version and parameters of STAR were run to align the reads with the V. vinifera genome, version 2.1. For the common grape data, expression was compared between the control and inoculated samples, the inoculated and mutant samples, and the control and mutant samples (SRR4345376-SRR4345378, SRR4345409-SRR4345411, and SRR4345433-SRR4345435) using the same adjusted p-value to call differentially expressed genes.
To compare the expression of genes in ‘Carlos’ muscadine to common grape, the results of the earlier OrthoFinder run were used. Orthogroups containing significantly differentially expressed genes from analyses were identified. Functional gene annotation of ‘Carlos’ was determined using EnTAP version 0.10.8 , which assigned functions based on sequence similarity, Gene Ontology (GO) term assignment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. The Muscadine proteins of interest were also functionally annotated using KEGG’s BlastKOALA and GhostKOALA functions . Impacted pathways were determined by running KEGG separately on up- and down-regulated genes.
The ‘Carlos’ raw PacBio reads and RNA reads are available in NCBI under Bioproject PRJNA935741. The genome assembly and annotation are available in DOI https://doi.org/10.5281/zenodo.7944874.
Benchmarking Universal Single-Copy Ortholog
Continuous Long Read
Cetyltrimethyl ammonium bromide
Genome-Wide Association Study
Kyoto Encyclopedia of Genes and Genomes
Muscadine Grape Skin Extracts
Multiple Sequence Alignment
Nucleolus Organizing Region
Pathogen-Associated Molecular Pattern
Quantitative Trait Nucleotides
- Xfsf :
Xylella fastidiosa subsp. fastidiosa
Hoffmann M. Muscadine grape production guide for the Southeast. 2020.
Hopkins DL, Mollenhauer HH, Mortensen JA. Tolerance to Pierce’s Disease and the Associated Rickettsia-like Bacterium in Muscadine Grape1. J Am Soc Hortic Sci. 1974;99:436–9.
Wan Y, Schwaninger HR, Baldo AM, Labate JA, Zhong G-Y, Simon CJ. A phylogenetic analysis of the grape genus (Vitis L.) reveals broad reticulation and concurrent diversification during neogene and quaternary climate change. BMC Evol Biol. 2013;13:141.
Reimer FC, Hume HH, Michels J, Smith RI, Curtis RS. Scuppernong and other Muscadine grapes: origin and importance. North Carolina Agricultural Experiment Station of the College of Agriculture and Mechanic Arts; 1906.
Mendonca P, Darwish AG, Tsolova V, El-Sharkawy I, Soliman KFA. The anticancer and antioxidant Effects of Muscadine grape extracts on racially different triple-negative breast Cancer cells. Anticancer Res. 2019;39:4043–53.
Luo J, Song S, Wei Z, Huang Y, Zhang Y, Lu J. The comparative study among different fractions of muscadine grape “Noble” pomace extracts regarding anti-oxidative activities, cell cycle arrest and apoptosis in breast cancer. Food Nutr Res. 2017;61:1412795.
Ignacio DN, Mason KD, Hackett-Morton EC, Albanese C, Ringer L, Wagner WD, et al. Muscadine grape skin extract inhibits prostate cancer cells by inducing cell-cycle arrest, and decreasing migration through heat shock protein 40. Heliyon. 2019;5:e01128.
Lu J, Schell L, Ramming DW, INTERSPECIFIC HYBRIDIZATION BETWEEN, VITIS ROTUNDIFOLIA AND VITIS VINIFERA AND EVALUATION OF THE HYBRIDS. Acta Hortic. 2000;:481–6.
Patel GI, Olmo HP. Cytogenetics of Vitis: I. The hybrid v. vinifera x v. Rotundifolia. Am J Bot. 1955;42:141–59.
Krivanek AF, Walker MA. Vitis Resistance to Pierce’s disease is characterized by Differential Xylella fastidiosa populations in Stems and Leaves. Phytopathology. 2005;95:44–52.
Blua MJ, Phillips PA, Redak RA. A new sharpshooter threatens both crops and ornamentals. Calif Agric. 1999;53:22–5.
Hopkins DL, Purcell AH. Xylella fastidiosa: cause of Pierce’s Disease of Grapevine and other Emergent Diseases. Plant Dis. 2002;86:1056–66.
Mollenhauer HH, Hopkins DL. Xylem morphology of Pierce’s disease-infected grapevines with different levels of tolerance. Physiol Plant Pathol. 1976;9:95–100.
Fry SM, Milholland RD. Response of resistant, tolerant, and susceptible grapevine tissues to invasion by the Pierce’s disease bacterium, Xylella fastidiosa. Phytopathology. 1990;80:66–9.
Fry SM, Milholland RD, Others. Multiplication and translocation of Xylella fastidiosa in petioles and stems of grapevine resistant, tolerant, and susceptible to Pierce’s disease. Phytopathology. 1990;80:61–5.
Basha SM, Mazhar H, Vasanthaiah HKN. Proteomics approach to identify unique xylem sap proteins in Pierce’s disease-tolerant Vitis species. Appl Biochem Biotechnol. 2010;160:932–44.
Rapicavoli JN, Blanco-Ulate B, Muszyński A, Figueroa-Balderas R, Morales-Cruz A, Azadi P, et al. Lipopolysaccharide O-antigen delays plant innate immune recognition of Xylella fastidiosa. Nat Commun. 2018;9:390.
Zaini PA, Nascimento R, Gouran H, Cantu D, Chakraborty S, Phu M et al. Molecular profiling of Pierce’s Disease outlines the response circuitry of Vitis vinifera to Xylella fastidiosa infection. Front Plant Sci. 2018;9.
Cochetel N, Minio A, Massonnet M, Vondras AM. Diploid chromosome-scale assembly of the Muscadinia rotundifolia genome supports chromosome fusion and disease resistance gene expansion during Vitis and ?. G3. 2021.
Blanc S, Wiedemann-Merdinoglu S, Dumas V, Mestre P, Merdinoglu D. A reference genetic map of Muscadinia rotundifolia and identification of Ren5, a new major locus for resistance to grapevine powdery mildew. Theor Appl Genet. 2012;125:1663–75.
Lewter J, Worthington ML, Clark JR, Varanasi AV, Nelson L, Owens CL, et al. High-density linkage maps and loci for berry color and flower sex in muscadine grape (Vitis rotundifolia). Theor Appl Genet. 2019;132:1571–85.
Park M, Vera D, Kambrianda D, Gajjar P, Cadle-Davidson L, Tsolova V, et al. Chromosome-level genome sequence assembly and genome-wide association study of Muscadinia rotundifolia reveal the genetics of 12 berry-related traits. Hortic Res. 2022. https://doi.org/10.1093/hr/uhab011.
Bayer PE, Golicz AA, Scheben A, Batley J, Edwards D. Plant pan-genomes are the new reference. Nat Plants. 2020;6:914–20.
Della Coletta R, Qiu Y, Ou S, Hufford MB, Hirsch CN. How the pan-genome is changing crop genomics and improvement. Genome Biol. 2021;22:3.
Seppey M, Manni M, Zdobnov EM. BUSCO: assessing Genome Assembly and Annotation Completeness. Methods Mol Biol. 2019;1962:227–45.
Keilwagen J, Hartung F, Grau J. GeMoMa: homology-based gene prediction utilizing intron position conservation and RNA-seq data. Methods Mol Biol. 2019;1962:161–77.
Chan PP, Lowe TM. tRNAscan-SE: searching for tRNA genes in genomic sequences. Methods Mol Biol. 2019;1962:1–14.
Lagesen K, Hallin P, Rødland EA, Staerfeldt H-H, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007;35:3100–8.
Jaillon O, Aury J-M, Noel B, Policriti A, Clepet C, Casagrande A, et al. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature. 2007;449:463–7.
Delame M, Prado E, Blanc S, Robert-Siegwald G, Schneider C, Mestre P, et al. Introgression reshapes recombination distribution in grapevine interspecific hybrids. Theor Appl Genet. 2019;132:1073–87.
Lovell JT, Sreedasyam A, Eric Schranz M, Wilson M, Carlson JW, Harkess A et al. GENESPACE tracks regions of interest and gene copy number variation across multiple genomes. eLife. 2022;11.
Feechan A, Anderson C, Torregrosa L, Jermakow A, Mestre P, Wiedemann-Merdinoglu S, et al. Genetic dissection of a TIR-NB-LRR locus from the wild north american grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine. Plant J. 2013;76:661–74.
Sharma B, Joshi D, Yadav PK, Gupta AK, Bhatt TK. Role of ubiquitin-mediated degradation system in Plant Biology. Front Plant Sci. 2016;7:806.
Christopher LP, Yao B, Ji Y. Lignin biodegradation with Laccase-Mediator Systems. Front Energy Res. 2014;2.
Muhammad Aslam M, Waseem M, Jakada BH, Okal EJ, Lei Z, Saqib HSA et al. Mechanisms of abscisic acid-mediated Drought stress responses in plants. Int J Mol Sci. 2022;23.
Ron M, Avni A. The receptor for the fungal elicitor ethylene-inducing xylanase is a member of a resistance-like gene family in tomato. Plant Cell. 2004;16:1604–15.
Kanehisa M, Sato Y, Morishima K. BlastKOALA and GhostKOALA: KEGG Tools for functional characterization of genome and metagenome sequences. J Mol Biol. 2016;428:726–31.
Singh B, Sharma RA. Plant terpenes: defense responses, phylogenetic analysis, regulation and clinical applications. 3 Biotech. 2015;5:129–51.
Clifford JC, Rapicavoli JN, Caroline Roper MA, Rhamnose-Rich O-Antigen. Mediates adhesion, virulence, and host colonization for the Xylem-Limited Phytopathogen Xylella fastidiosa. Mol Plant-Microbe Interactions®. 2013;26:676–85.
Li N, Zhao M, Liu T, Dong L, Cheng Q, Wu J, et al. A novel soybean Dirigent Gene GmDIR22 contributes to Promotion of Lignan Biosynthesis and enhances resistance to Phytophthora sojae. Front Plant Sci. 2017;8:1185.
Paniagua C, Bilkova A, Jackson P, Dabravolski S, Riber W, Didi V, et al. Dirigent proteins in plants: modulating cell wall metabolism during abiotic and biotic stress exposure. J Exp Bot. 2017;68:3287–301.
Katam R, Chibanguza K, Latinwo LM, Smith D. Proteome biomarkers in Xylem Reveal Pierce’s disease tolerance in grape. J Proteom Bioinform. 2015;8:217–24.
Hon T, Mars K, Young G, Tsai Y-C, Karalius JW, Landolin JM, et al. Highly accurate long-read HiFi sequencing data for five complex genomes. Sci Data. 2020;7:399.
Rayamajhi N, Cheng C-HC, Catchen JM. Evaluating Illumina-, Nanopore-, and PacBio-based genome assembly strategies with the bald notothen, Trematomus borchgrevinki. G3. 2022;12.
Mascher M, Wicker T, Jenkins J, Plott C, Lux T, Koh CS, et al. Long-read sequence assembly: a technical evaluation in barley. Plant Cell. 2021;33:1888–906.
Castro C, Carvalho A, Pavia I, Leal F, Moutinho-Pereira J, Lima-Brito J. Nucleolar activity and physical location of ribosomal DNA loci in Vitis vinifera L. by silver staining and sequential FISH. Sci Hortic. 2018;232:57–62.
Pierozzi NI. Karyotype and NOR-banding of mitotic chromosomes of some Vitis L. species. Revista Brasileira de Fruticultura. 2011;33:564–70.
Andersen PC, Sarkhosh A, Huff D, Breman J. The Muscadine grape (Vitis rotundifolia Michx): HS763/HS100, rev. 10/2020. EDIS. 2020.
Buck K, Worthington M. Genetic diversity of wild and cultivated muscadine grapes (Vitis rotundifolia Michx). Front Plant Sci. 2022;13:852130.
Cao S, Stringer S, Gunawan G, McGregor C, Conner PJ. Genetic diversity and pedigree analysis of muscadine grape using SSR markers. J Am Soc Hortic Sci. 2020;145:143–51.
Hufford MB, Seetharam AS, Woodhouse MR, Chougule KM, Ou S, Liu J, et al. De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes. Science. 2021;373:655–62.
Gao L, Gonda I, Sun H, Ma Q, Bao K, Tieman DM, et al. The tomato pan-genome uncovers new genes and a rare allele regulating fruit flavor. Nat Genet. 2019;51:1044–51.
Woodhouse MR, Cannon EK, Portwood JL 2nd, Harper LC, Gardiner JM, Schaeffer ML, et al. A pan-genomic approach to genome databases using maize as a model system. BMC Plant Biol. 2021;21:385.
Zhao Q, Feng Q, Lu H, Li Y, Wang A, Tian Q, et al. Pan-genome analysis highlights the extent of genomic variation in cultivated and wild rice. Nat Genet. 2018;50:278–84.
Zhou Y, Chebotarov D, Kudrna D, Llaca V, Lee S, Rajasekar S, et al. A platinum standard pan-genome resource that represents the population structure of asian rice. Sci Data. 2020;7:113.
Pandolfi V, Neto JRCF, da Silva MD, Amorim LLB, Wanderley-Nogueira AC, de Oliveira Silva RL, et al. Resistance (R) genes: applications and prospects for plant biotechnology and breeding. Curr Protein Pept Sci. 2017;18:323–34.
Morales-Cruz A, Aguirre-Liguori J, Massonnet M, Minio A, Zaccheo M, Cochetel N, et al. Multigenic resistance to Xylella fastidiosa in wild grapes (Vitis sps.) And its implications within a changing climate. Commun Biol. 2023;6:580.
Burbank LP, Ochoa J. Evidence for elicitation of an oxidative burst in Vitis vinifera by Xylella fastidiosa Cold shock protein peptide csp20. PhytoFrontiers™. 2022;2:339–41.
Almasia NI, Nahirñak V, Hopp HE, Vazquez-Rovere C. Potato Snakin-1: an antimicrobial player of the trade-off between host defense and development. Plant Cell Rep. 2020;39:839–49.
Cândido E, de Pinto S, Pelegrini MFS, Lima PB, Silva TB, Pogue ON. Plant storage proteins with antimicrobial activity: novel insights into plant defense mechanisms. FASEB J. 2011;25:3290–305.
Gao M, Yin X, Yang W, Lam SM, Tong X, Liu J, et al. GDSL lipases modulate immunity through lipid homeostasis in rice. PLoS Pathog. 2017;13:e1006724.
Pei Y, Li X, Zhu Y, Ge X, Sun Y, Liu N et al. GhABP19, a Novel Germin-Like protein from Gossypium hirsutum, plays an important role in the regulation of resistance to Verticillium and Fusarium Wilt Pathogens. Front Plant Sci. 2019;10.
Lee DS, Kim BK, Kwon SJ, Jin HC, Park OK. Arabidopsis GDSL lipase 2 plays a role in pathogen defense via negative regulation of auxin signaling. Biochem Biophys Res Commun. 2009;379:1038–42.
Mavrodi OV, Mavrodi DV, Stafne ET, Adamczyk JJ, Babiker EM. Draft genome sequences of Xylella fastidiosa subsp. fastidiosa strains OK3, VB11, and NOB1, isolated from bunch and muscadine grapes grown in Southern Mississippi. Microbiol Resour Announc. 2020;9.
Minsavage GV, Thompson CM, Hopkins DL, Leite R, Stall RE. Others. Development of a polymerase chain reaction protocol for detection of Xylella fastidiosa in plant tissue. Phytopathology. 1994;84:456–61.
Xiao C-L, Chen Y, Xie S-Q, Chen K-N, Wang Y, Han Y, et al. MECAT: fast mapping, error correction, and de novo assembly for single-molecule sequencing reads. Nat Methods. 2017;14:1072–4.
Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017;27:722–36.
Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE. 2014;9:e112963.
Alonge M, Soyk S, Ramakrishnan S, Wang X, Goodwin S, Sedlazeck FJ, et al. RaGOO: fast and accurate reference-guided scaffolding of draft genomes. Genome Biol. 2019;20:224.
Xu M, Guo L, Gu S, Wang O, Zhang R, Peters BA et al. TGS-GapCloser: a fast and accurate gap closer for large genomes with low coverage of error-prone long reads. Gigascience. 2020;9.
Bushnell B. BBTools: a suite of fast, multithreaded bioinformatics tools designed for analysis of DNA and RNA sequence data. Joint Genome Institute; 2018.
Smit AFA, Hubley R, Green P. RepeatModeler Open-1.0. 2008–2015. Seattle, USA: Institute for Systems Biology available from: httpwww repeatmasker org. Last Accessed May. 2015;1:2018.
Smit AFA, Hubley R, Green P. RepeatMasker Open-4.0. 2013–2015. 2015.
Jiang H, Lei R, Ding S-W, Zhu S. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics. 2014;15:182.
Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21.
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25:2078–9.
Caballero M, Wegrzyn J, gFACs. Gene Filtering, Analysis, and Conversion to unify genome annotations across alignment and gene prediction frameworks. Genomics Proteom Bioinf. 2019;17:305–10.
Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997;25:955–64.
Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34:3094–100.
Goel M, Sun H, Jiao W-B, Schneeberger K. SyRI: finding genomic rearrangements and local sequence differences from whole-genome assemblies. Genome Biol. 2019;20:277.
Goel M, Schneeberger K. Plotsr: Visualising structural similarities and rearrangements between multiple genomes. Bioinformatics. 2022. https://doi.org/10.1093/bioinformatics/btac196.
Huff M. parse_pangenome.py at main · MattHuff/GENESPACE-Companion-Script. Github; 2023.
Osuna-Cruz CM, Paytuvi-Gallart A, Di Donato A, Sundesha V, Andolfo G, Aiese Cigliano R, et al. PRGdb 3.0: a comprehensive platform for prediction and analysis of plant disease resistance genes. Nucleic Acids Res. 2018;46:D1197–201.
Emms DM, Kelly S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 2019;20:238.
Emms D. OrthoFinder best practices. OrthoFinder Tutorials. 2019. https://davidemms.github.io/orthofinder_tutorials/orthofinder-best-practices.html. Accessed 29 Nov 2022.
Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–80.
Huff M, Seaman J, Wu D, Zhebentyayeva T, Kelly LJ, Faridi N, et al. A high-quality reference genome for Fraxinus pennsylvanica for ash species restoration and research. Mol Ecol Resour. 2022;22:1284–302.
Staton M, Addo-Quaye C, Cannon N, Yu J, Zhebentyayeva T, Huff M et al. “A reference genome assembly and adaptive trait analysis of Castanea mollissima ‘Vanuxem,’ a source of resistance to chestnut blight in restoration breeding.” Tree Genet Genomes. 2020;16.
Hao Z, Lv D, Ge Y, Shi J, Weijers D, Yu G, et al. RIdeogram: drawing SVG graphics to visualize and map genome-wide data on the idiograms. PeerJ Comput Sci. 2020;6:e251.
Anders S, Pyl PT, Huber W. HTSeq: Analysing high-throughput sequencing data with Python. 2010.
Love M, Anders S, Huber W. Differential analysis of count data–the DESeq2 package. Genome Biol. 2014;15:10–186.
Hart AJ, Ginzburg S, Xu M (sam), Fisher CR, Rahmatpour N, Mitton JB EnTAP, et al. editors. Bringing faster and smarter functional annotation to non-model eukaryotic transcriptomes. Mol Ecol Resour. 2020;20:591–604.
Support was provided by USDA-ARS research project numbers 6062-21000-010-000-D and 6066-21310-005-00-D. This research used resources provided by the SCINet project of the USDA ARS project number 0500-00093-001-00-D. This work was funded by USDA ARS Non-Assistance Cooperative Agreement number 6062-21000-010-013.
The authors declare no competing interests.
Ethics approval and consent to participate
The ‘Carlos’ cultivar is commonly cultivated and available for sale through muscadine retailers. The ‘Carlos’ individual used for this study was from the collection at the USDA-ARS Thad Cochran Southern Horticultural Laboratory, Poplarville, MS, USA 39470. Research with this plant material complied with all relevant institutional, national, and international guidelines and legislation.
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Supplemental Figure S1: Foliage and pollinated flowers of Vitis rotundifolia cv. ‘Carlos’ vines. Picture taken in Poplarville, MS
Supplemental Figure S3: Riparian plot of synteny between the 4 gene sets of the Vitis pangenome. Riparian plot generated using GENESPACE version 0.9.4
Supplemental Tables S1-S13: Summary of Early Forms of the Vitis rotundifolia ‘Carlos’ genome assembly. “Reference-Guided Scaffolds” refers to assembly before gap-closing
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Huff, M., Hulse-Kemp, A.M., Scheffler, B.E. et al. Long-read, chromosome-scale assembly of Vitis rotundifolia cv. Carlos and its unique resistance to Xylella fastidiosa subsp. fastidiosa. BMC Genomics 24, 409 (2023). https://doi.org/10.1186/s12864-023-09514-y