Multiple deletions of candidate effector genes lead to the breakdown of partial grapevine resistance to downy mildew.

Grapevine downy mildew, caused by the oomycete Plasmopara viticola (P. viticola, Berk. & M. A. Curtis; Berl. & De Toni), is a global threat to Eurasian wine grapes Vitis vinifera. Although resistant grapevine varieties are becoming more accessible, P. viticola populations are rapidly evolving to overcome these resistances. We aimed to uncover avirulence genes related to Rpv3.1-mediated grapevine resistance. We sequenced the genomes and characterized the development of 136 P. viticola strains on resistant and sensitive grapevine cultivars. A genome-wide association study was conducted to identify genomic variations associated with resistant-breaking phenotypes. We identified a genomic region associated with the breakdown of Rpv3.1 grapevine resistance (avrRpv3.1 locus). A diploid-aware reassembly of the P. viticola INRA-Pv221 genome revealed structural variations in this locus, including a 30 kbp deletion. Virulent P. viticola strains displayed multiple deletions on both haplotypes at the avrRpv3.1 locus. These deletions involve two paralog genes coding for proteins with 800-900 amino acids and signal peptides. These proteins exhibited a structure featuring LWY-fold structural modules, common among oomycete effectors. When transiently expressed, these proteins induced cell death in grapevines carrying Rpv3.1 resistance, confirming their avirulence nature. This discovery sheds light on the genetic mechanisms enabling P. viticola to adapt to grapevine resistance, laying a foundation for developing strategies to manage this destructive crop pathogen.

The wild side of grape genomics.

With broad genetic diversity and as a source of key agronomic traits, wild grape species (Vitis spp.) are crucial to enhance viticulture's climatic resilience and sustainability. This review discusses how recent breakthroughs in the genome assembly and analysis of wild grape species have led to discoveries on grape evolution, from wild species' adaptation to environmental stress to grape domestication. We detail how diploid chromosome-scale genomes from wild Vitis spp. have enabled the identification of candidate disease-resistance and flower sex determination genes and the creation of the first Vitis graph-based pangenome. Finally, we explore how wild grape genomics can impact grape research and viticulture, including aspects such as data sharing, the development of functional genomics tools, and the acceleration of genetic improvement.

Identification and Pathogenicity of Fusarium Species Associated with Young Vine Decline in California.

Grapevine trunk diseases are caused by a broad diversity of fungal taxa that have serious impacts on the worldwide viticulture industry due to significant reductions in vineyards yield and lifespan. Field surveys carried out from 2018 to 2022 in California nurseries and young vineyards revealed a high incidence of Fusarium. Since Fusarium species are important pathogens of other perennial crops, the present study aimed to identify and determine the pathogenicity of the Fusarium species on grapevines. Morphology of the fungal colonies coupled with multilocus phylogenetic analyses using nucleotide sequences of the translation elongation factor 1-alpha (tef1) and the RNA polymerase II second largest subunit (rpb2) genes revealed the occurrence of 10 species clustering in six species complexes, namely F. fujikuroi (FFSC), F. oxysporum (FOSC), F. solani (FSSC), F. sambucinum (FSAMSC), F. incarnatum-equiseti (FIESC), and F. tricinctum (FTSC) species complexes. The species F. annulatum (FFSC) was the most prevalent in samples from both symptomatic young vineyards (73.5% incidence) and nursery propagation material (62.5% incidence). Pathogenicity of the 10 most frequent species was confirmed by fulfilling Koch's postulates on living woody tissue of 1103 Paulsen rootstocks. Our results suggest that Fusarium spp. are involved in the development of young vine decline, probably as opportunistic pathogens when grapevines are under stress conditions.

Comparative Pangenomic Insights into the Distinct Evolution of Virulence Factors Among Grapevine Trunk Pathogens.

The permanent organs of grapevines (Vitis vinifera L.), like those of other woody perennials, are colonized by various unrelated pathogenic ascomycete fungi secreting cell wall-degrading enzymes and phytotoxic secondary metabolites that contribute to host damage and disease symptoms. Trunk pathogens differ in the symptoms they induce and the extent and speed of damage. Isolates of the same species often display a wide virulence range, even within the same vineyard. This study focuses on Eutypa lata, Neofusicoccum parvum, and Phaeoacremonium minimum, causal agents of Eutypa dieback, Botryosphaeria dieback, and Esca, respectively. We sequenced 50 isolates from viticulture regions worldwide and built nucleotide-level, reference-free pangenomes for each species. Through examination of genomic diversity and pangenome structure, we analyzed intraspecific conservation and variability of putative virulence factors, focusing on functions under positive selection and recent gene family dynamics of contraction and expansion. Our findings reveal contrasting distributions of putative virulence factors in the core, dispensable, and private genomes of each pangenome. For example, carbohydrate active enzymes (CAZymes) were prevalent in the core genomes of each pangenome, whereas biosynthetic gene clusters were prevalent in the dispensable genomes of E. lata and P. minimum. The dispensable fractions were also enriched in Gypsy transposable elements and virulence factors under positive selection (polyketide synthase genes in E. lata and P. minimum, glycosyltransferases in N. parvum). Our findings underscore the complexity of the genomic architecture in each species and provide insights into their adaptive strategies, enhancing our understanding of the underlying mechanisms of virulence. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A super-pangenome of the North American wild grape species.

BACKGROUND: Capturing the genetic diversity of wild relatives is crucial for improving crops because wild species are valuable sources of agronomic traits that are essential to enhance the sustainability and adaptability of domesticated cultivars. Genetic diversity across a genus can be captured in super-pangenomes, which provide a framework for interpreting genomic variations. RESULTS: Here we report the sequencing, assembly, and annotation of nine wild North American grape genomes, which are phased and scaffolded at chromosome scale. We generate a reference-unbiased super-pangenome using pairwise whole-genome alignment methods, revealing the extent of the genomic diversity among wild grape species from sequence to gene level. The pangenome graph captures genomic variation between haplotypes within a species and across the different species, and it accurately assesses the similarity of hybrids to their parents. The species selected to build the pangenome are a great representation of the genus, as illustrated by capturing known allelic variants in the sex-determining region and for Pierce's disease resistance loci. Using pangenome-wide association analysis, we demonstrate the utility of the super-pangenome by effectively mapping short reads from genus-wide samples and identifying loci associated with salt tolerance in natural populations of grapes. CONCLUSIONS: This study highlights how a reference-unbiased super-pangenome can reveal the genetic basis of adaptive traits from wild relatives and accelerate crop breeding research.

First Report of Binucleate Rhizoctonia AG-G Causing Grapevine (Vitis Vinifera) Trunk Diseases in California Nurseries.

Grapevine Trunk Diseases (GTD) are caused by a consortium of fungal pathogens that affect the biological functions of the vascular system of mature and young grapevines (Gramaje et al. 2018). We conducted surveys to profile GTD pathogens in California grapevine nurseries and collected 784 cuttings of cvs. Cabernet Sauvignon and Chardonnay grafted on 1103P rootstock. Several vines exhibited wood necrotic lesions and cankers at the graft union and the root ball (Figure 1A). Symptomatic wood tissues were cultured on PDA medium and after two weeks of incubation at room temperature (22°C), several known GTD pathogens were recovered. We also identified Rhizoctonia from 42 of the 784 vines (5.3% incidence) based on the morphological characteristics of a brown pigmented mycelium (Figure 1B), hyphae branched at a right angle with constrictions at the branch point (Figure 1C) and absence of spores (González García et al., 2006). A subsample of four isolates (DCHG2B, DCSG22R, JCSG9B, and JCHG12B) were randomly selected for further DNA-based taxonomic identification and pathogenicity evaluation to grapevine. The ITS and beta tubulin regions were amplified using the ITS1/ITS4 and B36F/B12R primer sets, respectively (González et al. 2006), and sequences were deposited in the NCBI database (Accession numbers: OR052655, OR052656, OR052657, OR052658 and OR059207, OR059208, OR059209, OR059210). Sequences displayed >99% and >96% identity with the respective ITS and beta tubulin sequences of the binucleate Rhizoctonia AG-G specimen C-653 (González et al. 2006). A phylogenetic tree constructed using the Neighbor-Joining method indicated a 100% bootstrap support with the binucleate Rhizoctonia AG-G (Figure 2). Pathogenicity of the binucleate AG-G Rhizoctonia were confirmed on two separate technical replicates using standard methods. For each replicate, one-year-old rootstock 1103P were wounded with sterile drill bits and inoculated with a single 5 mm diameter agar plug collected from Rhizoctonia growing cultures, while control vines were inoculated with sterile agar. The first replicate lasted 28 weeks with (DCHG2B, DCSG22R) inoculated on seven vines. The second bioassay lasted 24 weeks with two additional isolates (JCSG9B, JCHG12B) inoculated on twelve vines. Rhizoctonia-inoculated vines developed wood symptoms similar to those observed on cuttings in nurseries, with necrotic lesions lengths significantly longer than the controls (First replicate: 3.5  0.4 cm vs. 1.3  0.6 cm; Second replicate: 6.8  0.8 cm vs. 1.1  0.2 cm), based on one-way ANOVA statistical test (P value < 0.05). Rhizoctonia isolates recovery from wood necrotic lesions were confirmed by ITS sequencing, thereby fulfilling Koch's postulate. Several binucleate Rhizoctonia anastomosis groups, including AG-G, have been found to cause root rot and stem necrosis in plant nurseries (Aiello et al., 2017; Rinehart et al., 2007). Rhizoctonia has also been reported to be associated with grapevine nurseries in Europe (Pintos et al., 2018), South Africa (Halleen et al., 2003) and Australia (Walker, 1992). However, the multinucleate Rhizoctonia solani was the only species confirmed to cause root rot on grapevine (Walker, 1992). Our data suggests that the binucleate Rhizoctonia from the AG-G anastomosis group also cause wood necrosis in grapevine. Those findings warrant further studies on the complexity of Rhizoctonia anastomosis groups in nursery and their aggressiveness to grapevine.

A multitiered haplotype strategy to enhance phased assembly and fine mapping of a disease resistance locus.

Fine mapping of quantitative trait loci (QTL) to dissect the genetic basis of traits of interest is essential to modern breeding practice. Here, we employed a multitiered haplotypic marker system to increase fine mapping accuracy by constructing a chromosome-level, haplotype-resolved parental genome, accurate detection of recombination sites, and allele-specific characterization of the transcriptome. In the first tier of this system, we applied the preexisting panel of 2,000 rhAmpSeq core genome markers that is transferable across the entire Vitis genus and provides a genomic resolution of 200 kb to 1 Mb. The second tier consisted of high-density haplotypic markers generated from Illumina skim sequencing data for samples enriched for relevant recombinations, increasing the potential resolution to hundreds of base pairs. We used this approach to dissect a novel Resistance to Plasmopara viticola-33 (RPV33) locus conferring resistance to grapevine downy mildew, narrowing the candidate region to only 0.46 Mb. In the third tier, we used allele-specific RNA-seq analysis to identify a cluster of 3 putative disease resistance RPP13-like protein 2 genes located tandemly in a nonsyntenic insertion as candidates for the disease resistance trait. In addition, combining the rhAmpSeq core genome haplotype markers and skim sequencing-derived high-density haplotype markers enabled chromosomal-level scaffolding and phasing of the grape Vitis × doaniana 'PI 588149' assembly, initially built solely from Pacific Biosciences (PacBio) high-fidelity (HiFi) reads, leading to the correction of 16 large-scale phasing errors. Our mapping strategy integrates high-density, phased genetic information with individual reference genomes to pinpoint the genetic basis of QTLs and will likely be widely adopted in highly heterozygous species.

Whole genome sequencing and analysis of multiple isolates of Ceratocystis destructans, the causal agent of Ceratocystis canker of almond in California.

Ceratocystis canker caused by Ceratocystis destructans is a severe disease of almond, reducing the longevity and productivity of infected trees. Once the disease has established in an individual tree, there is no cure, and management efforts are often limited to removing the infected area of cankers. In this study, we present the genome assemblies of five C. destructans isolates isolated from symptomatic almond trees. The genomes were assembled into a genome size of 27.2 ± 0.9 Mbp with an average of 6924 ± 135 protein-coding genes and an average GC content of 48.8 ± 0.02%. We concentrated our efforts on identifying putative virulence factors of canker pathogens. Analysis of the secreted carbohydrate-active enzymes showed that the genomes harbored 83.4 ± 1.8 secreted CAZymes. The secreted CAZymes covered all the known categories of CAZymes. AntiSMASH revealed that the genomes had at least 7 biosynthetic gene clusters, with one of the non-ribosomal peptide synthases encoding dimethylcoprogen, a conserved virulence determinant of plant pathogenic ascomycetes. From the predicted proteome, we also annotated cytochrome P450 monooxygenases, and transporters, these are well-established virulence determinants of canker pathogens. Moreover, we managed to identify 57.4 ± 2.1 putative effector proteins. Gene Ontology (GO) annotation was applied to compare gene content with two closely related species C. fimbriata, and C. albifundus. This study provides the first genome assemblies for C. destructans, expanding genomic resources for an important almond canker pathogen. The acquired knowledge provides a foundation for further advanced studies, such as molecular interactions with the host, which is critical for breeding for resistance.

MYB24 orchestrates terpene and flavonol metabolism as light responses to anthocyanin depletion in variegated grape berries.

Variegation is a rare type of mosaicism not fully studied in plants, especially fruits. We examined red and white sections of grape (Vitis vinifera cv. 'Béquignol') variegated berries and found that accumulation of products from branches of the phenylpropanoid and isoprenoid pathways showed an opposite tendency. Light-responsive flavonol and monoterpene levels increased in anthocyanin-depleted areas in correlation with increasing MYB24 expression. Cistrome analysis suggested that MYB24 binds to the promoters of 22 terpene synthase (TPS) genes, as well as 32 photosynthesis/light-related genes, including carotenoid pathway members, the flavonol regulator HY5 HOMOLOGUE (HYH), and other radiation response genes. Indeed, TPS35, TPS09, the carotenoid isomerase gene CRTISO2, and HYH were activated in the presence of MYB24 and MYC2. We suggest that MYB24 modulates ultraviolet and high-intensity visible light stress responses that include terpene and flavonol synthesis and potentially affects carotenoids. The MYB24 regulatory network is developmentally triggered after the onset of berry ripening, while the absence of anthocyanin sunscreens accelerates its activation, likely in a dose-dependent manner due to increased radiation exposure. Anthocyanins and flavonols in variegated berry skins act as effective sunscreens but for different wavelength ranges. The expression patterns of stress marker genes in red and white sections of 'Béquignol' berries strongly suggest that MYB24 promotes light stress amelioration but only partly succeeds during late ripening.

Identification of grapevine clones via high-throughput amplicon sequencing: a proof-of-concept study.

Wine cultivars are available to growers in multiple clonal selections with agronomic and enological differences. Phenotypic differences between clones originated from somatic mutations that accrued over thousands of asexual propagation cycles. Genetic diversity between grape cultivars remains unexplored, and tools to discriminate unequivocally clones have been lacking. This study aimed to uncover genetic variations among a group of clonal selections of 4 important Vitis vinifera cultivars: Cabernet sauvignon, Sauvignon blanc, Chardonnay, and Merlot, and use this information to develop genetic markers to discriminate the clones of these cultivars. We sequenced with short-read sequencing technology the genomes of 18 clones, including biological replicates for a total of 46 genomes. Sequences were aligned to their respective cultivar's reference genome for variant calling. We used reference genomes of Cabernet sauvignon, Chardonnay, and Merlot and developed a de novo genome assembly of Sauvignon blanc using long-read sequencing. On average, 4 million variants were detected for each clone, with 74.2% being single nucleotide variants and 25.8% being small insertions or deletions (InDel). The frequency of these variants was consistent across all clones. From these variants, we validated 46 clonal markers using high-throughput amplicon sequencing for 77.7% of the evaluated clones, most of them small InDel. These results represent an advance in grapevine genotyping strategies and will benefit the viticulture industry for the characterization and identification of the plant material.

Clonal reproduction of Moniliophthora roreri and the emergence of unique lineages with distinct genomes during range expansion.

The basidiomycete Moniliophthora roreri causes frosty pod rot of cacao (Theobroma cacao) in the western hemisphere. Moniliophthora roreri is considered asexual and haploid throughout its hemibiotrophic life cycle. To understand the processes driving genome modification, using long-read sequencing technology, we sequenced and assembled 5 high-quality M. roreri genomes out of a collection of 99 isolates collected throughout the pathogen's range. We obtained chromosome-scale assemblies composed of 11 scaffolds. We used short-read technology to sequence the genomes of 22 similarly chosen isolates. Alignments among the 5 reference assemblies revealed inversions, translocations, and duplications between and within scaffolds. Isolates at the front of the pathogens' expanding range tend to share lineage-specific structural variants, as confirmed by short-read sequencing. We identified, for the first time, 3 new mating type A locus alleles (5 in total) and 1 new potential mating type B locus allele (3 in total). Currently, only 2 mating type combinations, A1B1 and A2B2, are known to exist outside of Colombia. A systematic survey of the M. roreri transcriptome across 2 isolates identified an expanded candidate effector pool and provided evidence that effector candidate genes unique to the Moniliophthoras are preferentially expressed during the biotrophic phase of disease. Notably, M. roreri isolates in Costa Rica carry a chromosome segment duplication that has doubled the associated gene complement and includes secreted proteins and candidate effectors. Clonal reproduction of the haploid M. roreri genome has allowed lineages with unique genome structures and compositions to dominate as it expands its range, displaying a significant founder effect.

Multigenic resistance to Xylella fastidiosa in wild grapes (Vitis sps.) and its implications within a changing climate.

Xylella fastidiosa is a bacterium that infects crops like grapevines, coffee, almonds, citrus and olives. There is little understanding of the genes that contribute to plant resistance, the genomic architecture of resistance, and the potential role of climate in shaping resistance, in part because major crops like grapevines (Vitis vinifera) are not resistant to the bacterium. Here we study a wild grapevine species, V. arizonica, that segregates for resistance. Using genome-wide association, we identify candidate resistance genes. Resistance-associated kmers are shared with a sister species of V. arizonica but not with more distant species, suggesting that resistance evolved more than once. Finally, resistance is climate dependent, because individuals from low ( < 10 °C) temperature locations in the wettest quarter were typically susceptible to infection, likely reflecting a lack of pathogen pressure in colder climates. In fact, climate is as effective a predictor of resistance phenotypes as some genetic markers. We extend our climate observations to additional crops, predicting that increased pathogen pressure is more likely for grapevines and almonds than some other susceptible crops.

Priming grapevine with lipopolysaccharide confers systemic resistance to Pierce's disease and identifies a peroxidase linked to defense priming.

Priming is an adaptive mechanism that fortifies plant defense by enhancing activation of induced defense responses following pathogen challenge. Microorganisms have signature microbe-associated molecular patterns (MAMPs) that induce the primed state. The lipopolysaccharide (LPS) MAMP isolated from the xylem-limited pathogenic bacterium, Xylella fastidiosa, acts as a priming stimulus in Vitis vinifera grapevines. Grapevines primed with LPS developed significantly less internal tyloses and external disease symptoms than naive vines. Differential gene expression analysis indicated major transcriptomic reprogramming during the priming and postpathogen challenge phases. Furthermore, the number of differentially expressed genes increased temporally and spatially in primed vines, but not in naive vines during the postpathogen challenge phase. Using a weighted gene co-expression analysis, we determined that primed vines have more genes that are co-expressed in both local and systemic petioles than naive vines indicating an inherent synchronicity that underlies the systemic response to this vascular pathogen specific to primed plants. We identified a cationic peroxidase, VviCP1, that was upregulated during the priming and postpathogen challenge phases in an LPS-dependent manner. Transgenic expression of VviCP1 conferred significant disease resistance, thus, demonstrating that grapevine is a robust model for mining and expressing genes linked to defense priming and disease resistance.

Insights into the domestication of avocado and potential genetic contributors to heterodichogamy.

The domestication history of the avocado (Persea americana) remains unclear. We created a reference genome from the Gwen varietal, which is closely related to the economically dominant Hass varietal. Our genome assembly had an N50 of 3.37 megabases, a BUSCO score of 91%, and was scaffolded with a genetic map, producing 12 pseudo-chromosomes with 49,450 genes. We used the Gwen genome as a reference to investigate population genomics, based on a sample of 34 resequenced accessions that represented the 3 botanical groups of P. americana. Our analyses were consistent with 3 separate domestication events; we estimated that the Mexican group diverged from the Lowland (formerly known as "West Indian") and Guatemalan groups >1 million years ago. We also identified putative targets of selective sweeps in domestication events; within the Guatemalan group, putative candidate genes were enriched for fruit development and ripening. We also investigated divergence between heterodichogamous flowering types, providing preliminary evidence for potential candidate genes involved in pollination and floral development.

Botrytis cinerea infection accelerates ripening and cell wall disassembly to promote disease in tomato fruit.

Postharvest fungal pathogens benefit from the increased host susceptibility that occurs during fruit ripening. In unripe fruit, pathogens often remain quiescent and unable to cause disease until ripening begins, emerging at this point into destructive necrotrophic lifestyles that quickly result in fruit decay. Here, we demonstrate that one such pathogen, Botrytis cinerea, actively induces ripening processes to facilitate infections and promote disease in tomato (Solanum lycopersicum). Assessments of ripening progression revealed that B. cinerea accelerated external coloration, ethylene production, and softening in unripe fruit, while mRNA sequencing of inoculated unripe fruit confirmed the corresponding upregulation of host genes involved in ripening processes, such as ethylene biosynthesis and cell wall degradation. Furthermore, an enzyme-linked immunosorbent assay (ELISA)-based glycomics technique used to assess fruit cell wall polysaccharides revealed remarkable similarities in the cell wall polysaccharide changes caused by both infections of unripe fruit and ripening of healthy fruit, particularly in the increased accessibility of pectic polysaccharides. Virulence and additional ripening assessment experiments with B. cinerea knockout mutants showed that induction of ripening depends on the ability to infect the host and break down pectin. The B. cinerea double knockout Δbc polygalacturonase1 Δbc polygalacturonase2 lacking two critical pectin degrading enzymes was incapable of emerging from quiescence even long after the fruit had ripened at its own pace, suggesting that the failure to accelerate ripening severely inhibits fungal survival on unripe fruit. These findings demonstrate that active induction of ripening in unripe tomato fruit is an important infection strategy for B. cinerea.

HiFi chromosome-scale diploid assemblies of the grape rootstocks 110R, Kober 5BB, and 101-14 Mgt.

Cultivated grapevines are commonly grafted on closely related species to cope with specific biotic and abiotic stress conditions. The three North American Vitis species V. riparia, V. rupestris, and V. berlandieri, are the main species used for breeding grape rootstocks. Here, we report the diploid chromosome-scale assembly of three widely used rootstocks derived from these species: Richter 110 (110R), Kober 5BB, and 101-14 Millardet et de Grasset (Mgt). Draft genomes of the three hybrids were assembled using PacBio HiFi sequences at an average coverage of 53.1 X-fold. Using the tool suite HaploSync, we reconstructed the two sets of nineteen chromosome-scale pseudomolecules for each genome with an average haploid genome size of 494.5 Mbp. Residual haplotype switches were resolved using shared-haplotype information. These three reference genomes represent a valuable resource for studying the genetic basis of grape adaption to biotic and abiotic stresses, and designing trait-associated markers for rootstock breeding programs.

Haplotype-resolved powdery mildew resistance loci reveal the impact of heterozygous structural variation on NLR genes in Muscadinia rotundifolia.

Muscadinia rotundifolia cv. Trayshed is a valuable source of resistance to grape powdery mildew. It carries 2 powdery mildew resistance-associated genetic loci, Run1.2 on chromosome 12 and Run2.2 on chromosome 18. The purpose of this study was to identify candidate resistance genes associated with each haplotype of the 2 loci. Both haplotypes of each resistance-associated locus were identified, phased, and reconstructed. Haplotype phasing allowed the identification of several structural variation events between haplotypes of both loci. Combined with a manual refinement of the gene models, we found that the heterozygous structural variants affected the gene content, with some resulting in duplicated or hemizygous nucleotide-binding leucine-rich repeat genes. Heterozygous structural variations were also found to impact the domain composition of some nucleotide-binding leucine-rich repeat proteins. By comparing the nucleotide-binding leucine-rich repeat proteins at Run1.2 and Run2.2 loci, we discovered that the 2 loci include different numbers and classes of nucleotide-binding leucine-rich repeat genes. To identify powdery mildew resistance-associated genes, we performed a gene expression profiling of the nucleotide-binding leucine-rich repeat genes at Run1.2b and Run2.2 loci with or without powdery mildew present. Several nucleotide-binding leucine-rich repeat genes were constitutively expressed, suggesting a role in powdery mildew resistance. These first complete, haplotype-resolved resistance-associated loci and the candidate nucleotide-binding leucine-rich repeat genes identified by this study are new resources that can aid the development of powdery mildew-resistant grape cultivars.

Assembly of complete diploid-phased chromosomes from draft genome sequences.

De novo genome assembly is essential for genomic research. High-quality genomes assembled into phased pseudomolecules are challenging to produce and often contain assembly errors because of repeats, heterozygosity, or the chosen assembly strategy. Although algorithms that produce partially phased assemblies exist, haploid draft assemblies that may lack biological information remain favored because they are easier to generate and use. We developed HaploSync, a suite of tools that produces fully phased, chromosome-scale diploid genome assemblies, and performs extensive quality control to limit assembly artifacts. HaploSync scaffolds sequences from a draft diploid assembly into phased pseudomolecules guided by a genetic map and/or the genome of a closely related species. HaploSync generates a report that visualizes the relationships between current and legacy sequences, for both haplotypes, and displays their gene and marker content. This quality control helps the user identify misassemblies and guides Haplosync's correction of scaffolding errors. Finally, HaploSync fills assembly gaps with unplaced sequences and resolves collapsed homozygous regions. In a series of plant, fungal, and animal kingdom case studies, we demonstrate that HaploSync efficiently increases the assembly contiguity of phased chromosomes, improves completeness by filling gaps, corrects scaffolding, and correctly phases highly heterozygous, complex regions.

Glutathione S-transferase: a candidate gene for berry color in muscadine grapes (Vitis rotundifolia).

Muscadine grapes (Vitis rotundifolia Michx.) are a specialty crop cultivated in the southern United States. Muscadines (2n = 40) belong to the Muscadinia subgenus of Vitis, while other cultivated grape species belong to the subgenus Euvitis (2n = 38). The muscadine berry color locus was mapped to a 0.8 Mbp region syntenic with chromosome 4 of Vitis vinifera. In this study, we identified glutathione S-transferase4 as a likely candidate gene for anthocyanin transport within the berry color locus. PCR and Kompetitive allele-specific PCR genotyping identified a single intragenic SNP (C/T) marker corresponding to a proline to leucine mutation within the muscadine glutathione S-transferase4 (VrGST4) that differentiated black (CC and CT) from bronze (TT) muscadines in 126 breeding selections, 76 cultivars, and 359 progeny from 3 mapping populations. Anthocyanin profiling on a subset of the progeny indicated a dominant VrGST4 action. VrGST4 was expressed in skins of both black and bronze muscadines at similar levels. While nonsynonymous polymorphisms between black and bronze muscadines were discovered in VrGSTF12, another Type I GST-coding gene in the muscadine color locus, this gene was ruled out as a possible candidate for berry color because RNA sequencing indicated it is not expressed in berry skins at véraison from black or bronze genotypes. These results suggest that the bronze phenotype in muscadines is regulated by a mechanism distinct from the MybA gene cluster responsible for berry color variation in Vitis vinifera.