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Multiple grape powdery mildew (PM) genetic resistance (R) loci have been found in wild grape species. Little is known about the defense responses associated with eachRlocus. In this study, we compare the defense mechanisms associated with PM resistance in interspecific crosses segregating for a singleRlocus fromMuscadinia rotundifolia(Run1, Run1.2b, Run2.1, Run2.2),Vitis cinerea(Ren2),V. romanetii(Ren4DandRen4U), and the interspecific hybrid Villard blanc (Ren3). By combining optical microscopy, visual scoring, and biomass estimation, we show that the eightRloci confer resistance by limiting infection at different stages. We assessed the defense mechanisms triggered in response to PM at 1 and 5 days post inoculation (dpi) via RNA sequencing. To account for the genetic differences between species, we developed for each accession a diploid synthetic reference transcriptome by incorporating into thePN40024reference homozygous and heterozygous sequence variants andde novoassembled transcripts. Most of theRloci exhibited a higher number of differentially expressed genes (DEGs) associated with PM resistance at 1 dpi compared to 5 dpi, suggesting that PM resistance is mostly associated with an early transcriptional reprogramming. Comparison of the PM resistance-associated DEGs showed a limited overlap between pairs ofRloci, and nearly half of the DEGs were specific to a singleRlocus. The largest overlap of PM resistance-associated DEGs was found betweenRen3+,Ren4D+, andRen4U+genotypes at 1 dpi, and betweenRen4U+andRun1+accessions at 5 dpi. TheRen3+,Ren4D+, andRen4U+were also found to have the highest number ofRlocus-specific DEGs in response to PM. Both shared andRlocus-specific DEGs included genes from different defense-related categories, indicating that the presence ofE. necatortriggered distinct transcriptional responses in the eightRloci.

Xylella fastidiosais a bacterium that infects crops like grapevines, coffee, almonds, citrus and olives, causing economically devastating damage. There is, however, little understanding of the genes that contribute to resistance, the genomic architecture of resistance, and the potential role of climate in shaping resistance, in part because major crops like grapevines (V. vinifera) are not resistant to the bacterium. Here we studied a wild grapevine species,Vitis arizonica, that segregates for resistance toX. fastidiosa. Using genome-wide association, we identified candidate genes that mediate the host response toX. fastidiosainfection. We uncovered evidence that resistance requires genes from multiple genomic regions, based on data from breeding populations and from additionalVitisspecies. We also inferred that resistance evolved more than once in the wild, suggesting that wildVitisspecies may be a rich source for resistance alleles and mechanisms. Finally, resistance inV. arizonicawas 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 these climates. Surprisingly, climate was nearly as effective a predictor of resistance phenotypes as some genetic markers. This work underscores that pathogen pressure is likely to increase with climate, but it also provides genetic insight and tools for breeding and transforming resistant crops.

Pierce's disease (PD) in grapevine (Vitis vinifera) is caused by the bacterial pathogen Xylella fastidiosa. X. fastidiosa is limited to the xylem tissue and following infection induces extensive plant‐derived xylem blockages, primarily in the form of tyloses. Tylose‐mediated vessel occlusions are a hallmark of PD, particularly in susceptible V. vinifera. We temporally monitored tylose development over the course of the disease to link symptom severity to the level of tylose occlusion and the presence/absence of the bacterial pathogen at fine‐scale resolution. The majority of vessels containing tyloses were devoid of bacterial cells, indicating that direct, localized perception of X. fastidiosa was not a primary cause of tylose formation. In addition, we used X‐ray computed microtomography and machine‐learning to determine that X. fastidiosa induces significant starch depletion in xylem ray parenchyma cells. This suggests that a signalling mechanism emanating from the vessels colonized by bacteria enables a systemic response to X. fastidiosa infection. To understand the transcriptional changes underlying these phenotypes, we integrated global transcriptomics into the phenotypes we tracked over the disease spectrum. Differential gene expression analysis revealed that considerable transcriptomic reprogramming occurred during early PD before symptom appearance. Specifically, we determined that many genes associated with tylose formation (ethylene signalling and cell wall biogenesis) and drought stress were up‐regulated during both Phase I and Phase II of PD. On the contrary, several genes related to photosynthesis and carbon fixation were down‐regulated during both phases. These responses correlate with significant starch depletion observed in ray cells and tylose synthesis in vessels., During Pierce's disease, Xylella fastidiosa triggers transcriptional changes in Vitis vinifera grapevines and induces major physiological responses, including tylose formation and starch depletion.

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.

Muscadinia rotundifolia cv. Trayshed is a valuable source of resistance to grape powdery mildew. It carries two powdery mildew resistance-associated genetic loci, Run1.2 on chromosome 12 and Run2.2 on chromosome 18. In this study, we identified, phased, and reconstructed the two haplotypes of each resistance-associated locus. Haplotype phasing allowed the identification of several structural variation events between haplotypes of both loci. Combined with 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 (NLR) genes. Structural variations also impacted the domain composition of some NLRs. These findings emphasize the need of generating haplotype-resolved sequences instead of using consensus sequences for identifying haplotype-specific candidate genes. Comparison of the NLRs in the Run1.2 and Run2.2 loci indicated that the two loci are composed of a different number and classes of NLR genes. We provide a list of candidate NLR genes from the Run1.2b and Run2.2 loci, whose expression suggests a role in powdery mildew resistance in Trayshed. These first complete and haplotype-resolved resistance-associated loci, and their candidate NLR genes, represent new resources to develop powdery mildew-resistant grape cultivars.

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.

Structural variants (SVs) are a largely unexplored feature of plant genomes. Little is known about the type and size of SVs, their distribution among individuals and, especially, their population dynamics. Understanding these dynamics is critical for understanding both the contributions of SVs to phenotypes and the likelihood of identifying them as causal genetic variants in genome-wide associations. Here, we identify SVs and study their evolutionary genomics in clonally propagated grapevine cultivars and their outcrossing wild progenitors. To catalogue SVs, we assembled the highly heterozygous Chardonnay genome, for which one in seven genes is hemizygous based on SVs. Using an integrative comparison between Chardonnay and Cabernet Sauvignon genomes by whole-genome, long-read and short-read alignment, we extended SV detection to population samples. We found that strong purifying selection acts against SVs but particularly against inversion and translocation events. SVs nonetheless accrue as recessive heterozygotes in clonally propagated lineages. They also define outlier regions of genomic divergence between wild and cultivated grapevines, suggesting roles in domestication. Outlier regions include the sex-determination region and the berry colour locus, where independent large, complex inversions have driven convergent phenotypic evolution. This study identified structural variants in grapevine populations, including wild progenitors, using a de novo assembly and comparative genomics approach, and examined their evolutionary genomics and roles in domestication and phenotypic evolution.

Grapevine leafroll‐associated virus (GLRaV) infections are accompanied by symptoms influenced by host genotype, rootstock, environment, and which individual or combination of GLRaVs is present. Using a dedicated experimental vineyard, we studied the responses to GLRaVs in ripening berries from Cabernet Franc grapevines grafted to different rootstocks and with zero, one, or pairs of leafroll infection(s). RNA sequencing data were mapped to a high‐quality Cabernet Franc genome reference assembled to carry out this study and integrated with hormone and metabolite abundance data. This study characterized conserved and condition‐dependent responses to GLRaV infection(s). Common responses to GLRaVs were reproduced in two consecutive years and occurred in plants grafted to different rootstocks in more than one infection condition. Though different infections were inconsistently distinguishable from one another, the effects of infections in plants grafted to different rootstocks were distinct at each developmental stage. Conserved responses included the modulation of genes related to pathogen detection, abscisic acid (ABA) signalling, phenylpropanoid biosynthesis, and cytoskeleton remodelling. ABA, ABA glucose ester, ABA and hormone signalling‐related gene expression, and the expression of genes in several transcription factor families differentiated the effects of GLRaVs in berries from Cabernet Franc grapevines grafted to different rootstocks. These results support that ABA participates in the shared responses to GLRaV infection and differentiates the responses observed in grapevines grafted to different rootstocks., GLRaVs elicit changes in gene expression, ripening‐related metabolites, and hormone metabolism in berries; one set of responses is observed across infections and host genotypes while other responses differ based on rootstock. ​

Significance We studied the grape sex-determining region (SDR) in 12 Vitis genomes and demonstrated its conservation across 556 genotypes including 193 accessions from 47 world-wide wild grapevine species and 363 accessions of cultivated grapevine. Although the grape SDR is recombination free in all wild species, we found two distinct hermaphrodite (H) haplotypes (H1 and H2) among the cultivated grapevines, both chimeras of male (M) and female (f) haplotypes. The two independent recombinations carry different genetic signatures which long predate the domestication of grapevine, suggesting independent evolutions of this trait in wild European grapevine gene pools prior to human domestication., Hermaphroditic (perfect) flowers were a key trait in grapevine domestication, enabling a drastic increase in yields due to the efficiency of self-pollination in the domesticated grapevine (Vitis vinifera L. ssp. vinifera). In contrast, all extant wild Vitis species are dioecious, each plant having only male or female flowers. In this study, we identified the male (M) and female (f) haplotypes of the sex-determining region (SDR) in the wild grapevine species V. cinerea and confirmed the boundaries of the SDR. We also demonstrated that the SDR and its boundaries are precisely conserved across the Vitis genus using shotgun resequencing data of 556 wild and domesticated accessions from North America, East Asia, and Europe. A high linkage disequilibrium was found at the SDR in all wild grape species, while different recombination signatures were observed along the hermaphrodite (H) haplotype of 363 cultivated accessions, revealing two distinct H haplotypes, named H1 and H2. To further examine the H2 haplotype, we sequenced the genome of two grapevine cultivars, 'Riesling' and 'Chardonnay'. By reconstructing the first two H2 haplotypes, we estimated the divergence time between H1 and H2 haplotypes at ∼6 million years ago, which predates the domestication of grapevine (∼8,000 y ago). Our findings emphasize the important role of recombination suppression in maintaining dioecy in wild grape species and lend additional support to the hypothesis that at least two independent recombination events led to the reversion to hermaphroditism in grapevine.

Muscadinia rotundifolia, the muscadine grape, has been cultivated for centuries in the southeastern United States.M. rotundifoliais resistant to many of the pathogens that detrimentally affectVitis vinifera, the grape species commonly used for winemaking. For this reason,M. rotundifoliais a valuable genetic resource for breeding. Single-molecule real-time reads were combined with optical maps to reconstruct the two haplotypes of each of the 20M. rotundifoliacv. Trayshed chromosomes. The completeness and accuracy of the assembly were confirmed using a high-density linkage map ofM. rotundifolia.Protein-coding genes were annotated using an integrated and comprehensive approach. This included using Full-length cDNA sequencing (Iso-Seq) to improve gene structure and hypothetical spliced variant predictions. Our data strongly support thatMuscadiniachromosomes 7 and 20 are fused inVitisand pinpoint the location of the fusion in Cabernet Sauvignon and PN40024 chromosome 7. Disease-related gene numbers in Trayshed and Cabernet Sauvignon were similar, but their clustering locations were different. A dramatic expansion of the Toll/Interleukin-1 Receptor-like Nucleotide-Binding Site Leucine-Rich Repeat (TIR-NBS-LRR) class was detected on Trayshed chromosome 12 at theResistance to Uncinula necator 1(RUN1)/Resistance to Plasmopara viticola 1(RPV1) locus, which confers strong dominant resistance to powdery and downy mildews. A genome browser for Trayshed, its annotation, and an associated Blast tool are available atwww.grapegenomics.com.

We gathered genomic data from grapes (Vitis vinifera ssp. vinifera), a clonally propagated perennial crop, to address three ongoing mysteries about plant domestication. The first is the duration of domestication; archaeological evidence suggests that domestication occurs over millennia, but genetic evidence indicates that it can occur rapidly. We estimated that our wild and cultivated grape samples diverged ∼22,000 years ago and that the cultivated lineage experienced a steady decline in population size (Ne ) thereafter. The long decline may reflect low-intensity management by humans before domestication. The second mystery is the identification of genes that contribute to domestication phenotypes. In cultivated grapes, we identified candidate-selected genes that function in sugar metabolism, flower development, and stress responses. In contrast, candidate-selected genes in the wild sample were limited to abiotic and biotic stress responses. A genomic region of high divergence corresponded to the sex determination region and included a candidate male sterility factor and additional genes with sex-specific expression. The third mystery concerns the cost of domestication. Annual crops accumulate putatively deleterious variants, in part due to strong domestication bottlenecks. The domestication of perennial crops differs from that of annuals in several ways, including the intensity of bottlenecks, and it is not yet clear if they accumulate deleterious variants. We found that grape accessions contained 5.2% more deleterious variants than wild individuals, and these were more often in a heterozygous state. Using forward simulations, we confirm that clonal propagation leads to the accumulation of recessive deleterious mutations but without decreasing fitness.

Grapevine (Vitis vinifera) berry development involves a succession of physiological and biochemical changes reflecting the transcriptional modulation of thousands of genes. Although recent studies have investigated the dynamic transcriptome during berry development, most have focused on a single grapevine variety, so there is a lack of comparative data representing different cultivars. Here, we report, to our knowledge, the first genome-wide transcriptional analysis of 120 RNA samples corresponding to 10 Italian grapevine varieties collected at four growth stages. The 10 varieties, representing five red-skinned and five white-skinned berries, were all cultivated in the same experimental vineyard to reduce environmental variability. The comparison of transcriptional changes during berry formation and ripening allowed us to determine the transcriptomic traits common to all varieties, thus defining the core transcriptome of berry development, as well as the transcriptional dynamics underlying differences between red and white berry varieties. A greater variation among the red cultivars than between red and white cultivars at the transcriptome level was revealed, suggesting that anthocyanin accumulation during berry maturation has a direct impact on the transcriptomic regulation of multiple biological processes. The expression of genes related to phenylpropanoid/flavonoid biosynthesis clearly distinguished the behavior of red and white berry genotypes during ripening but also reflected the differential accumulation of anthocyanins in the red berries, indicating some form of cross talk between the activation of stilbene biosynthesis and the accumulation of anthocyanins in ripening berries.

It remains a major challenge to identify the genes and mutations that lead to plant sexual differentiation. Here, we study the structure and evolution of the sex-determining region (SDR) in Vitis species. We report an improved, chromosome-scale Cabernet Sauvignon genome sequence and the phased assembly of nine wild and cultivated grape genomes. By resolving twenty Vitis SDR haplotypes, we compare male, female, and hermaphrodite haplotype structures and identify sex-linked regions. Coupled with gene expression data, we identify a candidate male-sterility mutation in the VviINP1 gene and potential female-sterility function associated with the transcription factor VviYABBY3. Our data suggest that dioecy has been lost during domestication through a rare recombination event between male and female haplotypes. This work significantly advances the understanding of the genetic basis of sex determination in Vitis and provides the information necessary to rapidly identify sex types in grape breeding programs., Grapevine is one of a few ancestrally dioecious crops that are reverted to hermaphroditism during domestication. Here, the authors identify candidate genes related to male- and female-sterility in grapes and describe the genetic process that led to hermaphroditism during domestication.

Sex determination in grapevine evolved through a complex succession of switches in sexual systems. Phased genomes built with single molecule real-time sequencing reads were assembled for eleven accessions of cultivated hermaphrodite grapevines and dioecious males and females, including the ancestor of domesticated grapevine and other related wild species. By comparing the phased sex haplotypes, we defined the sex locus of theVitisgenus and identified polymorphisms spanning regulatory and coding sequences that are in perfect association with each sex-type throughout the genus. These findings identified a novel male-fertility candidate gene,INP1, and significantly refined the model of sex determination inVitisand its evolution.

Transcriptomics has been widely applied to study grape berry development. With few exceptions, transcriptomic studies in grape are performed using the available genome sequence, PN40024, as reference. However, differences in gene content among grape accessions, which contribute to phenotypic differences among cultivars, suggest that a single reference genome does not represent the species’ entire gene space. Though whole genome assembly and annotation can reveal the relatively unique or “private” gene space of any particular cultivar, transcriptome reconstruction is a more rapid, less costly, and less computationally intensive strategy to accomplish the same goal. In this study, we used single molecule-real time sequencing (SMRT) to sequence full-length cDNA (Iso-Seq) and reconstruct the transcriptome of Cabernet Sauvignon berries during berry ripening. In addition, short reads from ripening berries were used to error-correct low-expression isoforms and to profile isoform expression. By comparing the annotated gene space of Cabernet Sauvignon to other grape cultivars, we demonstrate that the transcriptome reference built with Iso-Seq data represents most of the expressed genes in the grape berries and includes 1,501 cultivar-specific genes. Iso-Seq produced transcriptome profiles similar to those obtained after mapping on a complete genome reference. Together, these results justify the application of Iso-Seq to identify cultivar-specific genes and build a comprehensive reference for transcriptional profiling that circumvents the necessity of a genome reference with its associated costs and computational weight.

A major goal of viticulture is to exert control over ripening and produce fruit of reproducible yield and quality. This implies developing effective viticultural practices, breeding cultivars with improved characteristics, and requires considering the numerous variables that can influence development and ripening, like cultivar-specific traits, regional climate, and stresses. Molecular tools aid these efforts. Among them, transcriptome measurements that capture expression across the genome allow monitoring which genomic features are transcribed given the aforementioned variables. The technologies used to study the transcriptome have rapidly improved and become less expensive since the early 2000s, increasing the feasibility of developing molecular marker-driven practices. This chapter briefly reviews the history and state of transcriptomic technologies since they have been applied to grapevine, reviews the seminal publications that have used these tools, and proposes a direction for this field in the future.

Structural variants (SVs) affect plant phenotypes, but they are a largely unexplored feature of plant genomes. Little is known about the type and size of SVs, their distribution among individuals or their evolutionary dynamics. Here we identify SVs and study their evolutionary dynamics in clonally propagated grapevine cultivars and their outcrossing wild relatives. To catalog SVs, we assembled the highly heterozygous Chardonnay genome, for which one in seven genes is hemizygous. Using genomic inference as the standard, we extended SV detection to population samples. We found that negative selection acts against SVs, but particularly against inversion and translocation events. SVs nonetheless accrue as recessive heterozygotes in clonal lineages. They also define outlier regions of genomic divergence between wild and cultivated grapevines, suggesting roles in domestication. Outlier regions include the sex determination region and the berry color locus, where independent large, complex inversions drive convergent phenotypic evolution.

The Ascomycete fungusPhaeoacremonium minimumis one of the primary causal agents of Esca, a widespread and damaging grapevine trunk disease. Variation in virulence amongPm. minimumisolates has been reported, but the underlying genetic basis of the phenotypic variability remains unknown. The goal of this study was to characterize intraspecific genetic diversity and explore its potential impact on virulence functions associated with secondary metabolism, cellular transport, and cell wall decomposition. We generated a chromosome-scale genome assembly, using single molecule real-time sequencing, and resequenced the genomes and transcriptomes of multiple isolates to identify sequence and structural polymorphisms. Numerous insertion and deletion events were found for a total of about 1 Mbp in each isolate. Structural variation in this extremely gene dense genome frequently caused presence/absence polymorphisms of multiple adjacent genes, mostly belonging to biosynthetic clusters associated with secondary metabolism. Because of the observed intraspecific diversity in gene content due to structural variation we concluded that a transcriptome reference developed from a single isolate is insufficient to represent the virulence factor repertoire of the species. We therefore compiled a pan-transcriptome reference ofPm. minimumcomprising a non-redundant set of 15,245 protein-coding sequences. Using naturally infected field samples expressing Esca symptoms, we demonstrated that mapping of meta-transcriptomics data on a multi-species reference that included thePm. minimumpan-transcriptome allows the profiling of an expanded set of virulence factors, including variable genes associated with secondary metabolism and cellular transport.

Significance We generated genomic data to estimate the population history of grapes, the most economically important horticultural crop in the world. Domesticated grapes experienced a protracted, 22,000-y population decline prior to domestication; we hypothesize that this decline reflects low-intensity cultivation by humans prior to domestication. Domestication altered the mating system of grapes. The sex determination region is detectable as a region of heightened genetic divergence between wild and cultivated accessions. Based on gene expression analyses, we propose candidate genes that alter sex determination. Finally, grapes contain more deleterious mutations in heterozygous states than do their wild ancestors. The accumulation of deleterious mutations is due in part to clonal propagation, which shelters deleterious recessive mutations., We gathered genomic data from grapes (Vitis vinifera ssp. vinifera), a clonally propagated perennial crop, to address three ongoing mysteries about plant domestication. The first is the duration of domestication; archaeological evidence suggests that domestication occurs over millennia, but genetic evidence indicates that it can occur rapidly. We estimated that our wild and cultivated grape samples diverged ∼22,000 years ago and that the cultivated lineage experienced a steady decline in population size (Ne) thereafter. The long decline may reflect low-intensity management by humans before domestication. The second mystery is the identification of genes that contribute to domestication phenotypes. In cultivated grapes, we identified candidate-selected genes that function in sugar metabolism, flower development, and stress responses. In contrast, candidate-selected genes in the wild sample were limited to abiotic and biotic stress responses. A genomic region of high divergence corresponded to the sex determination region and included a candidate male sterility factor and additional genes with sex-specific expression. The third mystery concerns the cost of domestication. Annual crops accumulate putatively deleterious variants, in part due to strong domestication bottlenecks. The domestication of perennial crops differs from that of annuals in several ways, including the intensity of bottlenecks, and it is not yet clear if they accumulate deleterious variants. We found that grape accessions contained 5.2% more deleterious variants than wild individuals, and these were more often in a heterozygous state. Using forward simulations, we confirm that clonal propagation leads to the accumulation of recessive deleterious mutations but without decreasing fitness.

We gathered genomic data from grapes (Vitis viniferassp.vinifera), a clonally propagated perennial crop, to address three ongoing mysteries about plant domestication. The first is the duration of domestication; archaeological evidence suggests that domestication occurs over millennia, but genetic evidence indicates it can occur rapidly. We estimated that our wild and cultivated grape samples diverged ~22,000 years ago and that the cultivated lineage experienced a steady decline in population size (Ne) thereafter. The long decline may reflect low intensity management by humans prior to domestication. The second mystery is the identification of genes that contribute to domestication phenotypes. In cultivated grapes, we identified candidate-selected genes that function in sugar metabolism, flower development and stress responses. In contrast, candidate selected genes in the wild sample were limited to abiotic and biotic stress responses. A genomic region of high divergence corresponded to the sex determination region and included a candidate male sterility factor and additional genes with sex-specific expression. The third mystery concerns the cost of domestication. Annual crops accumulate putatively deleterious variants, in part due to strong domestication bottlenecks. The domestication of perennial crops differs from annuals in several ways, including the intensity of bottlenecks, and it is not yet clear if they accumulate deleterious variants. We found that grape accessions contained 5.2% more deleterious variants than wild individuals, and these were more often in a heterozygous state. Using forward simulations, we confirm that clonal propagation leads to the accumulation of recessive deleterious mutations but without decreasing fitness.Significance StatementWe generated genomic data to estimate the population history of grapes, the most economically important horticultural crop in the world. Domesticated grapes experienced a protracted, 22,000 year population decline prior to domestication; we hypothesize that this decline reflects low intensity cultivation by humans prior to domestication. Domestication altered the mating system of grapes. The sex determination region is detectable as a region of heightened genetic divergence between wild and cultivated accessions. Based on gene expression analyses, we propose new candidate genes that alter sex determination. Finally, grapes contain more deleterious mutations in heterozygous states than their wild ancestors. The accumulation of deleterious mutations is due in part to clonal propagation, which shelters deleterious, recessive mutations.

Grapevine (Vitis vinifera) berry development involves a succession of physiological and biochemical changes reflecting the transcriptional modulation of thousands of genes. Although recent studies have investigated the dynamic transcriptome during berry development, most have focused on a single grapevine variety, so there is a lack of comparative data representing different cultivars. Here, we report, to our knowledge, the first genome-wide transcriptional analysis of 120 RNA samples corresponding to 10 Italian grapevine varieties collected at four growth stages. The 10 varieties, representing five red-skinned and five white-skinned berries, were all cultivated in the same experimental vineyard to reduce environmental variability. The comparison of transcriptional changes during berry formation and ripening allowed us to determine the transcriptomic traits common to all varieties, thus defining the core transcriptome of berry development, as well as the transcriptional dynamics underlying differences between red and white berry varieties. A greater variation among the red cultivars than between red and white cultivars at the transcriptome level was revealed, suggesting that anthocyanin accumulation during berry maturation has a direct impact on the transcriptomic regulation of multiple biological processes. The expression of genes related to phenylpropanoid/flavonoid biosynthesis clearly distinguished the behavior of red and white berry genotypes during ripening but also reflected the differential accumulation of anthocyanins in the red berries, indicating some form of cross talk between the activation of stilbene biosynthesis and the accumulation of anthocyanins in ripening berries.

The ascomycete Neofusicoccum parvum, one of the causal agents of Botryosphaeria dieback, is a destructive wood-infecting fungus and a serious threat to grape production worldwide. The capability to colonize woody tissue, combined with the secretion of phytotoxic compounds, is thought to underlie its pathogenicity and virulence. Here, we describe the repertoire of virulence factors and their transcriptional dynamics as the fungus feeds on different substrates and colonizes the woody stem. We assembled and annotated a highly contiguous genome using single-molecule real-time DNA sequencing. Transcriptome profiling by RNA sequencing determined the genome-wide patterns of expression of virulence factors both in vitro (potato dextrose agar or medium amended with grape wood as substrate) and in planta. Pairwise statistical testing of differential expression, followed by co-expression network analysis, revealed that physically clustered genes coding for putative virulence functions were induced depending on the substrate or stage of plant infection. Co-expressed gene clusters were significantly enriched not only in genes associated with secondary metabolism, but also in those associated with cell wall degradation, suggesting that dynamic co-regulation of transcriptional networks contributes to multiple aspects of N. parvum virulence. In most of the co-expressed clusters, all genes shared at least a common motif in their promoter region, indicative of co-regulation by the same transcription factor. Co-expression analysis also identified chromatin regulators with correlated expression with inducible clusters of virulence factors, suggesting a complex, multi-layered regulation of the virulence repertoire of N. parvum.