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Open-pollinated seeds from grapevines in Parlier and Davis (in California) and Geneva (in New York) were collected in 2016, 2017, and 2018. Seeds were subjected to a series of cold stratification treatments of varying lengths and germinated in incubators to compare germination rates. Two V. vinifera cultivars (Chardonnay and Cabernet Sauvignon) and three other cultivars (V. labrusca hybrids) with a similar genetic background were compared across three locations to test for maternal environmental effects on germination rates under different cold stratification durations. Two interspecific hybrids (‘Salamander’ and ‘Sovereign Rose’) and three genotypes each from two species, V. riparia and V. cinerea, were evaluated to compare germination rate variability at different cold stratification durations among and within species and hybrids. Large variability in germination rates was evident among and within grape species, with some accessions requiring little to no cold stratification, and others requiring 10 to 12 weeks. These differences could be useful for breeding grapevines with high or low dormancy requirements. The maternal plant environment impacted the seed weight and total seed germination across years and locations.

Background and Aims Condensed tannins, responsible for berry and wine astringency, may have been selected during grapevine domestication. This work examines the phylogenetic distribution of condensed tannins throughout the Vitaceae phylogenetic tree. Methods Green berries and mature leaves of representative true-to-type members of the Vitaceae were collected before ‘véraison’, freeze-dried and pulverized, and condensed tannins were measured following depolymerization by nucleophilic addition of 2-mercaptoethanol to the C4 of the flavan-3-ol units in an organic acidic medium. Reaction products were separated and quantified by ultrahigh pressure liquid chromatography/diode array detection/mass spectrometry. Key Results and Conclusions The original ability to incorporate epigallocatechin (EGC) into grapevine condensed tannins was lost independently in both the American and Eurasian/Asian branches of the Vitaceae, with exceptional cases of reversion to the ancestral EGC phenotype. This is particularly true in the genus Vitis, where we now find two radically distinct groups differing with respect to EGC content. While Vitis species from Asia are void of EGC, 50 % of the New World Vitis harbour EGC. Interestingly, the presence of EGC is tightly coupled with the degree of leaf margin serration. Noticeably, the rare Asian EGC-forming species are phylogenetically close to Vitis vinifera, the only remnant representative of Vitis in Eurasia. Both the wild ancestral V. vinifera subsp. sylvestris as well as the domesticated V. vinifera subsp. sativa can accumulate EGC and activate galloylation biosynthesis that compete for photoassimilates and reductive power.

Background Grafting is a horticultural practice used widely across woody perennial crop species to fuse together the root and shoot system of two distinct genotypes, the rootstock and the scion, combining beneficial traits from both. In grapevine, grafting is used in nearly 80% of all commercial vines to optimize fruit quality, regulate vine vigor, and enhance biotic and abiotic stress-tolerance. Rootstocks have been shown to modulate elemental composition, metabolomic profiles, and the shape of leaves in the scion, among other traits. However, it is currently unclear how rootstock genotypes influence shoot system gene expression as previous work has reported complex and often contradictory findings. Results In the present study, we examine the influence of grafting on scion gene expression in leaves and reproductive tissues of grapevines growing under field conditions for three years. We show that the influence from the rootstock genotype is highly tissue and time dependent, manifesting only in leaves, primarily during a single year of our three-year study. Further, the degree of rootstock influence on scion gene expression is driven by interactions with the local environment. Conclusions Our results demonstrate that the role of rootstock genotype in modulating scion gene expression is not a consistent, unchanging effect, but rather an effect that varies over time in relation to local environmental conditions.

The spotted lanternfly, a newly invasive insect in the U.S. that is a great concern for the grapevine industry, produces damage on its host plants through aggressive feeding, using a piercing and sucking method to feed on the phloem of plants. In the eastern US, adult SLF can invade vineyards through fruit ripening until the end of the growing season; however, it is still unclear how prolonged late-season SLF feeding can affect the health of grapevines, as well as the host responses to this extensive damage. Thus, we have performed a comprehensive genome-wide transcriptome analysis in grapevines heavily infested by the spotted lanternfly, as it occurs in Pennsylvania vineyards, and compared it to other relevant transcriptomes in grapes with different degrees to susceptibility to similar pests. Among a variety of plant responses, we highlight here a subset of relevant biological pathways that distinguish or are common to the spotted lanternfly and other phloem feeders in grapevine. The molecular interaction between spotted lanternfly and the vine begins with activation of signal transduction cascades mediated mainly by protein kinase genes. It also induces the expression of transcription factors in the nucleus, of other signaling molecules like phytohormones and secondary metabolites, and their downstream target genes responsible for defense and physiological functions, such as detoxification and photosynthesis. Grapevine responses furthermore include the activation of genes for cell wall strengthening via biosynthesis of major structural components. With this study, we hope to provide the regulatory network to explain effects that the invasive spotted lanternfly has on grapevine health with the goal to improve its susceptibility.

Premise As a leaf expands, its shape dynamically changes. Previously, we documented an allometric relationship between vein and blade area in grapevine leaves. Larger leaves have a smaller ratio of primary and secondary vein area relative to blade area compared to smaller leaves. We sought to use allometry as an indicator of leaf size and plasticity. Methods We measured the ratio of vein-to-blade area from the same 208 vines across four growing seasons (2013, 2015, 2016, and 2017). Matching leaves by vine and node, we analyzed the correlation between the size and shape of grapevine leaves as repeated measures with climate variables across years. Results The proportion of leaf area occupied by vein and blade exponentially decreased and increased, respectively, during leaf expansion making their ratio a stronger indicator of leaf size than area itself. Total precipitation and leaf wetness hours of the previous year but not the current showed strong negative correlations with vein-to-blade ratio, whereas maximum air temperature from the previous year was positively correlated. Conclusions Our results demonstrate that vein-to-blade ratio is a strong allometric indicator of leaf size and plasticity in grapevines measured across years. Grapevine leaf primordia are initiated in buds the year before they emerge, and we found that total precipitation and maximum air temperature of the previous growing season exerted the largest statistically significant effects on leaf morphology. Vein-to-blade ratio is a promising allometric indicator of relationships between leaf morphology and climate, the robustness of which should be explored further.

Cold injury limits distribution of perennial agricultural crops, though replacement of plants and other management practices may allow for some damage tolerance. However, winter damage to crops such as grapevines (Vitisspp.) can result in losses in yield the following year if buds are damaged, but over many years when vines must be replaced and reach maturity before fruiting. Despite risks, grapevines are cultivated at the edge of permissible climate and rely on cold hardiness monitoring programs to determine when cold damage mitigation and management practices are required. These monitoring programs represent a critical, but laborious process for tracking cold hardiness. To reduce the need for continuous monitoring, a model (WAUS.2) using cold hardiness data collected over many years from Washington state, USA, growers was published in 2014. Although the WAUS.2 model works well regionally, it underperforms in other regions. Therefore, the objective of this work was to develop a new model (NYUS.1) that incorporates recent knowledge of cold hardiness dynamics for better prediction outcomes. Cold hardiness data fromV. labruscana‘Concord’, andV. vinifera‘Cabernet Sauvignon’ and ‘Riesling’ from Geneva, NY, USA were used. Data were separated in calibration (~2/3) and validation (~1/3) datasets. The proposed model uses three functions to describe acclimation, and two functions to describe deacclimation, with a total of nine optimized parameters. A shared chill response between acclimation and deacclimation provides a phased integration where acclimation responses decrease over the course of winter and are overcome by deacclimation. The NYUS.1 model outperforms the WAUS.2 model, reducing RMSE by up to 37% depending on cultivar. The NYUS.1 model also tends to be more conservative in its prediction, slightly underpredicting cold hardiness, as opposed to the overprediction from the WAUS.2 model. Some optimized parameters were shared between cultivars, suggesting conserved physiology was captured by the new model.Highlights-Multi-year cold hardiness data from three grapevine cultivars were used for modeling-Cold hardiness was modeled based on daily temperature and accumulated chill-Phased acclimation and deacclimation processes result in cold hardiness predictions-The new model was compared to the currently available model for grapevines-The model proposed here outperforms the currently available model

The viticulture industry needs advanced grape cultivars with genes that enhance disease resistance and environmental stress tolerance to meet the challenges of a changing climate. To discover beneficial allelic variants of grape genes, we established an F1 mapping population from a cross between two North American grapevines, Vitis rupestris Scheele and Vitis riparia Michx. We generated genotyping-by-sequencing (GBS) markers and constructed parental linkage maps consisting of 1177 and 1115 GBS markers, respectively (LOD threshold ≥ 14), which were validated by mapping the sex-determining locus to chromosome 2. Taking advantage of loci heterozygous in both parents, we also constructed an integrated map containing 2583 markers. We mapped a major quantitative trait locus (QTL) for downy mildew (Plasmopara viticola) resistance to chromosome 10 of V. rupestris using both greenhouse- and in vitro-generated leaf resistance data. This QTL explains 66.5% of the phenotypic variance under greenhouse conditions, and its 2-LOD confidence interval corresponds to region 2,470,297 to 3,024,940 bp on chromosome 10 in the Vitis vinifera L. PN40024 reference genome sequence (assembly 12X.v2). We provide PN40024-projected positions of the GBS markers, which can be used as anchors to develop additional markers for the introgression of this V. rupestris haplotype into cultivated grape varieties.

Background Modern biological approaches generate volumes of multi-dimensional data, offering unprecedented opportunities to address biological questions previously beyond reach owing to small or subtle effects. A fundamental question in plant biology is the extent to which below-ground activity in the root system influences above-ground phenotypes expressed in the shoot system. Grafting, an ancient horticultural practice that fuses the root system of one individual (the rootstock) with the shoot system of a second, genetically distinct individual (the scion), is a powerful experimental system to understand below-ground effects on above-ground phenotypes. Previous studies on grafted grapevines have detected rootstock influence on scion phenotypes including physiology and berry chemistry. However, the extent of the rootstock's influence on leaves, the photosynthetic engines of the vine, and how those effects change over the course of a growing season, are still largely unknown. Results Here, we investigate associations between rootstock genotype and shoot system phenotypes using 5 multi-dimensional leaf phenotyping modalities measured in a common grafted scion: ionomics, metabolomics, transcriptomics, morphometrics, and physiology. Rootstock influence is ubiquitous but subtle across modalities, with the strongest signature of rootstock observed in the leaf ionome. Moreover, we find that the extent of rootstock influence on scion phenotypes and patterns of phenomic covariation are highly dynamic across the season. Conclusions These findings substantially expand previously identified patterns to demonstrate that rootstock influence on scion phenotypes is complex and dynamic and underscore that broad understanding necessitates volumes of multi-dimensional data previously unmet.

Grapevine 3D inflorescence architecture was comprehensively characterized among 10 wild Vitis species to reveal new phenotypic and evolutionary relationships., Inflorescence architecture provides the scaffold on which flowers and fruits develop, and consequently is a primary trait under investigation in many crop systems. Yet the challenge remains to analyse these complex 3D branching structures with appropriate tools. High information content datasets are required to represent the actual structure and facilitate full analysis of both the geometric and the topological features relevant to phenotypic variation in order to clarify evolutionary and developmental inflorescence patterns. We combined advanced imaging (X-ray tomography) and computational approaches (topological and geometric data analysis and structural simulations) to comprehensively characterize grapevine inflorescence architecture (the rachis and all branches without berries) among 10 wild Vitis species. Clustering and correlation analyses revealed unexpected relationships, for example pedicel branch angles were largely independent of other traits. We identified multivariate traits that typified species, which allowed us to classify species with 78.3% accuracy, versus 10% by chance. Twelve traits had strong signals across phylogenetic clades, providing insight into the evolution of inflorescence architecture. We provide an advanced framework to quantify 3D inflorescence and other branched plant structures that can be used to tease apart subtle, heritable features for a better understanding of genetic and environmental effects on plant phenotypes.

Understanding how root systems modulate shoot system phenotypes is a fundamental question in plant biology and will be useful in developing resilient agricultural crops. Grafting is a common horticultural practice that joins the roots (rootstock) of one plant to the shoot (scion) of another, providing an excellent method for investigating how these two organ systems affect each other. In this study, we used the French-American hybrid grapevine ‘Chambourcin’ (Vitis L.) as a model to explore the rootstock–scion relationship. We examined leaf shape, ion concentrations, and gene expression in ‘Chambourcin’ grown ungrafted as well as grafted to three different rootstocks (‘SO4’, ‘1103P’ and ‘3309C’) across 2 years and three different irrigation treatments. We found that a significant amount of the variation in leaf shape could be explained by the interaction between rootstock and irrigation. For ion concentrations, the primary source of variation identified was the position of a leaf in a shoot, although rootstock and rootstock by irrigation interaction also explained a significant amount of variation for most ions. Lastly, we found rootstock-specific patterns of gene expression in grafted plants when compared to ungrafted vines. Thus, our work reveals the subtle and complex effect of grafting on ‘Chambourcin’ leaf morphology, ionomics, and gene expression., Horticulture: dissecting the effects of grafting Researchers in the US have uncovered how rootstocks affect scions following grafting, which will help to develop more resilient crops. A team led by Allison Miller of Saint Louis University grafted the ‘Chambourcin’ grape variety onto three different rootstocks and investigated the effect on leaf shape, ion concentration, and gene expression in the scion. They discovered that the rootstock influenced how leaf shape changed in response to different irrigation conditions. A similar irrigation-dependent effect of rootstock was found for the ion composition in the shoot. The team also identified roughly 100 genes with altered expression in each of the grafted vines, including five genes which were altered by all three graft combinations. These findings are a first step toward understanding the relationship between rootstocks and scions and modulating it to produce crops better adapted to challenging environments.

Background and Aims Grapevine production in cool climates is limited by aspects of winter survival and frost risk. Cold hardiness‐related traits are key to future viticultural sustainability as climate variations, including acute cold events and frost, are predicted to increase even in traditional cultivation regions. This study examines the variation in dormant bud cold hardiness (supercooling) and dormancy (chilling requirement) in 43 different genotypes of the wild grapevine species Vitis riparia, the dominant wild species used to incorporate cold hardiness traits into new hybrid grapevine cultivars. Methods and Results Cold hardiness was evaluated bi‐weekly in 2 years using measures of low temperature exotherms. Whole winter responses were modelled to determine significant factors affecting cold hardiness and determine genotypic differences. Results demonstrate significant differences in supercooling ability and deacclimation rate (loss of cold hardiness) between genotypes. Conclusions This study determined that genotypic differences contribute to initial differences in cold hardiness. However, data modelling suggests that midwinter cold hardiness changes are driven by environment as all Vitis riparia tested in this study respond to temperature in the same manner during the endodormant period of winter. In contrast, responses to warming temperature during ecodormancy are significantly different by genotype. Significance of the Study This study has demonstrated that these two traits interact to determine differences in early versus late winter cold hardiness and help identify breeding germplasm with delayed loss of cold hardiness.

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.

Macro and micro nutrient accumulation affects all stages of plant growth and development. When nutrient deficiencies or excesses occur, normal plant growth is altered resulting in symptoms such as leaf chlorosis, plant stunting or death. In grapes, few genomic regions associated with nutrient accumulation or deficiencies have been identified. Our study evaluated micro and macro nutrient concentrations in Vitis vinifera L. to identify associated SNPs using an association approach with genotype by sequencing data. Nutrient concentrations and foliar symptoms (leaf chlorosis and stunting) were compared among 249 F1 Vitis vinifera individuals in 2015 and 2016. Foliar symptoms were consistent (≥90%) between years and correlated with changes in nutrient concentrations of magnesium (r = 0.65 and r = 0.38 in 2015 and 2016, respectively), aluminum (r = 0.24 and r = 0.49), iron (r = 0.21 and r = 0.49), and sodium (r = 0.32 and r = 0.21). Single nucleotide polymorphisms associated with symptoms, sodium, and magnesium were detected on each chromosome with the exception of 5, 7 and 17 depending on the trait and genome used for analyses explaining up to 40% of the observed variation. Symptoms and magnesium concentration were primarily associated with SNPs on chromosome 3, while SNPs associated with increased sodium content were primarily found on chromosomes 11 and 18. Mean concentrations for each nutrient varied between years in the population between symptomatic and asymptomatic plants, but relative relationships were mostly consistent. These data suggest a complex relationship among foliar symptoms and micro and macro nutrients accumulating in grapevines.

Premise Leaf morphology is dynamic, continuously deforming during leaf expansion and among leaves within a shoot. Here, we measured the leaf morphology of more than 200 grapevines (Vitis spp.) over four years and modeled changes in leaf shape along the shoot to determine whether a composite leaf shape comprising all the leaves from a single shoot can better capture the variation and predict species identity compared with individual leaves. Methods Using homologous universal landmarks found in grapevine leaves, we modeled various morphological features as polynomial functions of leaf nodes. The resulting functions were used to reconstruct modeled leaf shapes across the shoots, generating composite leaves that comprehensively capture the spectrum of leaf morphologies present. Results We found that composite leaves are better predictors of species identity than individual leaves from the same plant. We were able to use composite leaves to predict the species identity of previously unassigned grapevines, which were verified with genotyping. Discussion Observations of individual leaf shape fail to capture the true diversity between species. Composite leaf shape-an assemblage of modeled leaf snapshots across the shoot-is a better representation of the dynamic and essential shapes of leaves, in addition to serving as a better predictor of species identity than individual leaves.

SummaryIn many perennial crops, grafting the root system of one individual to the shoot system of another individual has become an integral part of propagation performed at industrial scales to enhance pest, disease, and stress tolerance and to regulate yield and vigor. Grafted plants offer important experimental systems for understanding the extent and seasonality of root system effects on shoot system biology.Using an experimental vineyard where a common scion ‘Chambourcin’ is growing ungrafted and grafted to three different rootstocks, we explore associations between root system genotype and leaf phenotypes in grafted grapevines across a growing season. We quantified five high-dimensional leaf phenotyping modalities: ionomics, metabolomics, transcriptomics, morphometrics, and physiology and show that rootstock influence is subtle but ubiquitous across modalities.We find strong signatures of rootstock influence on the leaf ionome, with unique signatures detected at each phenological stage. Moreover, all phenotypes and patterns of phenotypic covariation were highly dynamic across the season.These findings expand upon previously identified patterns to suggest that the influence of root system on shoot system phenotypes is complex and broad understanding necessitates volumes of high-dimensional, multi-scale data previously unmet.

Premise of studyLeaf morphology is dynamic, continuously deforming during leaf expansion and among leaves within a shoot. We measured leaf morphology from over 200 vines over four years, and modeled changes in leaf shape along the shoot to determine if a composite “shape of shapes” can better capture variation and predict species identity compared to individual leaves.MethodsUsing homologous universal landmarks found in grapevine leaves, we modeled various morphological features as a polynomial function of leaf node. The resulting functions are used to reconstruct modeled leaf shapes across shoots, generating composite leaves that comprehensively capture the spectrum of possible leaf morphologies.ResultsWe found that composite leaves are better predictors of species identity than individual leaves from the same plant. We were able to use composite leaves to predict species identity of previously unassigned vines, which were verified with genotyping.DiscussionObservations of individual leaf shape fail to capture the true diversity between species. Composite leaf shape—an assemblage of modeled leaf snapshots across the shoot—is a better representation of the dynamic and essential shapes of leaves, as well as serving as a better predictor of species identity than individual leaves.

Premise Leaf morphology is dynamic, continuously deforming during leaf expansion and among leaves within a shoot. Here, we measured the leaf morphology of more than 200 grapevines (Vitis spp.) over four years and modeled changes in leaf shape along the shoot to determine whether a composite leaf shape comprising all the leaves from a single shoot can better capture the variation and predict species identity compared with individual leaves. Methods Using homologous universal landmarks found in grapevine leaves, we modeled various morphological features as polynomial functions of leaf nodes. The resulting functions were used to reconstruct modeled leaf shapes across the shoots, generating composite leaves that comprehensively capture the spectrum of leaf morphologies present. Results We found that composite leaves are better predictors of species identity than individual leaves from the same plant. We were able to use composite leaves to predict the species identity of previously unassigned grapevines, which were verified with genotyping. Discussion Observations of individual leaf shape fail to capture the true diversity between species. Composite leaf shape—an assemblage of modeled leaf snapshots across the shoot—is a better representation of the dynamic and essential shapes of leaves, in addition to serving as a better predictor of species identity than individual leaves.

Vitis riparia, a critically important Native American grapevine species, is used globally in rootstock and scion breeding and contributed to the recovery of the French wine industry during the mid-19th century phylloxera epidemic. This species has abiotic and biotic stress tolerance and the largest natural geographic distribution of the North American grapevine species. Here we report an Illumina short-read 369X coverage, draft de novo heterozygous genome sequence of V. riparia Michx. ‘Manitoba 37’ with the size of ~495 Mb for 69,616 scaffolds and a N50 length of 518,740 bp. Using RNAseq data, 40,019 coding sequences were predicted and annotated. Benchmarking with Universal Single-Copy Orthologs (BUSCO) analysis of predicted gene models found 96% of the complete BUSCOs in this assembly. The assembly continuity and completeness were further validated using V. riparia ESTs, BACs, and three de novo transcriptome assemblies of three different V. riparia genotypes resulting in >98% of respective sequences/transcripts mapping with this assembly. Alignment of the V. riparia assembly and predicted CDS with the latest V. vinifera ‘PN40024’ CDS and genome assembly showed 99% CDS alignment and a high degree of synteny. An analysis of plant transcription factors indicates a high degree of homology with the V. vinifera transcription factors. QTL mapping to V. riparia ‘Manitoba 37’ and V. vinifera PN40024 has identified genetic relationships to phenotypic variation between species. This assembly provides reference sequences, gene models for marker development and understanding V. riparia’s genetic contributions in grape breeding and research.

PremiseAs a leaf expands, its shape dynamically changes. Previously, we documented an allometric relationship between vein and blade area in grapevine leaves. Larger leaves have a smaller ratio of primary and secondary vein area relative to blade area compared to smaller leaves. We sought to use allometry as an indicator of leaf size to measure the environmental effects of climate on grapevine leaf morphology.MethodsWe measure the ratio of vein-to-blade area in 8,412 leaves from the same 208 vines across four growing seasons (2013, 2015, 2016, and 2017) using 21 homologous landmarks. Matching leaves by vine and node, we correlate size and shape of grapevine leaves with climate variables.Key resultsVein-to-blade ratio varies strongly between years in ways that blade or vein area do not. Maximum daily temperature and to a lesser degree precipitation are the most strongly correlated climate variables with vein-to-blade ratio, indicating that smaller leaves are associated with heat waves and drought. Leaf count and overall leaf area of shoots and the vineyard population studied also diminish with heat and drought. Grapevine leaf primordia initiate in buds the year prior to when they emerge, and we find that climate during the previous growing season exerts the largest statistical effects over these relationships.ConclusionsOur results demonstrate the profound effects of heat and drought on the vegetative morphology of grapevines and show that vein-to-blade ratio is a strong allometric indicator of the effects of climate on grapevine leaf morphology.

‘Concord’, the most well-known juice grape with a parentage of the North American grape species Vitis labrusca L., possesses a special ‘foxy’ aroma predominantly resulted from the accumulation of methyl anthranilate (MA) in berries. This aroma, however, is often perceived as an undesirable attribute by wine consumers and rarely noticeable in the common table and wine grape species V. vinifera. Here we discovered homology-induced promoter indels as a major genetic mechanism for species-specific regulation of a key ‘foxy’ aroma gene, anthraniloyl-CoA:methanol acyltransferase (AMAT), that is responsible for MA biosynthesis. We found the absence of a 426-bp and/or a 42-bp sequence in AMAT promoters highly associated with high levels of AMAT expression and MA accumulation in ‘Concord’ and other V. labrusca-derived grapes. These promoter variants, all with direct and inverted repeats, were further confirmed in more than 1,300 Vitis germplasm. Moreover, functional impact of these indels was validated in transgenic Arabidopsis. Superimposed on the promoter regulation, large structural changes including exonic insertion of a retrotransposon were present at the AMAT locus in some V. vinifera grapes. Elucidation of the AMAT genetic regulation advances our understanding of the ‘foxy’ aroma trait and makes it genetically trackable and amenable in grapevine breeding.

Transferable DNA markers are essential for breeding and genetics. Grapevine (Vitis) breeders utilize disease resistance alleles from congeneric species ~20 million years divergent, but existing Vitis marker platforms have cross-species transfer rates as low as 2%. Here, we apply a marker strategy targeting the inferred Vitis core genome. Incorporating seven linked-read de novo assemblies and three existing assemblies, the Vitis collinear core genome is estimated to converge at 39.8 Mb (8.67% of the genome). Adding shotgun genome sequences from 40 accessions enables identification of conserved core PCR primer binding sites flanking polymorphic haplotypes with high information content. From these target regions, we develop 2,000 rhAmpSeq markers as a PCR multiplex and validate the panel in four biparental populations spanning the diversity of the Vitis genus, showing transferability increases to 91.9%. This marker development strategy should be widely applicable for genetic studies in many taxa, particularly those ~20 million years divergent., Trait introgression requires universal markers, but cross-species transferability of current SNP markers can be as low as 2%. Here, the authors use an AmpSeq haplotype strategy targeting the collinear core genome for marker development and show transferability increases to 91.4% in the Vitis genus.

PREMISE OF THE STUDY Grapes are one of the most economically important berry crops worldwide, with the vast majority of production derived from the domesticated Eurasian species Vitis vinifera. Expansion of production into new areas, development of new cultivars, and concerns about adapting grapevines for changing climates necessitate the use of wild grapevine species in breeding programs. Diversity within Vitis has long been a topic of study; however, questions remain regarding relationships between species. Furthermore, the identity of some living accessions is unclear. METHODS This study generated 11,020 single nucleotide polymorphism (SNP) markers for more than 300 accessions in the USDA-ARS grape germplasm repository using genotyping-by-sequencing. Resulting data sets were used to reconstruct evolutionary relationships among several North American and Eurasian Vitis species, and to suggest taxonomic labels for previously unidentified and misidentified germplasm accessions based on genetic distance. KEY RESULTS Maximum likelihood analyses of SNP data support the monophyly of Vitis, subg. Vitis, a Eurasian subg. Vitis clade, and a North American subg. Vitis clade. Data delineate species groups within North America. In addition, analysis of genetic distance suggested taxonomic identities for 20 previously unidentified Vitis accessions and for 28 putatively misidentified accessions. CONCLUSIONS This work advances understanding of Vitis evolutionary relationships and provides the foundation for ongoing germplasm enhancement. It supports conservation and breeding efforts by contributing to a growing genetic framework for identifying novel genetic variation and for incorporating new, unsampled populations into the germplasm repository system.

Background The technological advances of RNA-seq and de novo transcriptome assembly have enabled genome annotation and transcriptome profiling in highly heterozygous species such as grapevine (Vitis vinifera L.). This work is an attempt to utilize a de novo-assembled transcriptome of the V. vinifera cultivar ‘Riesling’ to improve annotation of the grapevine reference genome sequence. Results Here we show that the transcriptome assembly of a single V. vinifera cultivar is insufficient for a complete genome annotation of the grapevine reference genome constructed from V. vinifera PN40024. Further, we provide evidence that the gene models we identified cannot be completely anchored to the previously published V. vinifera PN40024 gene models. In addition to these findings, we present a computational pipeline for the de novo identification of lncRNAs. Our results demonstrate that, in grapevine, lncRNAs are significantly different from protein coding transcripts in such metrics as length, GC-content, minimum free energy, and length-corrected minimum free energy. Conclusions In grapevine, high-level heterozygosity necessitates that transcriptome characterization be based on cultivar-specific reference genome sequences. Our results strengthen the hypothesis that lncRNAs have thermodynamically different properties than protein-coding RNAs. The analyses of both coding and non-coding RNAs will be instrumental in uncovering inter-cultivar variation in wild and cultivated grapevine species. Electronic supplementary material The online version of this article (10.1186/s12864-017-4346-6) contains supplementary material, which is available to authorized users.

Grapevine (Vitisspp.) buds must survive winter temperatures in order to resume growth when suitable conditions return in spring. They do so by developing cold hardiness through deep supercooling, but the mechanistic process of supercooling in buds remains largely unknown. Here we use synchrotron X-ray phase contrast imaging to study cold hardiness-related characteristics ofV. amurensis, V. riparia, andV. viniferabuds: time-resolved 2D imaging was used to visualize freezing; and microtomography was used to evaluate morphological changes during deacclimation. Bud cold hardiness was determined (low temperature exotherms; LTEs) using needle thermocouples during 2D imaging as buds were cooled with a N2gas cryostream. Resolution in 2D imaging did not allow for ice crystal identification, but freezing was assessed due to movement of tissues coinciding with LTE values. Freezing was observed to propagate from the center of the bud toward the outer bud scales. The freezing events observed lasted several minutes. Additionally, loss of supercooling ability appears to be correlated with increases in bud tissue volume during the process of deacclimation, but major increases in volume occur after most of the supercooling ability is lost, suggesting growth resumption processes are limited by deacclimation state.HighlightX-ray phase contrast imaging shows freezing occurs over several minutes and propagates from center toward tip ofVitisspp. buds. Incremental increase in bud volume correlates with cold deacclimation

SummaryWe characterized grapevine inflorescence architecture (the rachis and all branches without berries) to describe variation among 10 wild Vitis species, assess phylogenetic signals underlying inflorescence architecture traits, and interpret this variation in the context of breeding objectives.Three-dimensional X-ray tomography scans of grapevine inflorescences were used to measure geometric traits and inflorescence topology using persistent homology, a mathematical approach that can comprehensively measure and compare shapes. We simulated potential space available for berry growth within a given inflorescence architecture by evaluating expanding spheres attached to pedicels, referred to as “berry potential.” Lastly, we performed phylogenetic analysis and mapped trait variation.We detected wide variation in inflorescence architecture features among Vitis species. Hierarchical clustering and correlation analyses revealed relationships among traits. Multivariate analyses identify traits contributing the most to variation and distinguish between species with high accuracy. Phylogenetic analyses revealed 12 morphological traits with strong phylogenetic signal.Morphometric analysis uncovered novel differences in inflorescence architecture among clades and between Vitis species. Cluster density is an important trait for assessing crop quality and forecasting yield; analyses presented here can be used to tease apart subtle, heritable features and environmental influences on this major agronomic trait.

HighlightFaster deacclimation and budbreak phenology is related to a faster regulon rather than higher expression of specific genes. ABA is a master regulator of deacclimation.AbstractDormancy release, loss of cold hardiness and budbreak are critical aspects of the annual cycle of deciduous perennial plants. Molecular control of these processes is not fully understood, and genotypic variation may be important for climate adaptation. Single-node cuttings from wild (Vitis amurensis, V. riparia) and cultivatedVitisgenotypes (V. vinifera‘Cabernet Sauvignon’, ‘Riesling’) were collected from the field during winter and placed under forcing conditions. Cold hardiness was measured daily, and buds were collected for RNA-Seq until budbreak. Field-collected single-node cuttings of ‘Riesling’ were treated with abscisic acid (ABA), and cold hardiness and budbreak at 7 °C were tracked. WildVitisgenotypes had faster deacclimation and budbreak thanV. vinifera. Temperature-sensing related genes were quickly and synchronously differentially expressed in all genotypes. ABA synthesis was down-regulated in all genotypes, and exogenous ABA prevented deacclimation. Ethylene- and oxidative stress-related genes were transiently up-regulated. Growth-related genes were up-regulated and showed staggering similar to deacclimation and budbreak of the four genotypes. The gene expression cascade that occurs during deacclimation and budburst phenology of fast (wild) and slow (cultivated) grapevines appears coordinated and temporally conserved. This may extend to other temperate woody species and suggest constraints on identification of process-specific keystone genes.

Spring frosts and subsequent crop losses threaten the economic sustainability of fruit crop producers all over the world. This study used a controlled-freezing technique to impose a post-budburst freezing stress to grapevine shoots forced from one-node cuttings [‘Albariño’, ‘Cabernet Franc’, ‘Cabernet Sauvignon’, and ‘Pinot Grigio’ (Vitis vinifera)] and whole plants [‘Noiret’ (Vitis hybrid)]. Our goal was to investigate the incidence of freeze injury among cultivars, stage of phenological development, and a potassium salt-based fertilizer (KDL) with potential cryoprotectant activity. Among the V. vinifera cultivars, the incidence of mortality of shoots exposed to −3.5 °C was highest for ‘Albariño’ (71%) and lowest for ‘Cabernet Sauvignon’ (51%). Cuttings sprayed with KDL 24 hours before cold temperature exposure exhibited 16% lower shoot mortality and lower osmotic potential (Ψs) (−0.92 MPa) than the unsprayed cuttings (−0.77 MPa). However, application of KDL did not impact shoot mortality for whole ‘Noiret’ vines. Mortality for ‘Noiret’ shoots greatly increased with the advancement of phenological development, ranging from 10% in wooly buds to 78% in shoots ≈10-cm long. The practical significance of KDL remains questionable; cultivar selection still appears to be a more reliable method for avoiding spring frost, by planting late bursting cultivars in more frost-prone areas.

The shape of leaves are dynamic, changing over evolutionary time between species, within a single plant producing different shaped leaves at successive nodes, during the development of a single leaf as it allometrically expands, and in response to the environment. Notably, strong correlations between the dissection and size of leaves with temperature and precipitation exist in both the paleorecord and extant populations. Yet, a morphometric model integrating evolutionary, developmental, and environmental effects on leaf shape is lacking. Here, we continue a morphometric analysis of >5,500 leaves representing 270 grapevines of multiple Vitis species between two growing seasons. Leaves are paired one-to-one, vine-to-vine accounting for developmental context, between growing seasons. Linear Discriminant Analysis reveals shape features that specifically define growing season, regardless of species or developmental context. The shape feature, a more pronounced distal sinus, is associated with the colder, drier growing season, consistent with patterns observed in the paleorecord. We discuss the implications of such plasticity in a long-lived woody perennial, such as grapevine, with respect to the evolution and functionality of plant morphology and changes in climate.

Bud dormancy and cold hardiness are critical adaptations for surviving winter cold stress for temperate perennial plant species. In grapevine, acquisition of cold hardiness requires dormancy induction in the early winter and careful maintenance of dormancy state throughout winter. With sufficient exposure to low, non-freezing temperatures (chilling requirement), grapevine buds transition between early (endodormant) and late winter (ecodormant) states. The objective of this study was to uncover the relationship between fulfilment of the chilling requirement and the effects of various temperatures on loss of cold hardiness (deacclimation). The relationship between chilling requirement and temperature as it affects the rate of deacclimation (kdeacc) was examined for dormant cuttings of Vitis vinifera, V. aestivalis, V. amurensis and V. riparia. The effect of temperature on kdeacc was exponential at low and logarithmic at high temperatures. Deacclimation rates also increased in magnitude as chilling accumulated demonstrating a change in deacclimation potential (Ψdeacc), following a logarithmic response. The combination of Ψdeacc and kdeacc indicates genotype-specific thermal efficiency for deacclimation and growth in Vitis that may be overlooked by simple growing degree-day computations. The Ψdeacc and kdeacc parameters are genotype-specific and will greatly increase the refinement of models predicting effects of climate change on phenology. Deacclimation rates represent a quantitative determinant of dormancy transition and budbreak in grapevine and will assist researchers in selecting germplasm for differences in chilling requirement and thermal efficiency., Perennial plants use bud dormancy as a strategy to survive stressful environments, such as cold temperatures during winter. This strategy is dependent on the ability of the dormant bud to track the length of winter, avoiding early spring budbreak. In this study, we demonstrate that as winter progresses, the sensitivity of grapevine buds to temperature increases. This demonstrates a link between dormancy state and loss of winter hardiness. The kinetics of the process also describe early bud phenology. With this understanding, we can now model the response of buds to current and future climate conditions.

2.AbstractBud dormancy and cold hardiness are critical adaptations for surviving winter cold stress for temperate perennial plant species, with shifting temperature-based responses during the winter. The objective of this study was to uncover the relationship between dormancy transition (chilling requirement) and temperature on the loss of cold hardiness and budbreak. Dormant cuttings ofVitis vinifera,V. aestivalis,V. amurensis, andV. ripariawere examined to determine the relationship between chilling requirement and temperature on rate of deacclimation (kdeacc). Differential thermal analysis was used to determinekdeaccusing mean low temperature exotherms. Effect of chill was evaluated as the deacclimation potential (ψdeacc), which was the change inkdeaccdue to chill accumulation. Budbreak was also evaluated in fully chilled buds at different temperatures. Results indicate that ψdeaccvaries dependent on dormancy state, following a logarithmic response to chill accumulation. The effect of temperature onkdeaccwas exponential at low and logarithmic at high temperatures. The combination of ψdeaccandkdeaccresulted in good prediction of deacclimation. Budbreak phenology was also explained by differences inkdeacc. Deacclimation rates can be used as a quantitative determinant of dormancy transition and budbreak, and to refine models predicting effects of climate change.

Low-temperature stresses limit the sustainability and productivity of grapevines when early spring frosts damage young grapevine leaves. Spring conditions often expose grapevines to low, but not damaging, chilling temperatures and these temperatures have been shown to increase freeze resistance in other model systems. In this study, we examined whole-transcriptome gene expression patterns of young leaf tissue from cuttings of five different grapevine cultivars, exposed to chill and freeze shock, in order to understand the underlying transcriptional landscape associated with cold stress response. No visible damage was observed when grapevine leaves were exposed to chilling temperatures while freeze temperatures resulted in variable damage in all cultivars. Significant differences in gene expression were observed between warm control conditions and all types of cold stress. Exposure to chill stress (4 °C) versus freezing stress (−3 °C) resulted in very different patterns of gene expression and enriched pathway responses. Genes from the ethylene signaling, ABA signaling, the AP2/ERF, WRKY, and NAC transcription factor families, and starch/sucrose/galactose pathways were among the most commonly observed to be differentially regulated. Preconditioning leaves to chill temperatures prior to freezing temperatures resulted in slight buffering of gene expression responses, suggesting that differences between chill and freeze shock perception complicates identification of candidate genes for cold resistance in grapevine. Overall, the transcriptional landscape contrasts observed between low temperature and freezing stresses demonstrate very different activation of candidate pathways impacting grapevine cold response., Plant stress responses: How cold is too cold for grapevines? An exploration of gene activity in grapevine (Vitis vinifera) suggests that these plants respond very differently to cold versus freezing temperatures. Low temperatures during the growing season (such as late spring frosts) can cause great damage to developing leaves of crops such as grapevines, particularly when grown outside their natural climatic zone. Jason Londo, of the US Department of Agriculture and Cornell University, and colleagues, explored the molecular response of young grapevine leaves to both cold (4 °C) and freezing (−3 °C) temperatures. They found very different responses in terms of genes and metabolic pathways that were activated or deactivated under the two regimes. In contrast to other species, exposure to cold did not predispose the vines to resist freezing temperatures. The research may help to identify potential targets for genetic manipulation to produce frost-resistant grapevine varieties.

Current morphometric methods that comprehensively measure shape cannot compare the disparate leaf shapes found in seed plants and are sensitive to processing artifacts. We explore the use of persistent homology, a topological method applied across the scales of a function, to overcome these limitations. The described method isolates subsets of shape features and measures the spatial relationship of neighboring pixel densities in a shape. We apply the method to the analysis of 182,707 leaves, both published and unpublished, representing 141 plant families collected from 75 sites throughout the world. By measuring leaves from throughout the seed plants using persistent homology, a defined morphospace comparing all leaves is demarcated. Clear differences in shape between major phylogenetic groups are detected and estimates of leaf shape diversity within plant families are made. This approach does not only predict plant family, but also the collection site, confirming phylogenetically invariant morphological features that characterize leaves from specific locations. The application of a persistent homology method to measure leaf shape allows for a unified morphometric framework to measure plant form, including shape and branching architectures.

Dormancy release, loss of cold hardiness and budbreak are critical aspects of the annual cycle of deciduous perennial plants. Molecular control of these processes is not fully understood, and genotypic variation may be important for climate adaptation. To gain greater understanding of these processes, single-node cuttings from wild (Vitis amurensis, V. riparia) and cultivated Vitis genotypes (V. vinifera ‘Cabernet Sauvignon’, ‘Riesling’) were collected from the vineyard during winter and placed under forcing conditions. Cold hardiness was measured daily, and buds were collected for gene expression analysis until budbreak. Wild Vitis genotypes had faster deacclimation and budbreak than V. vinifera. Temperature-sensing related genes were quickly and synchronously differentially expressed in all genotypes. Significant changes in the pattern of expression changes for eight major metabolic and hormone related pathways were seen across all genotypes. Downregulation of ABA synthesis appears to play an important role in loss of cold hardiness and budbreak in all genotypes. This role was validated through an observed halt in cold hardiness loss of ‘Riesling’ buds treated with exogenous ABA. The gene expression cascade that occurs during deacclimation and budbreak phenology of fast (wild) and slow (cultivated) grapevines appears coordinated and temporally conserved within these phenotypes.

Plants have an intriguing tripartite genetic system: Nuclear genome × Mitochondria × Plastids and their interactions may impact germplasm breeding. In grapevine, the study of cytoplasmic genomes has been limited, and their role with respect to grapevine germplasm diversity has yet to be elucidated. In the present study, the results of an analysis of the cytoplasmic diversity among 6073 individuals (comprising cultivars, interspecific hybrids and segregating progenies) are presented. Genotyping by sequencing (GBS) was used to elucidate plastid and mitochondrial DNA sequences, and results were analyzed using multivariate techniques. Single nucleotide polymorphism (SNP) effects were annotated in reference to plastid and mitochondrial genome sequences. The cytoplasmic diversity identified was structured according to synthetic domestication groups (wine and raisin/table grape types) and interspecific-hybridization-driven groups with introgression from North American Vitis species, identifying five cytoplasmic groups and four major clusters. Fifty-two SNP markers were used to describe the diversity of the germplasm. Ten organelle genes showed distinct SNP annotations and effect predictions, of which six were chloroplast-derived and three were mitochondrial genes, in addition to one mitochondrial SNP affecting a nonannotated open reading frame. The results suggest that the application of GBS will aid in the study of cytoplasmic genomes in grapevine, which will enable further studies on the role of cytoplasmic genomes in grapevine germplasm, and then allow the exploitation of these sources of diversity in breeding.

Marker-assisted selection (MAS) is often employed in crop breeding programs to accelerate and enhance cultivar development, via selection during the juvenile phase and parental selection prior to crossing. Next-generation sequencing and its derivative technologies have been used for genome-wide molecular marker discovery. To bridge the gap between marker development and MAS implementation, this study developed a novel practical strategy with a semi-automated pipeline that incorporates trait-associated single nucleotide polymorphism marker discovery, low-cost genotyping through amplicon sequencing (AmpSeq) and decision making. The results document the development of a MAS package derived from genotyping-by-sequencing using three traits (flower sex, disease resistance and acylated anthocyanins) in grapevine breeding. The vast majority of sequence reads (⩾99%) were from the targeted regions. Across 380 individuals and up to 31 amplicons sequenced in each lane of MiSeq data, most amplicons (83 to 87%) had

In winegrapes (Vitis spp.), fruit quality traits such as berry color, total soluble solids content (SS), malic acid content (MA), and yeast assimilable nitrogen (YAN) affect fermentation or wine quality, and are important traits in selecting new hybrid winegrape cultivars. Given the high genetic diversity and heterozygosity of Vitis species and their tendency to exhibit inbreeding depression, linkage map construction and quantitative trait locus (QTL) mapping has relied on F1 families with the use of simple sequence repeat (SSR) and other markers. This study presents the construction of a genetic map by single nucleotide polymorphisms identified through genotyping-by-sequencing (GBS) technology in an F2 mapping family of 424 progeny derived from a cross between the wild species V. riparia Michx. and the interspecific hybrid winegrape cultivar, 'Seyval'. The resulting map has 1449 markers spanning 2424 cM in genetic length across 19 linkage groups, covering 95% of the genome with an average distance between markers of 1.67 cM. Compared to an SSR map previously developed for this F2 family, these results represent an improved map covering a greater portion of the genome with higher marker density. The accuracy of the map was validated using the well-studied trait berry color. QTL affecting YAN, MA and SS related traits were detected. A joint MA and SS QTL spans a region with candidate genes involved in the malate metabolism pathway. We present an analytical pipeline for calling intercross GBS markers and a high-density linkage map for a large F2 family of the highly heterozygous Vitis genus. This study serves as a model for further genetic investigations of the molecular basis of additional unique characters of North American hybrid wine cultivars and to enhance the breeding process by marker-assisted selection. The GBS protocols for identifying intercross markers developed in this study can be adapted for other heterozygous species.