Wine production has deep historical roots in France, yet the biological foundations of early viticulture remain elusive. We report genome-wide ancient DNA data from 49 archaeological grape pips spanning ~4000 years, from the Bronze Age to the Medieval period, primarily focusing on France. Population genetic analyses reveal the genetic makeup of wild local grapevines in the Bronze Age and the early use of domesticated grapevines by ~625-500 BCE. Roman-era genomes reflect long-distance exchange from Iberian, Balkan, Levantine, and Caucasian domesticated varieties. Vegetative propagation, evidenced by genetically identical clones across sites and centuries, emerged by the mid-Iron Age and became a pillar of viticultural practice. Remarkably, one Medieval sample from Valenciennes is genetically identical to modern 'Pinot Noir', demonstrating clonal continuity over nearly 600 years. This study traces the introduction, integration and spread of diverse grapevine ancestries that have contributed to shape the varietal landscape of French viticulture.

Roman writers provide detailed accounts of viticulture practices in ancient Mediterranean civilisations. While records describe varieties, methods of vine management, and consumption traditions, major questions remain about how varieties were spread and whether viticulture traditions were impacted during Roman expansion. At the site of Cetamura del Chianti (Siena, Tuscany), waterlogged grape seeds from two wells dated between ca. 300 BCE and ca. 1200 CE provided an opportunity to integrate multiple methodologies to investigate Etruscan and Roman viticulture. Genetic testing of 80 grape seeds revealed that the deepest parts of the wells were associated with higher endogenous DNA content, and complementary near-infrared spectroscopy found patterns with well-preserved pips. Both genome-wide DNA data and geometric morphometric analysis indicated that most seeds originated from domesticated vines associated with winemaking, however, the latter suggested that some seeds with little DNA may derive from wild vines. Following enrichment of genetic markers, relatedness analyses demonstrated links to the past and present: one Cetamura seed was closely related to a Roman seed from Mont Ferrier in France while another pip had affinity to the modern Hungarian variety ‘Baratcsuha szurke’. Within the wells, genetic data and radiocarbon dating revealed continuity from Etruscan to Roman periods, with over a quarter of the seeds belonging to one clonal variety maintained for centuries. Furthermore, genetic markers associated with anthocyanin content indicated the dominant clonal variety likely produced white berries; however, red wine was presumably not unknown at Cetamura, as two other pips were consistent with a dark berry phenotype.

The grapevine (Vitis vinifera L.) is one of the most important horticultural crops, with thousands of varieties cultivated worldwide. In this study, we analyzed chloroplast SNV markers using a whole-genome shotgun sequencing approach to investigate the genetic diversity and phylogeny of 409 cultivated V. vinifera accessions originating from nine countries across Southeast and Central Europe, as well as a heterogeneous set of additional accessions maintained by INRAE. Shotgun sequencing allowed high coverage, enabling the detection of 93 SNVs across 24 chloroplast genes, including 11 non-synonymous variants. The ycf1 gene showed the highest variability, consistent with its role in species differentiation. Haplotype analysis revealed 102 distinct haplotypes, with clear geographic structuring: ATT predominated in the eastern Mediterranean, ATA in western Europe, and GTA mainly in a heterogeneous group of varieties from a French collection. To validate the shotgun approach, seven SNV markers were analyzed using target capture sequencing, confirming the accuracy of detected variants with only minimal discrepancies, which is mostly attributable to homopolymeric regions and low-frequency alleles. Phylogenetic analyses using both trees and networks delineated three major haplotype clusters, reflecting human-mediated dispersal of grapevine cultivars through historical viticultural practices. This study represents the largest chloroplast genome analysis of cultivated V. vinifera to date, providing a large cpDNA resource for assessing chloroplast diversity and maternal haplotype structure in cultivated grapevine. The results highlight the power of combining high-throughput sequencing and chloroplast genomics for population-level studies in perennial crops.

Pangenomes have now been developed for several horticultural crops, yet the extent to which genome diversity in sequence and organization contribute to plant adaptation and major agronomic traits remains poorly understood. Here, we assembled the genomes of 9 cultivated grapevine varieties and compared the genomes of 15 cultivated grapevine varieties for variation in gene and TE content. We found that genomic collinearity is highly conserved among varieties. We still observed substantial variation across genomes. Notably, we identified across varieties 55,662 orthologous genes, of which 55.3% appears to be dispensable. Dispensable genes are enriched for functions related to adaptation to biotic and abiotic constraints, suggesting that they may play a role in adaptation. Comparing our results with a recently published study, we found substantial differences with ∼12.6% of the genes we classified as core genes being classified as dispensable genes in this other study. We then constructed a pangenome graph and used it to performed genome-wide association studies for 3 important traits in grapevine production, which allowed us to include large structural variants as markers in the analyses. We identified 32 loci that we did not detect when we used the PN40024 genome as a reference, 20 of which are newly reported associations. Overall, our results indicates that despite recent advances in characterizing plant pangenomes, current gene classification into core and dispensable gene categories should be taken with caution. They also highlight the value of incorporating structural variants into GWAS, to better characterize the genetic architecture of agronomic traits.

In the face of climate change, new grapevine varieties will have to show an adaptive phenotypic plasticity to maintain production with erratic water resources, and still ensure the quality of the final product. Their selection requires a better knowledge of the genetic basis of those traits and of the elementary processes involved in their variability. 'PlastiVigne', an emblematic project of the Vinid'Occ key challenge, funded by the Occitanie Region (France), tackles this issue with innovative genomic and physiological tools implemented on a unique panel of grape genetic resources representing the genetic diversity of Vitis vinifera. A graph-pangenome is developed from a representative set of high-quality genomes to study the extent and impact of structural genome variations and chromosomal rearrangements in the rapid adaptation capacity of grapevine. We will characterize structural variants potentially related to differential expression or alternative spicing of candidate genes for stress tolerance in individual grape berries. Markers derived from structural variants mapped on the pangenome, as well as new sets of SNP markers, will allow the identification of genomic regions associated to leaf water and carbon balance under several water stress regimes, its plasticity, adaptation traits like phenology, genomic vulnerability, and to some traits related to the aromatic potential of grape berries. They represent new tools for grape breeding. More detailed functional analysis of leaf and berry phenotypic plasticity in response to water deficit will be then conducted, on a subset of contrasted varieties. We will present the project strategy and highlight a few preliminary results.

Recent excavations of Late Antiquity settlements in the Negev Highlands of southern Israel uncovered a society that established commercial -scale viticulture in an arid environment [D. Fuks et al., Proc. Natl. Acad. Sci. U.S.A. 117, 19780-19791 (2020)]. We applied target-enriched genome- wide sequencing and radiocarbon dating to examine grapevine pips that were excavated at three of these sites. Our analyses revealed centuries long and continuous grape cultivation in the Southern Levant. The genetically diverse pips also provided clues to ancient cultivation strategies aimed at improving agricultural productivity and ensuring food security. Applying genomic prediction analysis, a pip dated to the eighth century CE was determined to likely be from a white grape, to date the oldest to be identified. In a kinship analysis, another pip was found to be descendant from a modern Greek cultivar and was thus linked with several popular historic wines that were once traded across the Byzantine Empire. These findings shed light on historical Byzantine trading networks and on the genetic contribution of Levantine varieties to the classic Aegean landscape.

We elucidate grapevine evolution and domestication histories with 3525 cultivated and wild accessions worldwide. In the Pleistocene, harsh climate drove the separation of wild grape ecotypes caused by continuous habitat fragmentation. Then, domestication occurred concurrently about 11,000 years ago in Western Asia and the Caucasus to yield table and wine grapevines. The Western Asia domesticates dispersed into Europe with early farmers, introgressed with ancient wild western ecotypes, and subsequently diversified along human migration trails into muscat and unique western wine grape ancestries by the late Neolithic. Analyses of domestication traits also reveal new insights into selection for berry palatability, hermaphroditism, muscat flavor, and berry skin color. These data demonstrate the role of the grapevines in the early inception of agriculture across Eurasia.

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 'veraison', 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.

To cope with the challenges facing agriculture, speeding-up breeding programs is a worthy endeavor, especially for perennial species such as grapevine, but requires understanding the genetic architecture of target traits. To go beyond the mapping of quantitative trait loci in bi-parental crosses, we exploited a diversity panel of 279 Vitis vinifera L. cultivars planted in 5 blocks in the vineyard. This panel was phenotyped over several years for 127 traits including yield components, organic acids, aroma precursors, polyphenols, and a water stress indicator. The panel was genotyped for 63k single nucleotide polymorphisms by combining an 18K microarray and genotyping-by-sequencing. The experimental design allowed to reliably assess the genotypic values for most traits. Marker densification via genotyping-by-sequencing markedly increased the proportion of genetic variance explained by single nucleotide polymorphisms, and 2 multi-single nucleotide polymorphism models identified quantitative trait loci not found by a single nucleotide polymorphism-by-single nucleotide polymorphism model. Overall, 489 reliable quantitative trait loci were detected for 41% more response variables than by a single nucleotide polymorphism-by-single nucleotide polymorphism model with microarray-only single nucleotide polymorphisms, many new ones compared with the results from bi-parental crosses. A prediction accuracy higher than 0.42 was obtained for 50% of the response variables. Our overall approach as well as quantitative trait locus and prediction results provide insights into the genetic architecture of target traits. New candidate genes and the application into breeding are discussed.

The Near East and the Caucasus are commonly regarded as the original domestication centres of Vitis vinifera (grapevine), and the region continues to be home to a high diversity of wild and cultivated grapevines, particularly within Georgia. The earliest chemical evidence for wine making was recorded in Georgian Neolithic sites (6000–5800 bc ) and grape pips, possibly of the domesticated morphotype, have been reported from several sites of about the same period. We performed geometric morphometric and palaeogenomic investigations of grape pip samples in order to identify the appearance of domesticated grapevine and explore the changes in cultivated diversity in relation to modern varieties. We systematically investigated charred and uncharred grape pip samples from Georgian archaeological sites. Their chronology was thoroughly assessed by direct radiocarbon dating. More than 500 grape pips from 14 sites from the Middle Bronze Age to modern times were selected for geometric morphometric studies. The shapes of the ancient pips were compared to hundreds of modern wild individuals and cultivated varieties. Degraded DNA was isolated from three pips from two sites, converted to Illumina libraries, sequenced at approximately 10,000 single nucleotide polymorphism (SNP) sites, and compared to a large public database of grapevine diversity. The most ancient pip dates from the Middle Bronze Age (1900–1500 cal bc ) and the domesticated morphotype is identified from ca. 1000 bc onwards. A great diversity of domesticated shapes was regularly seen in the samples. Most are close to modern cultivars from the Caucasian, southwest Asian and Balkan areas, which suggests that the modern local vine diversity is deeply rooted in early viticulture. DNA was successfully recovered from historic pips and genome-wide analyses found close parental relationships to modern Georgian cultivars.

The pip, as the most common grapevine archaeological remain, is extensively used to document past viticulture dynamics. This paper uses state of the art morphological analyses to analyse the largest reference collection of modern pips to date, representative of the present-day diversity of the domesticated grapevine from Western Eurasia. We tested for a costructure between the form of the modern pips and the: destination use (table/wine), geographical origins, and populational labels obtained through two molecular approaches. Significant structuring is demonstrated for each of these cofactors and for the first time it is possible to infer properties of varieties without going through the parallel with modern varieties. These results provide a unique tool that can be applied to archaeological pips in order to reconstruct the spatio-temporal dynamics of grape diversity on a large scale and to better understand viticulture history. The models obtained were then used to infer the affiliations with archaeobotanical remains recovered in Mas de Vignoles XIV (Nîmes, France). The results show a twofold shift between the Late Iron Age and the Middle Ages, from table to wine grape varieties and from eastern to western origins which correlates with previous palaeogenomic results.

Vitis vinifera L. can be divided into two subspecies, V. vinifera subsp. vinifera, the cultivated grapevine, and its wild ancestor, V. vinifera subsp. sylvestris. Three flower types have been described: hermaphrodite and female in some varieties of vinifera, and male or female flowers in sylvestris. We have conducted an expression analysis of the functional genes candidate to sex determination in the newly defined sex locus described by Picq et al (2014) using four flower types. The candidate gene Ethylene overproducer-1 (ETO1) localized in the sex locus region and which inhibits the enzyme activity of the enzyme ACS (1-aminocyclopropane-1-carboxylic acid synthase) was showed highly significantly different expression pattern according to the sex flower. Other genes studied in the sex locus do not reveal significant different expression patterns. For genes located outside of the sex locus, only the SAUR (Small auxin up RNAs) protein and the ACS gene showed different expression among sex flowers. Therefore, as ETO1 is only expressed in female and hermaphrodite flowers, it could be a good candidate for the recessive female fertility mutation and ACS copy could be implied in the reaction cascade leading to the inhibition of stamens in female flowers. However, the ETO1 only negatively interacts with type 2 ACS and our ACS phylogeny analysis confirmed that the VviACS copy is not type 2. Therefore, it is unlikely that there is such molecular interaction in grapevine. Another hypothesis could be that the molecular mechanisms that regulated the activity of VviACS2 are induced by the VvETO1 protein regulating the activity of both families of ACS type I and type 2. The last gene showing differential expression according to sex is the SAUR protein. This gene consists in early auxin response genes family playing key role in hormonal and environmental signals. Our results pointed out that one gene (ETO1) inside of the flower sex locus region and two genes (ACS, SAUR) located outside of the sex locus region, could be considered as putative candidate genes for the control of sexual traits in grapevine.

Background A key step in domestication of the grapevine was the transition from separate sexes (dioecy) in wild Vitis vinifera ssp. sylvestris ( V. sylvestris ) to hermaphroditism in cultivated Vitis vinifera ssp. sativa ( V. vinifera ). It is known that V. sylvestris has an XY system and V. vinifera a modified Y haplotype (Yh) and that the sex locus is small, but it has not previously been precisely characterized. Results We generate a high-quality de novo reference genome for V. sylvestris , onto which we map whole-genome re-sequencing data of a cross to locate the sex locus. Assembly of the full X, Y, and Yh haplotypes of V. sylvestris and V. vinifera sex locus and examining their gene content and expression profiles during flower development in wild and cultivated accessions show that truncation and deletion of tapetum and pollen development genes on the X haplotype likely causes male sterility, while the upregulation of a Y allele of a cytokinin regulator ( APRT3 ) may cause female sterility. The downregulation of this cytokinin regulator in the Yh haplotype may be sufficient to trigger reversal to hermaphroditism. Molecular dating of X and Y haplotypes is consistent with the sex locus being as old as the Vitis genus, but the mechanism by which recombination was suppressed remains undetermined. Conclusions We describe the genomic and evolutionary characterization of the sex locus of cultivated and wild grapevine, providing a coherent model of sex determination in the latter and for transition from dioecy to hermaphroditism during domestication.

Grapevine has a major economical and cultural importance since antiquity. A key step in domestication was the transition from separate sexes (dioecy) in wild Vitis vinifera ssp. sylvestris ( V. sylvestris ) to hermaphroditism in cultivated Vitis vinifera ssp. vinifera . While the grapevine sex locus is known to be small, its precise boundaries, gene content and the sex-determining genes are unknown. Here we obtained a high-quality de novo reference genome for V. sylvestris and whole-genome resequencing data of a cross. Studying SNP segregation patterns, gene content and expression in wild and cultivated accessions allowed us to build a model for sex determination in grapevine. In this model, up- and down-regulation of a cytokinin regulator is sufficient to cause female sterility and reversal to hermaphroditism, respectively. This study highlights the importance of neo-functionalization of Y alleles in sex determination and provides a resource for studying genetic diversity in V. sylvestris and the genomic processes of grapevine domestication.

The Eurasian grapevine (Vitis vinifera) has long been important for wine production as well as being a food source. Despite being clonally propagated, modern cultivars exhibit great morphological and genetic diversity, with thousands of varieties described in historic and contemporaneous records. Through historical accounts, some varieties can be traced to the Middle Ages, but the genetic relationships between ancient and modern vines remain unknown. We present target-enriched genome-wide sequencing data from 28 archaeological grape seeds dating to the Iron Age, Roman era and medieval period. When compared with domesticated and wild accessions, we found that the archaeological samples were closely related to western European cultivars used for winemaking today. We identified seeds with identical genetic signatures present at different Roman sites, as well as seeds sharing parent–offspring relationships with varieties grown today. Furthermore, we discovered that one seed dated to ~1100 ce was a genetic match to ‘Savagnin Blanc’, providing evidence for 900 years of uninterrupted vegetative propagation. Thousands of varieties of wine grapes have been recorded and described in historical accounts, some going back as far as the Middle Ages, but genetic relationships between ancient and modern varieties were unknown. Genomic sequencing of 28 seeds, dating back as far as the Iron Age, finds close relationships to today’s cultivars, including a genetic match to Savagnin Blanc from 1100 ce.

Summary If grapevine phenotypic changes occurred during domestication and diversification processes are quite well known, particularly seed morphology used in archaeobiological studies, the functional causes and consequences behind these variations are still poorly understood. This study clarifies many aspects of size and shape (co)variation between pip and berry in both wild and domesticated Vitis vinifera. The covariation of berry size, number of seeds per berry (‘piposity’), pip size and pip shape were explored on 49 grapevine accessions sampled among Euro-Mediterranean traditional cultivars and wild grapevines. Consequences for archaeobotanical studies are discussed through the prediction of berry dimensions from archaeological pips from a Southern France Roman site. For wild grapevine, the higher the piposity, the bigger the berry and the longer the pip. For wild and domesticated grapevine, the longer is the pip, the more it has a « domesticated » shape. Such covariations allow to infer the berry dimensions in archaeological context where only the pips are recovered. This systematic exploration sheds light on new aspects of pip-berry covariation in both size and shape, draws the curtain on grapevine eco-evo-devo changes during domestication, and invites to explore the impact of cultivation practices on pip shape.

The grapevine intra-specific variability captured an increasing interest during the last decades, as demonstrated by the number of recently funded European projects focused on the grapevine biodiversity preservation. However, nowadays, crop plants are mainly characterized by genotyping methods. The present work summarizes the phenotype data collected among 20 ampelographic collections spread over 15 countries, covering most of the viticultural areas in the Euro-Asiatic region: from Portugal to Armenia and from Cyprus to Luxembourg. Together with agro-climatic characterization of the experimental site, over two years about 2,400 accessions were described. A common experimental protocol mainly focused on the carpological and oenological traits was followed, obtaining a general overview of the distribution of the considered phenotypic traits in the cultivated Vitis vin Vera species. The most replicated cultivars were selected and, for the subset of these reference cultivars, their behavior in the different environmental conditions over sites and years was described by ANOVA methods.