Species relationships within the genus Vitis based on molecular and morphological data.

The grape genus Vitis L. includes the domesticated V. vinifera, which is one of the most important fruit crop, and also close relatives recognized as valuable germplasm resources for improving cultivars. To resolve some standing problems in the species relationships within the Vitis genus we analyzed diversity in a set of 90 accessions comprising most of Vitis species and some putative hybrids. We discovered single nucleotide polymorphisms (SNPs) in SANGER sequences of twelve loci and genotyped accessions at a larger number of SNPs using a previously developed SNP array. Our phylogenic analyses consistently identified: three clades in North America, one in East Asia, and one in Europe corresponding to V. vinifera. Using heterozygosity measurement, haplotype reconstruction and chloroplast markers, we identified the hybrids existing within and between clades. The species relationships were better assessed after discarding these hybrids from analyses. We also studied the relationships between phylogeny and morphological traits and found that several traits significantly correlated with the phylogeny. The American clade that includes important species such as V. riparia and V. rupestris showed a major divergence with all other clades based on both DNA polymorphisms and morphological traits.

A genome-wide association and prediction study in grapevine deciphers the genetic architecture of multiple traits and identifies genes under many new QTLs.

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.

Across-population genomic prediction in grapevine opens up promising prospects for breeding.

Crop breeding involves two selection steps: choosing progenitors and selecting individuals within progenies. Genomic prediction, based on genome-wide marker estimation of genetic values, could facilitate these steps. However, its potential usefulness in grapevine (Vitis vinifera L.) has only been evaluated in non-breeding contexts mainly through cross-validation within a single population. We tested across-population genomic prediction in a more realistic breeding configuration, from a diversity panel to ten bi-parental crosses connected within a half-diallel mating design. Prediction quality was evaluated over 15 traits of interest (related to yield, berry composition, phenology and vigour), for both the average genetic value of each cross (cross mean) and the genetic values of individuals within each cross (individual values). Genomic prediction in these conditions was found useful: for cross mean, average per-trait predictive ability was 0.6, while per-cross predictive ability was halved on average, but reached a maximum of 0.7. Mean predictive ability for individual values within crosses was 0.26, about half the within-half-diallel value taken as a reference. For some traits and/or crosses, these across-population predictive ability values are promising for implementing genomic selection in grapevine breeding. This study also provided key insights on variables affecting predictive ability. Per-cross predictive ability was well predicted by genetic distance between parents and when this predictive ability was below 0.6, it was improved by training set optimization. For individual values, predictive ability mostly depended on trait-related variables (magnitude of the cross effect and heritability). These results will greatly help designing grapevine breeding programs assisted by genomic prediction.

Genetic diversity and population structure in Vitis species illustrate phylogeographic patterns in eastern North America.

Geographical distribution and diversity of current plant species have been strongly shaped by climatic oscillations during the Quaternary. Analysing the resulting divergence among species and differentiation within species is crucial to understand the evolution of taxa like the Vitis genus, which provides very useful genetic resources for grapevine improvement and might reveal original recolonization patterns due to growth habit and dispersal mode. Here, we studied the genetic structure in natural populations of three species from eastern North America: Vitis aestivalis, V. cinerea and V. riparia using different marker types. Vitis aestivalis and V. cinerea showed higher diversity than V. riparia. The two former species are less differentiated, confirming an earlier divergence of V. riparia. V. aestivalis and V. riparia exhibited different genetic groups on both sides of the Appalachian Mountains that could mirror different recolonization routes from southern refugia. Genetic structure was stronger in V. cinerea, for which two varieties (var. berlandieri and var. cinerea) are morphologically recognized. Our results confirm this distinction and suggest the existence of three other lineages within var. cinerea. These discontinuities appear linked to adaptation of var. berlandieri to dry and limy areas of Texas and partially to the Mississippi River Valley. Rapid range expansions from refugia upon climate warming are also suggested by the low linkage disequilibrium values observed. Furthermore, large variation for downy mildew resistance was observed in the three species. Our findings appeared consistent with the vegetation history of eastern North America.

Understanding the phyllosphere microbiome assemblage in grape species (Vitaceae) with amplicon sequence data structures.

Impacts of plant genotype on microbial assemblage in the phyllosphere (above-ground parts of plants, which predominantly consists of the set of photosynthetic leaves) of Vitis vinifera cultivars have been studied previously but the impact of grape species (under the grape family Vitaceae) was never investigated. Considering the fact, that the phyllosphere microbiome may have profound effects on host plant health and its performance traits, studying the impact of grape species in microbial taxa structuring in the phyllosphere could be of crucial importance. We performed 16S and ITS profiling (for bacteria and fungi respectively) to access genus level characterization of the microflora present in the leaf phyllosphere of five species within this plant family, sampled in two successive years from the repository situated in the Mediterranean. We also performed α and ß-diversity analyses with robust statistical estimates to test the impacts of grape species and growing year, over a two-year period. Our results indicated the presence of complex microbial diversity and assemblages in the phyllosphere with a significant effect of both factors (grape species and growing year), the latter effect is being more pronounced. We also compared separate normalization methods for high-throughput microbiome data-sets followed by differential taxa abundance analyses. The results suggested the predominance of a particular normalization method over others. This also indicated the need for more robust normalization methods to study the differential taxa abundance among groups in microbiome research.

A novel high-density grapevine (Vitis vinifera L.) integrated linkage map using GBS in a half-diallel population.

KEY MESSAGE: A half-diallel population involving five elite grapevine cultivars was generated and genotyped by GBS, and highly-informative segregation data was used to construct a high-density genetic map for Vitis vinifera L. Grapevine is one of the most relevant fruit crops in the world. Deeper genetic knowledge could assist modern grapevine breeding programs to develop new wine grape varieties able to face climate change effects. To assist in the rapid identification of markers for crop yield components, grape quality traits and adaptation potential, we generated a large Vitis vinifera L. population (N = 624) by crossing five red wine cultivars in a half-diallel scheme, which was subsequently sequenced by an efficient GBS procedure. A high number of fully informative genetic variants was detected using a novel mapping approach capable of reconstructing local haplotypes from adjacent biallelic SNPs, which were subsequently used to construct the densest consensus genetic map available for the cultivated grapevine to date. This 1378.3-cM map integrates 10 bi-parental consensus maps and orders 4437 markers in 3353 unique positions on 19 chromosomes. Markers are well distributed all along the grapevine reference genome, covering up to 98.8% of its genomic sequence. Additionally, a good agreement was observed between genetic and physical orders, adding confidence in the quality of this map. Collectively, our results pave the way for future genetic studies (such as fine QTL mapping) aimed to understand the complex relationship between genotypic and phenotypic variation in the cultivated grapevine. In addition, the method used (which efficiently delivers a high number of fully informative markers) could be of interest to other outbred organisms, notably perennial fruit crops.

Genotype-Environment Interaction Shapes the Microbial Assemblage in Grapevine's Phyllosphere and Carposphere: An NGS Approach.

Plant surface or phyllosphere is the habitat of hyperdiverse microbial communities and it is always exposed to the fluctuating environmental factors, which is thought to be one of the potential drivers of microbial community structuring. Impact of grapevine genotypes in variable environmental factors (i.e., at different geographic locations) on the phyllosphere has never been studied and is the main objective of this report. Using high throughput short amplicon sequencing of 16S rRNA genes and internal transcribed spacer (ITS), we analyzed the impacts of genotypes of Vitis Vinifera (coming from three genetic pool), on the microbial (bacterial and fungal) assemblage in the phyllosphere. First, we performed the analysis of the phyllosphere microbiome while using fifteen genotypes that were chosen to maximize intra-specific diversity and grown in two Mediterranean vineyards. Then, the same analysis was performed on five commercially important varieties of Vitis vinifera that were sampled from three different French agro-climatic zones (or terroir: a combination of climate, soils, and human practices). Our study revealed that, at a particular geographic location, genotypes have an impact on microbial assemblage in the phyllosphere and carposphere of leaf and fruit (or berries), respectively, which is more prominent on the carposphere but the effect of terroir was much stronger than the genotype when the leaf phyllosphere of five grapevine varieties grown in different agro-climatic zones was compared. Impacts of the season and exterior plant organs (leaf and berries) on microbial taxa structuring in the phyllosphere was also assessed and presented in this report.

Vitis vinifera L. Fruit Diversity to Breed Varieties Anticipating Climate Changes.

The wine industry is facing critical issues due to climate changes since production is established on very tight Genotype × Environment interaction bases. While, some cultivation practices may reduce adverse effects of abiotic stresses on the vines, e.g., the use of irrigation to mitigate drought, the deleterious impacts of warming on fruit development are difficult to manage. Elevated temperature alters grapevine fruit growth and composition, with a critical increase of the sugars/organic acids ratio. Select grapes with improved metabolite balances to offset high temperature effects is a valuable option to sustain viticulture. Unfortunately, the lack of knowledge about the genetic diversity for fruit traits impacted by temperature impairs the design of breeding programs. This study aimed to assess the variation in berry volume, main sugars and organic acids amounts in genetic resources. Fruit phenotyping focused on two critical stages of development: the end of green lag phase when organic acidity reaches its maximum, and the ripe stage when sugar unloading and water uptake stop. For that purpose, we studied a panel of 33 genotypes, including 12 grapevine varieties and 21 microvine offspring. To determine the date of sampling for each critical stage, fruit texture and growth were carefully monitored. Analyses at both stages revealed large phenotypic variation for malic and tartaric acids, as well as for sugars and berry size. At ripe stage, fruit fresh weight ranged from 1.04 to 5.25 g and sugar concentration from 751 to 1353 mmol.L-1. The content in organic acids varied both in quantity (from 80 to 361 meq.L-1) and in composition, with malic to tartaric acid ratio ranging from 0.13 to 3.62. At the inter-genotypic level, data showed no link between berry growth and osmoticum accumulation per fruit unit, suggesting that berry water uptake is not dependent only on fruit osmotic potential. Diversity among varieties for berry size, sugar accumulation and malic to tartaric acid ratio could be exploited through cross-breeding. This provides interesting prospects for improving grapevine to mitigate some adverse effects of climate warming on grapevine fruit volume and quality.

Extended diversity analysis of cultivated grapevine Vitis vinifera with 10K genome-wide SNPs.

Grapevine is a very important crop species that is mainly cultivated worldwide for fruits, wine and juice. Identification of the genetic bases of performance traits through association mapping studies requires a precise knowledge of the available diversity and how this diversity is structured and varies across the whole genome. An 18k SNP genotyping array was evaluated on a panel of Vitis vinifera cultivars and we obtained a data set with no missing values for a total of 10207 SNPs and 783 different genotypes. The average inter-SNP spacing was ~47 kbp, the mean minor allele frequency (MAF) was 0.23 and the genetic diversity in the sample was high (He = 0.32). Fourteen SNPs, chosen from those with the highest MAF values, were sufficient to identify each genotype in the sample. Parentage analysis revealed 118 full parentages and 490 parent-offspring duos, thus confirming the close pedigree relationships within the cultivated grapevine. Structure analyses also confirmed the main divisions due to an eastern-western gradient and human usage (table vs. wine). Using a multivariate approach, we refined the structure and identified a total of eight clusters. Both the genetic diversity (He, 0.26-0.32) and linkage disequilibrium (LD, 28.8-58.2 kbp) varied between clusters. Despite the short span LD, we also identified some non-recombining haplotype blocks that may complicate association mapping. Finally, we performed a genome-wide association study that confirmed previous works and also identified new regions for important performance traits such as acidity. Taken together, all the results contribute to a better knowledge of the genetics of the cultivated grapevine.

Cultivar Diversity of Grape Skin Polyphenol Composition and Changes in Response to Drought Investigated by LC-MS Based Metabolomics.

Phenolic compounds represent a large family of plant secondary metabolites, essential for the quality of grape and wine and playing a major role in plant defense against biotic and abiotic stresses. Phenolic composition is genetically driven and greatly affected by environmental factors, including water stress. A major challenge for breeding of grapevine cultivars adapted to climate change and with high potential for wine-making is to dissect the complex plant metabolic response involved in adaptation mechanisms. A targeted metabolomics approach based on ultra high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QqQ-MS) analysis in the Multiple Reaction Monitoring (MRM) mode has been developed for high throughput profiling of the phenolic composition of grape skins. This method enables rapid, selective, and sensitive quantification of 96 phenolic compounds (anthocyanins, phenolic acids, stilbenoids, flavonols, dihydroflavonols, flavan-3-ol monomers, and oligomers…), and of the constitutive units of proanthocyanidins (i.e., condensed tannins), giving access to detailed polyphenol composition. It was applied on the skins of mature grape berries from a core-collection of 279 Vitis vinifera cultivars grown with or without watering to assess the genetic variation for polyphenol composition and its modulation by irrigation, in two successive vintages (2014-2015). Distribution of berry weights and δ13C values showed that non irrigated vines were subjected to a marked water stress in 2014 and to a very limited one in 2015. Metabolomics analysis of the polyphenol composition and chemometrics analysis of this data demonstrated an influence of water stress on the biosynthesis of different polyphenol classes and cultivar differences in metabolic response to water deficit. Correlation networks gave insight on the relationships between the different polyphenol metabolites and related biosynthetic pathways. They also established patterns of polyphenol response to drought, with different molecular families affected either positively or negatively in the different cultivars, with potential impact on grape and wine quality.

Quantitative Assessment of Grapevine Wood Colonization by the Dieback Fungus Eutypa lata.

Eutypa lata is a fungal pathogen causing severe dieback in vineyards worldwide. This fungus colonizes vines through pruning wounds, eventually causing a brown sectorial necrosis in wood as well as stunted vegetative growth. Several years may pass between infection and the expression of external symptoms, hindering the rapid evaluation of both grapevine cultivars susceptibility and E. lata variation in aggressiveness. We aimed to develop a rapid quantitative method for the assessment of wood colonization after inoculation of cuttings in controlled conditions. We used several grape cultivars varying in susceptibility in the vineyard and fungal isolates with different levels of aggressiveness to monitor wood colonization during a maximum period of 2 months. Re-isolation allowed demonstration of the effects of both cultivars and fungal isolates on the rate of wood colonization. We also developed a real-time PCR method that was efficient in measuring fungal biomass, which was found to be correlated with isolate aggressiveness based on foliar symptom severity. The real-time PCR approach appears to be a useful technology to evaluate grapevine susceptibility to E. lata, and could be adapted to other pathogens associated with grapevine trunk diseases.

A large set of 26 new reference transcriptomes dedicated to comparative population genomics in crops and wild relatives.

We produced a unique large data set of reference transcriptomes to obtain new knowledge about the evolution of plant genomes and crop domestication. For this purpose, we validated a RNA-Seq data assembly protocol to perform comparative population genomics. For the validation, we assessed and compared the quality of de novo Illumina short-read assemblies using data from two crops for which an annotated reference genome was available, namely grapevine and sorghum. We used the same protocol for the release of 26 new transcriptomes of crop plants and wild relatives, including still understudied crops such as yam, pearl millet and fonio. The species list has a wide taxonomic representation with the inclusion of 15 monocots and 11 eudicots. All contigs were annotated using BLAST, prot4EST and Blast2GO. A strong originality of the data set is that each crop is associated with close relative species, which will permit whole-genome comparative evolutionary studies between crops and their wild-related species. This large resource will thus serve research communities working on both crops and model organisms. All the data are available at http://arcad-bioinformatics.southgreen.fr/.

Genetic diversity, linkage disequilibrium and power of a large grapevine (Vitis vinifera L) diversity panel newly designed for association studies.

BACKGROUND: As for many crops, new high-quality grapevine varieties requiring less pesticide and adapted to climate change are needed. In perennial species, breeding is a long process which can be speeded up by gaining knowledge about quantitative trait loci linked to agronomic traits variation. However, due to the long juvenile period of these species, establishing numerous highly recombinant populations for high resolution mapping is both costly and time-consuming. Genome wide association studies in germplasm panels is an alternative method of choice, since it allows identifying the main quantitative trait loci with high resolution by exploiting past recombination events between cultivars. Such studies require adequate panel design to represent most of the available genetic and phenotypic diversity. Assessing linkage disequilibrium extent and panel power is also needed to determine the marker density required for association studies. RESULTS: Starting from the largest grapevine collection worldwide maintained in Vassal (France), we designed a diversity panel of 279 cultivars with limited relatedness, reflecting the low structuration in three genetic pools resulting from different uses (table vs wine) and geographical origin (East vs West), and including the major founders of modern cultivars. With 20 simple sequence repeat markers and five quantitative traits, we showed that our panel adequately captured most of the genetic and phenotypic diversity existing within the entire Vassal collection. To assess linkage disequilibrium extent and panel power, we genotyped single nucleotide polymorphisms: 372 over four genomic regions and 129 distributed over the whole genome. Linkage disequilibrium, measured by correlation corrected for kinship, reached 0.2 for a physical distance between 9 and 458 Kb depending on genetic pool and genomic region, with varying size of linkage disequilibrium blocks. This panel achieved reasonable power to detect associations between traits with high broad-sense heritability (> 0.7) and causal loci with intermediate allelic frequency and strong effect (explaining > 10 % of total variance). CONCLUSIONS: Our association panel constitutes a new, highly valuable resource for genetic association studies in grapevine, and deserves dissemination to diverse field and greenhouse trials to gain more insight into the genetic control of many agronomic traits and their interaction with the environment.

MybA1 gene diversity across the Vitis genus.

The MybA1 gene in the genus Vitis encodes a transcription factor, belonging to the R2R3 Myb family, that controls the last steps in the anthocyanins biosynthesis pathway. Polymorphism within MybA1 has been associated with color variation in berries of V. vinifera and other Vitis species. In this work, we analyzed the sequence variation in MybA1 both in the subg. Muscadinia and in an extended set of Asian, American and European genotypes of subg. Vitis. Our aims were to infer the evolution of this gene during the speciation process and to identify polymorphisms that could potentially generate changes in gene regulation. The results show that MybA1 experienced many insertions and deletions in non-coding regions but also in the third exon sequence. Owing to the larger set of Vitis species compared here, new indels were identified and the origin of previously described indels was reconsidered. A large number of single nucleotide polymorphisms were found in non-coding regions but also in the sequence coding for the R2R3 domain and the C terminal part of the protein. Some of these changes led to amino acid substitutions and therefore could have modified MybA1 protein activity. Bayesian phylogenetic analysis of all polymorphisms did not provide a consensus tree depicting the geographical partitioning of the species but allowed highlighting several species relationships within subgenus Vitis. Finally, the evolutionary events described could be useful to gain more insight into the role of MybA1 for anthocyanin biosynthesis in grapevine.

New stable QTLs for berry weight do not colocalize with QTLs for seed traits in cultivated grapevine (Vitis vinifera L.).

BACKGROUND: In grapevine, as in other fruit crops, fruit size and seed content are key components of yield and quality; however, very few Quantitative Trait Loci (QTLs) for berry weight and seed content (number, weight, and dry matter percentage) have been discovered so far. To identify new stable QTLs for marker-assisted selection and candidate gene identification, we performed simultaneous QTL detection in four mapping populations (seeded or seedless) with various genetic backgrounds. RESULTS: For berry weight, we identified five new QTLs, on linkage groups (LGs) 1, 8, 11, 17 and 18, in addition to the known major QTL on LG 18. The QTL with the largest effect explained up to 31% of total variance and was found in two genetically distant populations on LG 17, where it colocalized with a published putative domestication locus. For seed traits, besides the major QTLs on LG 18 previously reported, we found four new QTLs explaining up to 51% of total variance, on LGs 4, 5, 12 and 14. The previously published QTL for seed number on LG 2 was found related in fact to sex. We found colocalizations between seed and berry weight QTLs only for the major QTL on LG 18 in a seedless background, and on LGs 1 and 13 in a seeded background. Candidate genes belonging to the cell number regulator CNR or cytochrome P450 families were found under the berry weight QTLs on LGs 1, 8, and 17. The involvement of these gene families in fruit weight was first described in tomato using a QTL-cloning approach. Several other interesting candidate genes related to cell wall modifications, water import, auxin and ethylene signalling, transcription control, or organ identity were also found under berry weight QTLs. CONCLUSION: We discovered a total of nine new QTLs for berry weight or seed traits in grapevine, thereby increasing more than twofold the number of reliable QTLs for these traits available for marker assisted selection or candidate gene studies. The lack of colocalization between berry and seed QTLs suggests that these traits may be partly dissociated.

Genetic structure in cultivated grapevines is linked to geography and human selection.

BACKGROUND: Grapevine (Vitis vinifera subsp. vinifera) is one of the most important and ancient horticultural plants in the world. Domesticated about 8-10,000 years ago in the Eurasian region, grapevine evolved from its wild relative (V. vinifera subsp. sylvestris) into very diverse and heterozygous cultivated forms. In this work we study grapevine genetic structure in a large sample of cultivated varieties, to interpret the wide diversity at morphological and molecular levels and link it to cultivars utilization, putative geographic origin and historical events. RESULTS: We analyzed the genetic structure of cultivated grapevine using a dataset of 2,096 multi-locus genotypes defined by 20 microsatellite markers. We used the Bayesian approach implemented in the STRUCTURE program and a hierarchical clustering procedure based on Ward's method to assign individuals to sub-groups. The analysis revealed three main genetic groups defined by human use and geographic origin: a) wine cultivars from western regions, b) wine cultivars from the Balkans and East Europe, and c) a group mainly composed of table grape cultivars from Eastern Mediterranean, Caucasus, Middle and Far East countries. A second structure level revealed two additional groups, a geographic group from the Iberian Peninsula and Maghreb, and a group comprising table grapes of recent origins from Italy and Central Europe. A large number of admixed genotypes were also identified. Structure clusters regrouped together a large proportion of family-related genotypes. In addition, Ward's method revealed a third level of structure, corresponding either to limited geographic areas, to particular grape use or to family groups created through artificial selection and breeding. CONCLUSIONS: This study provides evidence that the cultivated compartment of Vitis vinifera L. is genetically structured. Genetic relatedness of cultivars has been shaped mostly by human uses, in combination with a geographical effect. The finding of a large portion of admixed genotypes may be the trace of both large human-mediated exchanges between grape-growing regions throughout history and recent breeding.

Large-scale parentage analysis in an extended set of grapevine cultivars (Vitis vinifera L.).

Inheritance of nuclear microsatellite markers (nSSR) has been proved to be a powerful tool to verify or uncover the parentage of grapevine cultivars. The aim of the present study was to undertake an extended parentage analysis using a large sample of Vitis vinifera cultivars held in the INRA "Domaine de Vassal" Grape Germplasm Repository (France). A dataset of 2,344 unique genotypes (i.e. cultivars without synonyms, clones or mutants) identified using 20 nSSR was analysed with FAMOZ software. Parentages showing a logarithm of odds score higher than 18 were validated in relation to the historical data available. The analysis first revealed the full parentage of 828 cultivars resulting in: (1) 315 original full parentages uncovered for traditional cultivars, (2) 100 full parentages confirming results established with molecular markers in prior papers and 32 full parentages that invalidated prior results, (3) 255 full parentages confirming pedigrees as disclosed by the breeders and (4) 126 full parentages that invalidated breeders' data. Second, incomplete parentages were determined in 1,087 cultivars due to the absence of complementary parents in our cultivar sample. Last, a group of 276 genotypes showed no direct relationship with any other cultivar in the collection. Compiling these results from the largest set of parentage data published so far both enlarges and clarifies our knowledge of the genetic constitution of cultivated V. vinifera germplasm. It also allows the identification of the main genitors involved in varietal assortment evolution and grapevine breeding.

SNiPlay: a web-based tool for detection, management and analysis of SNPs. Application to grapevine diversity projects.

BACKGROUND: High-throughput re-sequencing, new genotyping technologies and the availability of reference genomes allow the extensive characterization of Single Nucleotide Polymorphisms (SNPs) and insertion/deletion events (indels) in many plant species. The rapidly increasing amount of re-sequencing and genotyping data generated by large-scale genetic diversity projects requires the development of integrated bioinformatics tools able to efficiently manage, analyze, and combine these genetic data with genome structure and external data. RESULTS: In this context, we developed SNiPlay, a flexible, user-friendly and integrative web-based tool dedicated to polymorphism discovery and analysis. It integrates:1) a pipeline, freely accessible through the internet, combining existing softwares with new tools to detect SNPs and to compute different types of statistical indices and graphical layouts for SNP data. From standard sequence alignments, genotyping data or Sanger sequencing traces given as input, SNiPlay detects SNPs and indels events and outputs submission files for the design of Illumina's SNP chips. Subsequently, it sends sequences and genotyping data into a series of modules in charge of various processes: physical mapping to a reference genome, annotation (genomic position, intron/exon location, synonymous/non-synonymous substitutions), SNP frequency determination in user-defined groups, haplotype reconstruction and network, linkage disequilibrium evaluation, and diversity analysis (Pi, Watterson's Theta, Tajima's D).Furthermore, the pipeline allows the use of external data (such as phenotype, geographic origin, taxa, stratification) to define groups and compare statistical indices.2) a database storing polymorphisms, genotyping data and grapevine sequences released by public and private projects. It allows the user to retrieve SNPs using various filters (such as genomic position, missing data, polymorphism type, allele frequency), to compare SNP patterns between populations, and to export genotyping data or sequences in various formats. CONCLUSIONS: Our experiments on grapevine genetic projects showed that SNiPlay allows geneticists to rapidly obtain advanced results in several key research areas of plant genetic diversity. Both the management and treatment of large amounts of SNP data are rendered considerably easier for end-users through automation and integration. Current developments are taking into account new advances in high-throughput technologies.SNiPlay is available at: http://sniplay.cirad.fr/.

Patterns of sequence polymorphism in the fleshless berry locus in cultivated and wild Vitis vinifera accessions.

BACKGROUND: Unlike in tomato, little is known about the genetic and molecular control of fleshy fruit development of perennial fruit trees like grapevine (Vitis vinifera L.). Here we present the study of the sequence polymorphism in a 1 Mb grapevine genome region at the top of chromosome 18 carrying the fleshless berry mutation (flb) in order, first to identify SNP markers closely linked to the gene and second to search for possible signatures of domestication. RESULTS: In total, 62 regions (17 SSR, 3 SNP, 1 CAPS and 41 re-sequenced gene fragments) were scanned for polymorphism along a 3.4 Mb interval (85,127-3,506,060 bp) at the top of the chromosome 18, in both V. vinifera cv. Chardonnay and a genotype carrying the flb mutation, V. vinifera cv. Ugni Blanc mutant. A nearly complete homozygosity in Ugni Blanc (wild and mutant forms) and an expected high level of heterozygosity in Chardonnay were revealed. Experiments using qPCR and BAC FISH confirmed the observed homozygosity. Under the assumption that flb could be one of the genes involved into the domestication syndrome of grapevine, we sequenced 69 gene fragments, spread over the flb region, representing 48,874 bp in a highly diverse set of cultivated and wild V. vinifera genotypes, to identify possible signatures of domestication in the cultivated V. vinifera compartment. We identified eight gene fragments presenting a significant deviation from neutrality of the Tajima's D parameter in the cultivated pool. One of these also showed higher nucleotide diversity in the wild compartments than in the cultivated compartments. In addition, SNPs significantly associated to berry weight variation were identified in the flb region. CONCLUSIONS: We observed the occurrence of a large homozygous region in a non-repetitive region of the grapevine otherwise highly-heterozygous genome and propose a hypothesis for its formation. We demonstrated the feasibility to apply BAC FISH on the very small grapevine chromosomes and provided a specific probe for the identification of chromosome 18 on a cytogenetic map. We evidenced genes showing putative signatures of selection and SNPs significantly associated with berry weight variation in the flb region. In addition, we provided to the community 554 SNPs at the top of chromosome 18 for the development of a genotyping chip for future fine mapping of the flb gene in a F2 population when available.