BackgroundWorldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented.Principal FindingsWe estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before).ConclusionsSanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.

In grapes, stilbene synthesis occurs in the skin, and it is induced by biotic and abiotic stresses. To date, experimental evidence of a constitutive production of resveratrols in healthy grape is scarce and not conclusive. The aim of the present work was to investigate stilbene biosynthesis in healthy grapes both at biochemical and molecular levels. By measuring the concentration of resveratrols in ripe berries of 78 Vitis vinifera varieties for 3 years, we could identify significant differences among genotypes, providing the first tentative varietal classification based on resveratrol content. Furthermore, an increasing stilbene accumulation from veraison to ripening phase was also observed. Using real-time reverse transcription polymerase chain reaction and a berry-specific cDNA array, gene expression analysis was carried out on two distinct pools of berries belonging to the high and low resveratrol producers and on three berry developmental stages. The stilbene synthase, phenylalanine ammonia-lyase, and 4-coumarate-CoA ligase expression profiles showed an increasing concentration of these transcripts from véraison to maturity and a higher accumulation in the grape of high resveratrol producers. Macroarray data analysis revealed that high resveratrol levels are also accompanied by the up-regulation of genes involved in plant defense and the concomitant underexpression of genes related to the ripening process and to indole alkaloid synthesis.

Muscat flavor is a relevant trait both in winemaking and in fresh grape consumption. From a chemical point of view, it is strongly related to the accumulation of monoterpenes in berries. However, knowledge of the genetic mechanisms underlying its regulation is still limited. The objective of this study was to dissect the genetic determinism of aroma in grapevine by applying the analysis of quantitative trait loci (QTL) and the candidate gene (CG) approach. Two F(1) segregating progenies were evaluated through high-resolution gas chromatography-mass spectrometry (HRGC-MS) for the amounts of individual monoterpenes over 3 and 2 years. In the Italia x Big Perlon cross 34 CGs, chosen according to gene ontology (GO) terms, were placed on a complete map and tested for linkage with QTLs for linalool, nerol and geraniol levels. Two CGs mapped within a QTL for linalool content on LG 10. A third one co-localized with a major QTL for the level of the three monoterpenes on LG 5; this gene encodes 1-deoxy-D: -xylulose 5-phosphate synthase (DXS), which is the first enzyme in the plastidial pathway of terpene biosynthesis. Depending on these findings, we report the first in silico analysis of grapevine DXS genes based on the whole genome sequence. Further research on the functional significance of these associations might help to understand the genetic control of Muscat flavor.

The construction of a dense genetic map for Vitis vinifera and its anchoring to a BAC-based physical map is described: it includes 994 loci mapped onto 19 linkage groups, corresponding to the basic chromosome number of Vitis. Spanning 1245 cM with an average distance of 1.3 cM between adjacent markers, the map was generated from the segregation of 483 single-nucleotide polymorphism (SNP)-based genetic markers, 132 simple sequence repeats (SSRs), and 379 AFLP markers in a mapping population of 94 F 1 individuals derived from a V. vinifera cross of the cultivars Syrah and Pinot Noir. Of these markers, 623 were anchored to 367 contigs that are included in a physical map produced from the same clone of Pinot Noir and covering 352 Mbp. On the basis of contigs containing two or more genetically mapped markers, region-dependent estimations of physical and recombinational distances are presented. The markers used in this study include 118 SSRs common to an integrated map derived from five segregating populations of V. vinifera. The positions of these SSR markers in the two maps are conserved across all Vitis linkage groups. The addition of SNP-based markers introduces polymorphisms that are easy to database, are useful for evolutionary studies, and significantly increase the density of the map. The map provides the most comprehensive view of the Vitis genome reported to date and will be relevant for future studies on structural and functional genomics and genetic improvement.

Expressed sequence tags (ESTs) are providing a valuable approach to sampling organism-expressed genomes, especially when studying large genomes such as those of many plants. We report on the comparison of 8,647 ESTs generated from six different grape (Vitis vinifera L.) organs: berry, root, leaf, bud, shoot and inflorescence. Clustering and assembly of these ESTs resulted in 4,203 unique sequences and revealed that at this level of EST sampling, each organ shares a low percentage of transcripts with the others. To define organ relationships based on EST counts, we calculated a distance matrix of pairwise correlation coefficients between the libraries which indicated bud, inflorescence and shoot as a group distinct from the other organs considered in this study. A putative function was identified for about 85% of the unique sequences. By assigning them to specific functional classes, we were able to highlight strong differences between organs in the metabolism, protein biosynthesis and photosynthesis categories. This grape EST collection has also proven to be a valuable source for the development of 'functional' simple sequence repeats (SSRs) markers: a total of 405 SSRs have been identified. EST sequences and annotation results have been organised in the IASMA-grape database, freely available at the address http://genomics.iasma.it.

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Ultradeep sequencing of genomes permits the detection of very low-level genomic instability in non-neoplastic tissues of patients with the most common form of inherited colorectal cancer.