Diploid genome assembly of the Malbec grapevine cultivar enables haplotype-aware analysis of transcriptomic differences underlying clonal phenotypic variation.

To preserve their varietal attributes, established grapevine cultivars (Vitis vinifera L. ssp. vinifera) must be clonally propagated, due to their highly heterozygous genomes. Malbec is a France-originated cultivar appreciated for producing high-quality wines and is the offspring of cultivars Prunelard and Magdeleine Noire des Charentes. Here, we have built a diploid genome assembly of Malbec, after trio binning of PacBio long reads into the two haploid complements inherited from either parent. After haplotype-aware deduplication and corrections, complete assemblies for the two haplophases were obtained with a very low haplotype switch-error rate (<0.025). The haplophase alignment identified > 25% of polymorphic regions. Gene annotation including RNA-seq transcriptome assembly and ab initio prediction evidence resulted in similar gene model numbers for both haplophases. The annotated diploid assembly was exploited in the transcriptomic comparison of four clonal accessions of Malbec that exhibited variation in berry composition traits. Analysis of the ripening pericarp transcriptome using either haplophases as a reference yielded similar results, although some differences were observed. Particularly, among the differentially expressed genes identified only with the Magdeleine-inherited haplotype as reference, we observed an over-representation of hypothetically hemizygous genes. The higher berry anthocyanin content of clonal accession 595 was associated with increased abscisic acid responses, possibly leading to the observed overexpression of phenylpropanoid metabolism genes and deregulation of genes associated with abiotic stress response. Overall, the results highlight the importance of producing diploid assemblies to fully represent the genomic diversity of highly heterozygous woody crop cultivars and unveil the molecular bases of clonal phenotypic variation.

New Technologies and Strategies for Grapevine Breeding Through Genetic Transformation.

Grapevine, as other woody perennials, has been considered a recalcitrant crop to produce transgenic plants. Since the production of transgenic and/or edited plants requires the ability to regenerate plants from transformed tissues, this step is often the biggest bottleneck in the process. The objective of this work is to review the state of the art technologies and strategies for the improvement of grapevine transformation and regeneration, focusing on three aspects: (i) problems associated with grapevine transformation; (ii) genes that promote grapevine regeneration; and (iii) vehicles for gene delivery. Concerning the first aspect, it is well documented that one of the main factors explaining the low success rate in obtaining transgenic plants is the regeneration process. After transgenic integration into receptor cells, tissue culture is required to regenerate transgenic seedlings from transformed cells. This process is time consuming and often requires the addition of environmentally damaging reagents (antibiotics and herbicides) to the culture medium to select transgenic plants. On the other hand, the expression of genes such as the so-called developmental regulators (DR), which induce specific development programs, can be used to avoid traditional tissue culture methods. The ectopic expression of specific combinations of DR in somatic cells has the potential to induce de novo meristems in diverse crops, including grapevine. Successful genome editing by de novo reprogramming of plant meristems in somatic tissues has been reported. Moreover, it has been shown that the expression of certain transcription factors can increase the regeneration efficiency in wheat, citrus, and rice. Finally, recent reports showed the use of nanoparticles, such as carbon dots (CDs), as an attractive alternative to Agrobacterium- and biolistic-mediated plant genetic transformation. In this way, the use of antibiotics in culture media is avoided, overcoming the loss of viability of plant tissues and accelerating the regeneration processes. It has been shown that CDs can act as a vehicle to transport plasmids to plant cells in transient transformation in several crops without negative impacts on photosynthesis or growth. Based on these advances, it is possible to combine these new available strategies and technologies to overcome the regeneration problems of species such as grapevine and other crops considered as recalcitrant.

Whole genome resequencing and custom genotyping unveil clonal lineages in 'Malbec' grapevines (Vitis vinifera L.).

Grapevine cultivars are clonally propagated to preserve their varietal attributes. However, genetic variations accumulate due to the occurrence of somatic mutations. This process is anthropically influenced through plant transportation, clonal propagation and selection. Malbec is a cultivar that is well-appreciated for the elaboration of red wine. It originated in Southwestern France and was introduced in Argentina during the 1850s. In order to study the clonal genetic diversity of Malbec grapevines, we generated whole-genome resequencing data for four accessions with different clonal propagation records. A stringent variant calling procedure was established to identify reliable polymorphisms among the analyzed accessions. The latter procedure retrieved 941 single nucleotide variants (SNVs). A reduced set of the detected SNVs was corroborated through Sanger sequencing, and employed to custom-design a genotyping experiment. We successfully genotyped 214 Malbec accessions using 41 SNVs, and identified 14 genotypes that clustered in two genetically divergent clonal lineages. These lineages were associated with the time span of clonal propagation of the analyzed accessions in Argentina and Europe. Our results show the usefulness of this approach for the study of the scarce intra-cultivar genetic diversity in grapevines. We also provide evidence on how human actions might have driven the accumulation of different somatic mutations, ultimately shaping the Malbec genetic diversity pattern.

Insights into long non-coding RNA regulation of anthocyanin carrot root pigmentation.

Carrot (Daucus carota L.) is one of the most cultivated vegetable in the world and of great importance in the human diet. Its storage organs can accumulate large quantities of anthocyanins, metabolites that confer the purple pigmentation to carrot tissues and whose biosynthesis is well characterized. Long non-coding RNAs (lncRNAs) play critical roles in regulating gene expression of various biological processes in plants. In this study, we used a high throughput stranded RNA-seq to identify and analyze the expression profiles of lncRNAs in phloem and xylem root samples using two genotypes with a strong difference in anthocyanin production. We discovered and annotated 8484 new genes, including 2095 new protein-coding and 6373 non-coding transcripts. Moreover, we identified 639 differentially expressed lncRNAs between the phenotypically contrasted genotypes, including certain only detected in a particular tissue. We then established correlations between lncRNAs and anthocyanin biosynthesis genes in order to identify a molecular framework for the differential expression of the pathway between genotypes. A specific natural antisense transcript linked to the DcMYB7 key anthocyanin biosynthetic transcription factor suggested how the regulation of this pathway may have evolved between genotypes.

Catastrophic Unbalanced Genome Rearrangements Cause Somatic Loss of Berry Color in Grapevine.

Grape (Vitis vinifera) color somatic variants that can be used to develop new grapevine cultivars occasionally appear associated with deletion events of uncertain origin. To understand the mutational mechanisms generating somatic structural variation in grapevine, we compared the Tempranillo Blanco (TB) white berry somatic variant with its black berry ancestor, Tempranillo Tinto. Whole-genome sequencing uncovered a catastrophic genome rearrangement in TB that caused the hemizygous deletion of 313 genes, including the loss of the functional copy for the MYB transcription factors required for anthocyanin pigmentation in the berry skin. Loss of heterozygosity and decreased copy number delimited interspersed monosomic and disomic regions in the right arm of linkage groups 2 and 5. At least 11 validated clustered breakpoints involving intrachromosomal and interchromosomal translocations between three linkage groups flanked the deleted fragments, which, according to segregation analyses, are phased in a single copy of each of the affected chromosomes. These hallmarks, along with the lack of homology between breakpoint joins and the randomness of the order and orientation of the rearranged fragments, are all consistent with a chromothripsis-like pattern generated after chromosome breakage and illegitimate rejoining. This unbalanced genome reshuffling has additional consequences in reproductive development. In TB, lack of sexual transmission of rearranged chromosomes associates with low gamete viability, which compromises fruit set and decreases fruit production. Our findings show that catastrophic genome rearrangements arise spontaneously and stabilize during plant somatic growth. These dramatic rearrangements generate new interesting phenotypes that can be selected for the improvement of vegetatively propagated plant species.

Expression of grapevine AINTEGUMENTA-like genes is associated with variation in ovary and berry size.

Fruit size is a highly important trait for most fruit and vegetable crops. This trait has been a main selection target and could be involved in divergent selection processes leading to the differentiation between modern table and wine cultivars. Even though its determination is highly influenced by cultural practices, several regions within the grapevine genome have been identified affecting berry size, either directly or indirectly through their effect on seed content. Using grapevine seeded cultivars, we have analyzed the relationship between ovary cell number and the final size of ovaries and berry fruits. We also performed the characterization of the grapevine AINTEGUMENTA-LIKE family, since it is well reported in Arabidopsis that AINTEGUMENTA (ANT) regulates cell proliferation and organ growth in flower organ primordia by maintaining the meristematic competence of cells during organogenesis. Here we show that orthologous grapevine gene expression associate with flower developmental stages suggesting a similar biological role for this gene family in this species. Moreover, we detected a correlation between those organs size and the level of expression of VviANT1 the grapevine homolog of AtANT. This grapevine gene also co-localizes in linkage group 18 with the confidence interval of a previously detected QTL for berry size. Thus our results suggest the involvement of ANT in the regulation of berry size in grapevine.

Fruit-localized photoreceptors increase phenolic compounds in berry skins of field-grown Vitis vinifera L. cv. Malbec.

Sunlight exposure has multiple effect on fruits, as it affects the light climate perceived by fruit photoreceptors and fruit tissue temperature. In grapes (Vitis vinifera L.), light exposure can have a strong effect on fruit quality and commercial value; however, the mechanisms of light action are not well understood. The role of fruit-localized photoreceptors in the control of berry quality traits was evaluated under field conditions in a commercial vineyard in Mendoza (Argentina). Characterization of the diurnal dynamics of the fruit light environment in a vertical trellis system indicated that clusters were shaded by leaves during most of the photoperiod. Supplementation of the fruit light environment from 20 days before veraison until technological harvest showed that red (R, 660 nm) and blue (B, 470 nm) light strongly increased total phenolic compound levels at harvest in the berry skins without affecting sugar content, acidity or berry size. Far-red (FR, 730 nm) and green (G, 560 nm) light supplementation had relatively small effects. The stimulation of berry phytochromes and cryptochromes favored accumulation of flavonoid and non-flavonoid compounds, including anthocyanins, flavonols, flavanols, phenolic acids and stilbenes. These results demonstrate that the chemical composition of grape berries is modulated by the light quality received by the clusters under field conditions, and that fruit photoreceptors are not saturated even in areas of high insolation and under management systems that are considered to result in a relatively high exposure of fruits to solar radiation. Therefore, manipulation of the light environment or the light sensitivity of fruits could have significant effects on critical grape quality traits.

Dissecting the transcriptional response to elicitors in Vitis vinifera cells.

The high effectiveness of cyclic oligosaccharides like cyclodextrins in the production of trans-resveratrol in Vitis vinifera cell cultures is enhanced in the presence of methyl jasmonate. In order to dissect the basis of the interactions among the elicitation responses triggered by these two compounds, a transcriptional analysis of grapevine cell cultures treated with cyclodextrins and methyl jasmonate separately or in combination was carried out. The results showed that the activation of genes encoding enzymes from phenylpropanoid and stilbene biosynthesis induced by cyclodextrins alone was partially enhanced in the presence of methyl jasmonate, which correlated with their effects on trans-resveratrol production. In addition, protein translation and cell cycle regulation were more highly repressed in cells treated with cyclodextrins than in those treated with methyl jasmonate, and this response was enhanced in the combined treatment. Ethylene signalling was activated by all treatments, while jasmonate signalling and salicylic acid conjugation were activated only in the presence of methyl jasmonate and cyclodextrins, respectively. Moreover, the combined treatment resulted in a crosstalk between the signalling cascades activated by cyclodextrins and methyl jasmonate, which, in turn, provoked the activation of additional regulatory pathways involving the up-regulation of MYB15, NAC and WRKY transcription factors, protein kinases and calcium signal transducers. All these results suggest that both elicitors cause an activation of the secondary metabolism in detriment of basic cell processes like the primary metabolism or cell division. Crosstalk between cyclodextrins and methyl jasmonate-induced signalling provokes an intensification of these responses resulting in a greater trans-resveratrol production.

Relationships among gene expression and anthocyanin composition of Malbec grapevine clones.

Anthocyanin profiles are commonly used for grapevine cultivar identification because it is currently accepted that this trait is closely related to their genetic characteristics. Nevertheless, the extent of the variation for the anthocyanin profiles among clones of the same cultivar has not yet been studied in depth. The relative concentration of anthocyanins of 131 Malbec clones grown in the same vineyard was investigated by HPLC-DAD and the use of comprehensive statistic procedures. Complementarily, the expression level of structural and regulatory genes was studied via real time polymerase chain reaction. Significant variation was identified among the profiles of the clones, mainly due to variations in the amounts of malvidin derivatives. Finally, the differential expression in F3'5'H, OMT1 and AM2 genes seems to be related to the malvidin content variation. This work shows the existence of variation for the anthocyanin profiles among clones from the same grapevine cultivar and the putative involvement of genes related to hydroxylation, methylation, and transport of anthocyanins on the basis of such variation.

Thermotolerance responses in ripening berries of Vitis vinifera L. cv Muscat Hamburg.

Berry organoleptic properties are highly influenced by ripening environmental conditions. In this study, we used grapevine fruiting cuttings to follow berry ripening under different controlled conditions of temperature and irradiation intensity. Berries ripened at higher temperatures showed reduced anthocyanin accumulation and hastened ripening, leading to a characteristic drop in malic acid and total acidity. The GrapeGen GeneChip® combined with a newly developed GrapeGen 12Xv1 MapMan version were utilized for the functional analysis of berry transcriptomic differences after 2 week treatments from veraison onset. These analyses revealed the establishment of a thermotolerance response in berries under high temperatures marked by the induction of heat shock protein (HSP) chaperones and the repression of transmembrane transporter-encoding transcripts. The thermotolerance response was coincident with up-regulation of ERF subfamily transcription factors and increased ABA levels, suggesting their participation in the maintenance of the acclimation response. Lower expression of amino acid transporter-encoding transcripts at high temperature correlated with balanced amino acid content, suggesting a transcriptional compensation of temperature effects on protein and membrane stability to allow for completion of berry ripening. In contrast, the lower accumulation of anthocyanins and higher malate metabolization measured under high temperature might partly result from imbalance in the expression and function of their specific transmembrane transporters and expression changes in genes involved in their metabolic pathways. These results open up new views to improve our understanding of berry ripening under high temperatures.

Berry flesh and skin ripening features in Vitis vinifera as assessed by transcriptional profiling.

BACKGROUND: Ripening of fleshy fruit is a complex developmental process involving the differentiation of tissues with separate functions. During grapevine berry ripening important processes contributing to table and wine grape quality take place, some of them flesh- or skin-specific. In this study, transcriptional profiles throughout flesh and skin ripening were followed during two different seasons in a table grape cultivar 'Muscat Hamburg' to determine tissue-specific as well as common developmental programs. METHODOLOGY/PRINCIPAL FINDINGS: Using an updated GrapeGen Affymetrix GeneChip® annotation based on grapevine 12×v1 gene predictions, 2188 differentially accumulated transcripts between flesh and skin and 2839 transcripts differentially accumulated throughout ripening in the same manner in both tissues were identified. Transcriptional profiles were dominated by changes at the beginning of veraison which affect both pericarp tissues, although frequently delayed or with lower intensity in the skin than in the flesh. Functional enrichment analysis identified the decay on biosynthetic processes, photosynthesis and transport as a major part of the program delayed in the skin. In addition, a higher number of functional categories, including several related to macromolecule transport and phenylpropanoid and lipid biosynthesis, were over-represented in transcripts accumulated to higher levels in the skin. Functional enrichment also indicated auxin, gibberellins and bHLH transcription factors to take part in the regulation of pre-veraison processes in the pericarp, whereas WRKY and C2H2 family transcription factors seems to more specifically participate in the regulation of skin and flesh ripening, respectively. CONCLUSIONS/SIGNIFICANCE: A transcriptomic analysis indicates that a large part of the ripening program is shared by both pericarp tissues despite some components are delayed in the skin. In addition, important tissue differences are present from early stages prior to the ripening onset including tissue-specific regulators. Altogether, these findings provide key elements to understand berry ripening and its differential regulation in flesh and skin.

Enhanced extracellular production of trans-resveratrol in Vitis vinifera suspension cultured cells by using cyclodextrins and methyljasmonate.

In this work, the effect of different inducing factors on trans-resveratrol extracellular production in Monastrell grapevine suspension cultured cells is evaluated. A detailed analysis provides the optimal concentrations of cyclodextrins, methyljasmonate and UV irradiation dosage, optimal cell density, elicitation time and sucrose content in the culture media. The results indicate that trans-resveratrol production decreases as the initial cell density increases for a constant elicitor concentration in Monastrell suspension cultured cells treated with cyclodextrins individually or in combination with methyljasmonate; the decrease observed in cell cultures elicited with cyclodextrins alone is far more drastic than those observed in the combined treatment. trans-Resveratrol extracellular production observed by the joint use of cyclodextrins and methyljasmonate (1,447.8 ± 60.4 µmol trans-resveratrol g(-1) dry weight) is lower when these chemical compounds are combined with UV light short exposure (669.9 ± 45.2 µmol trans-resveratrol g(-1) dry weight). Likewise, trans-resveratrol production is dependent on levels of sucrose in the elicitation medium with the maximal levels observed with 20 g l(-1) sucrose and the joint action of cyclodextrins and 100 µM methyljasmonate. The sucrose concentration did not seem to limit the process although it affects significantly the specific productivity since the lowest sucrose concentration is 10 g l(-1), the highest productivity is reached (100.7 ± 5.8 µmol trans-resveratrol g(-1) dry weight g(-1) sucrose) using cyclodextrins and 25 µM methyljasmonate.

A 48 SNP set for grapevine cultivar identification.

BACKGROUND: Rapid and consistent genotyping is an important requirement for cultivar identification in many crop species. Among them grapevine cultivars have been the subject of multiple studies given the large number of synonyms and homonyms generated during many centuries of vegetative multiplication and exchange. Simple sequence repeat (SSR) markers have been preferred until now because of their high level of polymorphism, their codominant nature and their high profile repeatability. However, the rapid application of partial or complete genome sequencing approaches is identifying thousands of single nucleotide polymorphisms (SNP) that can be very useful for such purposes. Although SNP markers are bi-allelic, and therefore not as polymorphic as microsatellites, the high number of loci that can be multiplexed and the possibilities of automation as well as their highly repeatable results under any analytical procedure make them the future markers of choice for any type of genetic identification. RESULTS: We analyzed over 300 SNP in the genome of grapevine using a re-sequencing strategy in a selection of 11 genotypes. Among the identified polymorphisms, we selected 48 SNP spread across all grapevine chromosomes with allele frequencies balanced enough as to provide sufficient information content for genetic identification in grapevine allowing for good genotyping success rate. Marker stability was tested in repeated analyses of a selected group of cultivars obtained worldwide to demonstrate their usefulness in genetic identification. CONCLUSIONS: We have selected a set of 48 stable SNP markers with a high discrimination power and a uniform genome distribution (2-3 markers/chromosome), which is proposed as a standard set for grapevine (Vitis vinifera L.) genotyping. Any previous problems derived from microsatellite allele confusion between labs or the need to run reference cultivars to identify allele sizes disappear using this type of marker. Furthermore, because SNP markers are bi-allelic, allele identification and genotype naming are extremely simple and genotypes obtained with different equipments and by different laboratories are always fully comparable.

A DIGE-based quantitative proteomic analysis of grape berry flesh development and ripening reveals key events in sugar and organic acid metabolism.

Grapevine (Vitis vinifera L.) is an economically important fruit crop. Quality-determining grape components, such as sugars, acids, flavours, anthocyanins, tannins, etc., are accumulated during the different grape berry development stages. Thus, correlating the proteomic profiles with the biochemical and physiological changes occurring in grape is of paramount importance to advance the understanding of the berry development and ripening processes. Here, the developmental analysis of V. vinifera cv. Muscat Hamburg berries is reported at protein level, from fruit set to full ripening. A top-down proteomic approach based on differential in-gel electrophoresis (DIGE) followed by tandem mass spectrometry led to identification and quantification of 156 and 61 differentially expressed proteins in green and ripening phases, respectively. Two key points in development, with respect to changes in protein level, were detected: end of green development and beginning of ripening. The profiles of carbohydrate metabolism enzymes were consistent with a net conversion of sucrose to malate during green development. Pyrophosphate-dependent phosphofructokinase is likely to play a key role to allow an unrestricted carbon flow. The well-known change of imported sucrose fate at the beginning of ripening from accumulation of organic acid (malate) to hexoses (glucose and fructose) was well correlated with a switch in abundance between sucrose synthase and soluble acid invertase. The role of the identified proteins is discussed in relation to their biological function, grape berry development, and to quality traits. Another DIGE experiment comparing fully ripe berries from two vintages showed very few spots changing, thus indicating that protein changes detected throughout development are specific.

Transcriptome changes in grapevine (Vitis vinifera L.) cv. Malbec leaves induced by ultraviolet-B radiation.

BACKGROUND: Ultraviolet-B radiation (UV-B, 280-315 nm) is a natural component of sunlight, which has numerous regulatory effects on plant physiology. The nature of the response to UV-B is dependent on fluence rate, dose, duration and wavelength of the UV-B treatment. Some reports have analyzed the changes in gene expression caused by UV-B light on several plant species using microarray technology. However, there is no information on the transcriptome response triggered by UV-B in grapevine. In this paper we investigate the gene expression responses of leaves from in vitro cultured Vitis vinifera cv. Malbec plants subjected to the same dose of biologically effective UV-B radiation (4.75 kJ m-2 d-1) administered at two different fluence rates (16 h at ≅ 8.25 µW cm-2, 4 h at ≅ 33 µW cm-2) using a new custom made GrapeGen Affymetrix GeneChip®. RESULTS: The number of genes modulated by high fluence rate UV-B doubled the number of genes modulated by low fluence UV-B. Their functional analyses revealed several functional categories commonly regulated by both UV-B treatments as well as categories more specifically modulated depending on UV-B fluence rate. General protective responses, namely the induction of pathways regulating synthesis of UV-B absorbing compounds such as the Phenylpropanoid pathway, the induction of different antioxidant defense systems and the activation of pathways commonly associated with pathogen defense and abiotic stress responses seem to play critical roles in grapevine responses against UV-B radiation. Furthermore, high fluence rate UV-B seemed to specifically modulate additional pathways and processes in order to protect grapevine plantlets against UV-B-induced oxidative stress, stop the cell cycle progression, and control protein degradation. On the other hand, low fluence rate UV-B regulated the expression of specific responses in the metabolism of auxin and abscisic acid as well as in the modification of cell walls that could be involved in UV-B acclimation-like processes. CONCLUSION: Our results show the UV-B radiation effects on the leaf transcriptome of grapevine (Vitis vinifera cv. Malbec) plantlets. Functional categories commonly modulated under both UV-B treatments as well as transcripts specifically regulated in an UV-B-intensity dependent way were identified. While high fluence rate UV-B had regulatory effects mainly on defense or general multiple-stress responses pathways, low fluence rate UV-B promoted the expression of genes that could be involved in UV-B protection or the amelioration of the UV-B-induced damage. This study also provides an extensive list of genes regulating multiple metabolic pathways involved in the response of grapevine to UV-B that can be used for future researches.

Genome-wide analysis of MIKCC-type MADS box genes in grapevine.

MIKC(C)-type MADS box genes encode transcription factors that play crucial roles in plant growth and development. Analysis of the grapevine (Vitis vinifera) genome revealed up to 38 MIKC(C)-type genes. We report here a complete analysis of this gene family regarding their phylogenetic relationships with homologous genes identified in other sequenced dicot genomes, their genome location, and gene structure and expression. The grapevine genes cluster in 13 subfamilies with their Arabidopsis (Arabidopsis thaliana) and poplar (Populus trichocarpa) counterparts. The lack of recent whole genome duplications in grapevine allows assigning the gene diversification processes observed within each subfamily either to an ancestral polyploidization event predating the divergence of those three species or to later duplication events within each lineage. Expression profiles of MIKC(C)-type genes in vegetative and reproductive organs as well as during flower and tendril development show conserved expression domains for specific subfamilies but also reflect characteristic features of grapevine development. Expression analyses in latent buds and during flower development reveal common features previously described in other plant systems as well as possible new roles for members of some subfamilies during flowering transition. The analysis of MIKC(C)-type genes in grapevine helps in understanding the origin of gene diversification within each subfamily and provides the basis for functional analyses to uncover the role of these MADS box genes in grapevine development.

Synergistic effect of methyljasmonate and cyclodextrin on stilbene biosynthesis pathway gene expression and resveratrol production in Monastrell grapevine cell cultures.

BACKGROUND: Plant cell cultures have been shown as feasible systems for the production of secondary metabolites, being the elicitation with biotic or abiotic stimuli the most efficient strategy to increase the production of those metabolites. Vitaceae phytoalexins constitute a group of molecules belonging to the stilbene family which are derivatives of the trans-resveratrol structure and are produced by plants and cell cultures as a response to biotic and abiotic stresses. The potential benefits of resveratrol on human health have made it one of the most thoroughly studied phytochemical molecules. The aim of this study was to evaluate the elicitor effect of both cyclodextrin (CD) and methyljasmonate (MeJA) on grapevine cell cultures by carrying out a quantitative analysis of their role on resveratrol production and on the expression of stilbene biosynthetic genes in Vitis vinifera cv Monastrell albino cell suspension cultures. FINDINGS: MeJA and CD significantly but transiently induced the expression of stilbene biosynthetic genes when independently used to treat grapevine cells. This expression correlated with resveratrol production in CD-treated cells but not in MeJA-treated cells, which growth was drastically affected. In the combined treatment of CD and MeJA cell growth was similarly affected, however resveratrol production was almost one order of magnitude higher, in correlation with maximum expression values for stilbene biosynthetic genes. CONCLUSION: The effect of MeJA on cell division combined with a true and strong elicitor like CD could be responsible for the observed synergistic effect of both compounds on resveratrol production and on the expression of genes in the stilbene pathway.

High throughput SNP discovery and genotyping in grapevine (Vitis vinifera L.) by combining a re-sequencing approach and SNPlex technology.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) are the most abundant type of DNA sequence polymorphisms. Their higher availability and stability when compared to simple sequence repeats (SSRs) provide enhanced possibilities for genetic and breeding applications such as cultivar identification, construction of genetic maps, the assessment of genetic diversity, the detection of genotype/phenotype associations, or marker-assisted breeding. In addition, the efficiency of these activities can be improved thanks to the ease with which SNP genotyping can be automated. Expressed sequence tags (EST) sequencing projects in grapevine are allowing for the in silico detection of multiple putative sequence polymorphisms within and among a reduced number of cultivars. In parallel, the sequence of the grapevine cultivar Pinot Noir is also providing thousands of polymorphisms present in this highly heterozygous genome. Still the general application of those SNPs requires further validation since their use could be restricted to those specific genotypes. RESULTS: In order to develop a large SNP set of wide application in grapevine we followed a systematic re-sequencing approach in a group of 11 grape genotypes corresponding to ancient unrelated cultivars as well as wild plants. Using this approach, we have sequenced 230 gene fragments, what represents the analysis of over 1 Mb of grape DNA sequence. This analysis has allowed the discovery of 1573 SNPs with an average of one SNP every 64 bp (one SNP every 47 bp in non-coding regions and every 69 bp in coding regions). Nucleotide diversity in grape (pi = 0.0051) was found to be similar to values observed in highly polymorphic plant species such as maize. The average number of haplotypes per gene sequence was estimated as six, with three haplotypes representing over 83% of the analyzed sequences. Short-range linkage disequilibrium (LD) studies within the analyzed sequences indicate the existence of a rapid decay of LD within the selected grapevine genotypes. To validate the use of the detected polymorphisms in genetic mapping, cultivar identification and genetic diversity studies we have used the SNPlextrade mark genotyping technology in a sample of grapevine genotypes and segregating progenies. CONCLUSION: These results provide accurate values for nucleotide diversity in coding sequences and a first estimate of short-range LD in grapevine. Using SNPlextrade mark genotyping we have shown the application of a set of discovered SNPs as molecular markers for cultivar identification, linkage mapping and genetic diversity studies. Thus, the combination a highly efficient re-sequencing approach and the SNPlextrade mark high throughput genotyping technology provide a powerful tool for grapevine genetic analysis.

Generation of ESTs in Vitis vinifera wine grape (Cabernet Sauvignon) and table grape (Muscat Hamburg) and discovery of new candidate genes with potential roles in berry development.

We report the generation and analysis of a total of 77,583 expressed sequence tags (ESTs) from two grapevine (Vitis vinifera L.) cultivars, Cabernet Sauvignon (wine grape) and Muscat Hamburg (table grape) with a focus on EST sequence quality and assembly optimization. The majority of the ESTs were derived from normalized cDNA libraries representing berry pericarp and seed developmental series, pooled non-berry tissues including root, flower, and leaf in Cabernet Sauvignon, and pooled tissues of berry, seed, and flower in Muscat Hamburg. EST and unigene sequence quality were determined by computational filtering coupled with small-scale contig reassembly, manual review, and BLAST analyses. EST assembly was optimized to better discriminate among closely related paralogs using two independent grape sequence sets, a previously published set of Vitis spp. gene families and our EST dataset derived from pooled leaf, flower, and root tissues of Cabernet Sauvignon. Sequence assembly within individual libraries indicated that those prepared from pooled tissues contributed the most to gene discovery. Annotations based upon searches against multiple databases including tomato and strawberry sequences helped to identify putative functions of ESTs and unigenes, particularly with respect to fleshy fruit development. Sequence comparison among the three wine grape libraries identified a number of genes preferentially expressed in the pericarp tissue, including transcription factors, receptor-like protein kinases, and hexose transporters. Gene ontology (GO) classification in the biological process aspect showed that GO categories corresponding to 'transport' and 'cell organization and biogenesis', which are associated with metabolite movement and cell wall structural changes during berry ripening, were higher in pericarp than in other tissues in the wine grape studied. The sequence data were used to characterize potential roles of new genes in berry development and composition.