Ontology-oriented retrieval of putative microRNAs in Vitis vinifera via GrapeMiRNA: a web database of de novo predicted grape microRNAs

Background  

Two complete genome sequences are available for Vitis vinifera Pinot noir. Based on the sequence and gene predictions produced by the IASMA, we performed an in silico detection of putative microRNA genes and of their targets, and collected the most reliable microRNA predictions in a web database. The application is available at .     

Genetic transformation of Vitis vinifera via organogenesis

Background  

Efficient transformation and regeneration methods are a priority for successful application of genetic engineering to vegetative propagated plants such as grape. The current methods for the production of transgenic grape plants are based on Agrobacterium-mediated transformation followed by regeneration from embryogenic callus. However, grape embryogenic calli are laborious to establish and the phenotype of the regenerated plants can be altered.     

Genetic structure and differentiation in cultivated grape,Vitis vinifera L

222 cultivated (Vitis vinifera) and 22 wild (V. vinifera ssp. sylvestris) grape accessions were analysed for genetic diversity and differentiation at eight microsatellite loci. A total of 94 alleles were detected, with extensive polymorphism among the accessions. Multivariate relationships among accessions revealed 16 genetic groups structured into three clusters, supporting the classical eco-geographic grouping of grape cultivars: occidentalis, pontica and orientalis. French cultivars appeared to be distinct and showed close affinity to the wild progenitor, ssp. sylvestris from south-western France (Pyrenees) and Tunisia, probably reflecting the origin and domestication history of many of the old wine cultivars from France. There was appreciable level of differentiation between table and wine grape cultivars, and the Muscat types were somewhat distinct within the wine grapes. Contingency chi2 analysis indicated significant heterogeneity in allele frequencies among groups at all loci. The observed heterozygosities for different groups ranged from 0.625 to 0.9 with an overall average of 0.771. Genetic relationships among groups suggested hierarchical differentiation within cultivated grape. The gene diversity analysis indicated narrow divergence among groups and that most variation was found within groups (approximately 85%). Partitioning of diversity suggested that the remaining variation is somewhat structured hierarchically at different levels of differentiation. The overall organization of genetic diversity suggests that the germplasm of cultivated grape represents a single complex gene pool and that its structure is determined by strong artificial selection and a vegetative mode of reproduction.     

Anthocyanin production in selected cell lines of grape (Vitis vinifera L.)

Summary Anthocyanin production of two lines ofVitis vinifera cell cultures, i.e., 5.4 and 13.1, which were obtained from the same starting material after 20 and 37 mo. of clonal selection, respectively, was investigated. Cell suspension cultures of lines 5.4 and 13.1 maintained an anthocyanin content of 0.44 ± 0.15 and 1.02 ± 0.31 mg·g⁻¹ fresh weight during 50 and 32 weekly maintenance subcultures, respectively. Under anthocyanin-promoting culture conditions, both lines showed an enhancement of their anthocyanin level by approximately fourfold. While line 5.4 accumulated peonidin 3-glucoside and cyanidin 3-glucoside in decreasing order, line 13.1 accumulated primarily peonidin 3-p-coumaroylglucoside with lesser amounts of malvidin monoglucoside. Results show that while the anthocyanin content was improved during the course of repeated selections, the anthocyanin composition was modified markedly favoring the accumulation of more metabolically-advanced anthocyanins.     

MAST2-like proteinkinase from grape Vitis vinifera: Cloning of catalytic domain cDNA

During the work on the project on the identification of proteinkinases that phosphorylate protein microtubules of plants, we revealed with the help of bioinformatics the genes of assumed homologues of proteinkinase MAST2 that is associated with microtubules in animal cells. Respectively, the gene of the closest homologue of MAST2, the assumed protein that we have named GMLK (Grape MAST2-Like Kinase, A7NTE9_VITVI) was identified in the genome of grape Vitis vinifera. This study presents the results of the successful cloning of protein GMLK (A7NTE9_VITVI) cDNA from the leaves of the Pinot Noir grape.     

RAPD analysis of selected local Turkish grape cultivars (Vitis vinifera)

Turkey is very rich in local grape varieties. The solution to the problem of identifying local cultivars, which is considered an important deficiency for the region, will only be possible when they can be defined with molecular markers. Forty-nine local grapevine cultivars from ?anl?urfa (Turkey) were characterized with RAPD markers. Twenty-five decamer primers selected from 60 primers were used in this analysis. A total of 171 bands were obtained with the 25 primers, of which 112 were polymorphic; the level of DNA polymorphism was 65.49% in these local cultivars. Among the selected primers, OPA-18, OPO-07 and P-123 gave the maximum number of polymorphic bands (seven). Genetic relationships among these cultivars were determined with a similarity index and using a dendogram. Among the grape cultivars, the lowest similarity ratio (0.578) was observed among the Külahi-K?z?lbanki cultivars and the highest similarity ratio (0.908) was observed for the ?ilorut-D?külgen combination. The high similarity ratio among the grape cultivars of ?anl?urfa Province was also reflected in the dendogram. In general, no relationships were encountered between the genetic identification of the cultivars and their ampelographic properties.     

Volatile components of grape pomaces from different cultivars of Sicilian Vitis vinifera L

The volatile components of grape pomace coming from the processing of some of the most important varieties of grape (Vitis vinifera L.) cultivated in Sicily, namely Nero d'Avola, Nerello Mascalese, Frappato and Cabernet Sauvignon, have been determined by gas-chromatography (GC) and gas-chromatography-mass spectrometry (GC-MS). According to the winemaking procedure that entails the removal of stalks before fermentation, two kinds of grape pomace are obtained. The first consists of skins, pulp residues and seeds, the proper grape pomace, which is partially used for grappa, a typical Italian spirit, and alcohol production, the second consists almost exclusively of stalks. On the whole, 38 components have been characterized in the samples of grape pomaces, with Frappato cv. showing the richest composition; instead, 88 components have been detected in the stalks of Frappato, Nero d'Avola, Nerello Mascalese and Cabernet Sauvignon varieties. In order to make a comparison between the grape varieties easier, the volatile components detected in the two sets of samples (grape pomaces and stalks) have been grouped in different classes. Significant differences among varieties have been detected and statistical treatment of data is also reported. This study is part of a wider project aimed at the possible exploitation of the main agro-industrial by-products. At the same time it is one of the first reports on the volatile components of this waste material.     

An efficient leaf-disc culture method for the regeneration via somatic embryogenesis and transformation of grape (Vitis vinifera L.)

By manipulating hormone levels, light intensities and temperature, we have developed an efficient leaf-disc method for the regeneration of plants via embryogenesis and for transformation in four genotypes of Vitis vinifera L. In MS basal medium supplemented with 1 mg l^-1 6-benzylaminopurine (BAP) and 0.1 mg l^-1 2,4-dichlorophenoxyacetic acid, leaf discs cultured for 2 weeks under dark conditions produced calli in over 80% of the cultures. These subsequently differentiated into pro-embryos and embryos only if kept under conditions of low light intensity (15 µE m^-2 s^-1) for 2 weeks before being transferred to conditions of high light intensity (60 µE m^-2 s^-1). If the calli were directly transferred to high light intensity, the differentiation into embryos was blocked and the calli turned pink. The somatic embryos germinated at a frequency of about 10% on NN basal medium and about 32% on NN medium supplemented with 1 mg l^-1BAP and 0.1 mg l^-1 indole-3-butyric acid. The embryos, however, germinated when pre-exposed to a low temperature of 4°C for 2 weeks. If they were transferred directly to room temperature under conditions of high light intensity (60 µE m^-2 s^-1), shoot buds were produced, whereas under conditions of low light intensity (15 µE m^-2 s^-1) secondary embryogenesis was induced. About 90-95% of the in vitro grown plantlets could be successfully transferred to soil. The above method was also applicable for developing transgenic embryos whose transgenic nature was monitored using #-glucuronidase as a reporter gene.     

Accumulation of anthocyanins enhanced by a high osmotic potential in grape (Vitis vinifera L.) cell suspensions

A cell suspension of grape, Vitis vinifera L. cv Gamay Fréaux, was grown under different conditions of water stress (high external osmotic potential) induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Best growth (cell density) was achieved in the low osmotic potential medium. Increasing the osmotic potential of the medium from –0.5 MPa to –0.9 MPa medium resulted in a significant increase in accumulation of anthocyanins in pigmented cells. Regulation of the osmotic potential of culture medium may be useful in controlling anthocyanin production.     

The application of SSRs characterized for grape (Vitis vinifera) to conservation studies in Vitaceae

The use of microsatellite loci developed from a single plant species across a number of related taxa is becoming increasingly widespread. This approach can provide highly informative markers even for species for which microsatellites have not been characterized. As a number of expressed sequence tag (EST)-derived and enrichment-derived microsatellites are available for grape (Vitis vinifera), this study set out to assess transferability of nine such loci across 25 species from five different Vitaceae genera. Intergeneric transfer success in Vitaceae was high (51.1%) and EST-derived loci performed better than enrichment-derived loci. Six loci were further tested across two Australian native species, Cissus hypoglauca and C. sterculiifolia, in order to assess the conservation of microsatellite repeats and their flanking sequences. Polymorphism of these selected loci was successfully tested for each species across a small, isolated rain forest population from northern New South Wales (Australia). Results from this preliminary investigation suggest that it is possible to use grape-derived simple sequence repeats (SSR) loci for population studies on Vitaceae. As Vitaceae are an important component of rain forest flora, the availability of such highly informative loci will be beneficial to future studies aimed at determining the genetic consequences of rain forest fragmentation.     

Phenology of flowering and starch accumulation in grape (Vitis vinifera L.) cuttings and vines

BACKGROUND AND AIMS: A reliable protocol for flowering and fruiting in cuttings was developed with the aim of (a) studying inflorescence and flower development in grapevine cuttings and field plants, and (b) assisting haploid plant production. METHODS: Inflorescence and flower development was studied in 'Gewurztraminer' (GW) and 'Pinot Noir' (PN) grape vines and cuttings grown in a glasshouse, along with variations in starch in the flowers. As there is a strong relationship between flower development and starch, the starch content of reproductive structures was estimated. KEY RESULTS: Inflorescence and flower development were similar in the vines and cuttings with consistent differences between the two cultivars. Indeed, the ontogenesis of male and female organs is not synchronous in GW and PN, with both female and male meiosis occurring earlier in PN than in GW. Moreover, changes of starch reserves were similar in the two plant types. CONCLUSIONS: Cuttings have a similar reproductive physiology to vines, and can be used to study grape physiology and to develop haploid plants.     

InterDom: a database of putative interacting protein domains for validating predicted protein interactions and complexes

Advances in proteomics technology have enabled new proteins to be discovered at an unprecedented speed, and high throughput experimental methods have been developed to detect protein interactions and complexes en masse. Such bottom-up, data-driven approach has resulted in data that may be uninformative or potentially errorful, requiring further validation and annotation. The InterDom database focuses on providing supporting evidence for the detected protein interactions based on putative protein domain interactions. Using an integrative approach, InterDom derives potential domain interactions by combining data from multiple sources, ranging from domain fusions, protein interactions and complexes, to scientific literature. The InterDom database is available at http://InterDom.lit.org.sg.     

Microsatellite analysis of ancient alpine grape cultivars: pedigree reconstruction of Vitis vinifera L. 'Cornalin du Valais'

Ancient and closely related grape cultivars from the Alps were analyzed with 50 microsatellite markers: 'Cornalin', 'Humagne Rouge' and 'Goron' from Valais (Switzerland); 'Cornalin', 'Petit Rouge' and 'Mayolet' from the Aosta Valley (Italy). Our results confirmed previous studies showing that the 'Cornalin' cultivars from Switzerland and Italy are distinct, and that 'Humagne Rouge' is identical to 'Cornalin' from the Aosta Valley. We propose the nomenclature 'Cornalin du Valais' and 'Cornalin d'Aoste' in order to prevent further confusion. At each locus, 'Goron', 'Petit Rouge', 'Mayolet' and 'Cornalin d'Aoste' all share at least one allele with 'Cornalin du Valais', strongly suggesting parent/offspring relationships. Alleles at 49 out of 50 microsatellite loci are consistent with 'Cornalin du Valais' being the progeny of 'Petit Rouge' and 'Mayolet'. The exception is a 10-base pair discrepancy at one locus, most likely the result of somatic mutation in one of the parents, since this parentage is supported by high likelihood ratios and historical data. We hypothesize that 'Cornalin du Valais' originated in the Aosta Valley through a natural cross and was then introduced into Valais centuries ago, probably via the Great St. Bernard Pass. Furthermore, 'Cornalin du Valais' is likely to be one of the parents of both 'Goron' and 'Cornalin d'Aoste', the respective second parents remaining unknown. This pedigree provides a convincing explanation for the allele-sharing patterns and is strongly supported by historical data. The present work is the first grapevine parentage study to deal with a multiple repeat unit discrepancy at a microsatellite locus. We suggest that the use of increasingly large numbers of loci in making parentage determinations leads to a corresponding increase in the probability of encountering a locus with intra-cultivar variability during the analysis. We therefore assume that a sole multiple repeat unit discrepancy is not sufficient to discard a parentage hypothesis.     

Abiotic and oxidative stress-dependent regulation of expression of the thioredoxin h multigenic family in grape Vitis vinifera

Thioredoxins (Trxs) are small ubiquitous oxidoreductases that participate in dithiol-disulphide exchange reactions. They are present in all organisms from prokaryotes to higher eukaryotes and involved in a variety of cellular processes in higher plants. The expression pattern of the multigenic family of grape Trxs_h was analysed in different tissues by semiquantitative RT-PCR. The grape Trx_h genes were approximately expressed in all tissues, but with different expression patterns than each other. The in silico analysis of the promoter regions of grape Trxs _h demonstrated the presence of a number of potential cis-acting elements to respond to environmental signals, suggesting that Trxs_h may respond to a variety of environmental signals, including dehydration, salinity, heat, cold, heavy metals, light, pathogens, and plant hormones. The grape Trx h genes were also found to be differentially induced under abiotic stress conditions due to the presence of different putative regulatory elements in their promoters. Collectively, the distinct expression patterns and different responses to environmental stress conditions suggest that each isoform of the multigenic family of VvTrxh may have a specific and non-redundant function in grape.     

Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.).

Four new simple sequence repeat (SSR) loci (designated VVMD5, VVMD6, VVMD7, and VVMD8) were characterized in grape and analyzed by silver staining in 77 cultivars of Vitis vinifera. Amplification products ranged in size from 141 to 263 base pairs (bp). The number of alleles observed per locus ranged from 5 to 11 and the number of diploid genotypes per locus ranged from 13 to 27. At each locus at least 75% of the cultivars were heterozygous. Alleles differing in length by only 1 bp could be distinguished by silver staining, and size estimates were within 1 or 2 bp, depending on the locus, of those obtained by fluorescence detection at previously reported loci. Allele frequencies were generally similar in wine grapes and table grapes, with some exceptions. Some alleles were found only in one of the two groups of cultivars. All 77 cultivars were distinguished by the four loci with the exception of four wine grapes considered to be somatic variants of the same cultivar, 'Pinot noir', 'Pinot gris', 'Pinot blanc', and 'Meunier'; two table grapes that are known to be synonymous, 'Keshmesh' and 'Thompson Seedless'; and three table grapes, 'Dattier', 'Rhazaki Arhanon', and 'Markandi', the first two of which have been suggested to be synonymous. Although the high polymorphism at grape SSR loci suggests that very few loci would theoretically be needed to separate all cultivars, the economic and legal significance of grape variety identification requires the increased resolution that can be provided by a larger number of loci. The ease with which SSR markers and data can be shared internationally should encourage their broad use, which will in turn increase the power of these markers for both identification and genetic analysis of grape. Key words : grape, Vitis, microsatellite, simple sequence repeat, DNA typing, identification.     

Evaluating the influence of temperature on proanthocyanidin biosynthesis in developing grape berries (Vitis vinifera L.)

Molecular Biology Reports - The variability in grape (Vitis vinifera L.) proanthocyanidin content is largely attributable to viticultural and environmental conditions. However, the particular...     

Vitis vinifera culture in a non-conventional bioreactor: the reciprocating plate bioreactor

This paper is concerned with the potential use of a reciprocating plate bioreactor (RPB) for suspended plant cell cultures. The agitation mechanism of the RPB system, a plate stack, was first evaluated in pure water and in pseudocells medium of 20, 40 and 60% of PCV. As the pseudocell concentration increases, the oxygen mass transfer coefficient, K_La, significantly decreases. Correlations were established for each plate stack and concentration with good prediction of K_La. Three fermentations were performed with Vitis vinifera cells, two in the RPB system and one in shake flasks. Shake flask cultures showed better performance whereas the first fermentation performed with the RPB showed the lowest performance. The lower growth observed was attributed to the operating conditions for aeration and the dissolved oxygen control strategy. CO₂ stripping in the initial portion of the fermentation led to lower biomass growth. The second fermentation, with more appropriate operating conditions, appears to follow the trend of shake flask cultures but was terminated after 5 days due to contamination. The RPB has the potential to be used for suspended plant cell cultures but significant research needs to be performed to find optimal operating conditions but, more importantly, to make appropriate modifications to ensure the sterility of the bioreactor over long time periods.