AFLP analysis of genetic variation within the two economically important Anatolian grapevine (Vitis vinifera L.) varietal groups.

The Anatolian region of modern-day Turkey is believed to have played an important role in the history of grapevine (Vitis vinifera L.) domestication and spread. Despite this, the rich grape germplasm of this region is virtually uncharacterized genetically. In this study, the amplified fragment length polymorphisms (AFLP)-based genetic relations of the grapevine accessions belonging to the 2 economically important Anatolian table grape varietal groups known as V. vinifera 'Misket' (Muscat) and V. vinifera 'Parmak' were studied. Thirteen AFLP primer combinations used in the analyses revealed a total of 1495 (35.5% polymorphic) and 1567 (34.6% polymorphic) DNA fragments for the 'Misket' and 'Parmak' varietal groups, respectively. The unweighted pair-group method with arthimetic averaging (UPGMA) cluster analysis and principal coordinate analysis (PCA) conducted on polymorphic AFLP markers showed that both varietal groups contain a number of synonymous (similar genotypes known by different names) as well as homony mous (genetically different genotypes known by the same name) accessions. Our results also showed that 6 of the Anatolian 'Misket' genotypes were genetically very similar to V. vinifera 'Muscat of Alexandria', implying that these genotypes might have played some role in the formation of this universally known grape cultivar. Finally, the close genetic similarities found here between 'Muscat of Alexandria' and V. vinifera 'Muscat of Hamburg' support the recent suggestion that 'Muscat of Hamburg' probably originated from 'Muscat of Alexandria' through spontaneous hybridizations. Overall, the results of this study have implications for not only preservation and use of the Anatolian grape germplasm, but also better understanding of the historical role that this region has played during the domestication of grapes.     

Molecular linkage maps of Vitis vinifera L. and Vitis riparia Mchx

Two linkage maps for grape (Vitis spp.) have been developed based on 81 F(1) plants derived from an interspecific cross between the wine cultivar Moscato bianco (Vitis vinifera L.) and a Vitis riparia Mchx. accession, a donor of pathogen resistance traits. The double pseudotest-cross mapping strategy was applied using three types of molecular markers. The efficiency of SSRs to anchor homologous linkage groups from different Vitis maps and the usefulness of AFLPs in saturating molecular linkage maps were evaluated. Moreover, the SSCP technique was developed based on sequence information in public databases concerning genes involved in flavonoid and stilbene biosynthesis. For the maternal genetic map a total of 338 markers were assembled in 20 linkage groups covering 1,639 cM, whereas 429 loci defined the 19 linkage groups of the paternal map which covers 1,518 cM. The identification of 14 linkage groups common to both maps was possible based on 21 SSR and 19 AFLP loci. The position of SSR loci in the maps presented here was consistent with other published mapping experiments in Vitis.     

Application of AFLPs to the characterization of grapevine Vitis vinifera L. genetic resources. A case study with accessions from Rioja (Spain)

 AFLPs were used to characterize 67 different grapevine accessions from a collection of D.O.Ca. Rioja in Spain. A correct selection of primers and selective nucleotides allowed us to maximize the number of amplified fragments analyzed per reaction yielding an average of 100 per reaction, 49% of which were polymorphic. Based on the presence or absence of amplified fragments for each genotype resulting from a reaction with two primer combinations, we have established the genetic similarity between the different accessions in the collection. These results allowed us to resolve different genotypes maintained under the same name (homonyms) and to identify the same genotype under different names (synonyms) thus permitting the elimination of redundant germplasm. Furthermore, by providing information on more than 50 polymorphic loci per reaction, a few reactions were sufficient to identify distinct AFLP patterns characteristic of specific clones, with different agronomic and organoleptic features, belonging to the same cultivar. The possibility for clonal identification, shown here for grapevines, can have important implications in the protection and management of clonal selections. Received: 1 February 1998/Accepted: 25 February 1998     

Evidence for a geranyl-diphosphate synthase located within the plastids of Vitis vinifera L. cultivated in vitro

Intact plastids from cell suspensions of Vitis vinifera L. cv. Muscat de Frontignan, free of detectable contamination by other particles as judged by the distribution of organelle-specific marker enzymes and by electron microscopy, exhibit geranyl-diphosphate synthase activity (EC 2.5.1.1). This synthase activity remains stable after tryptic digestion of unlysed organelles and is enhanced by plastid disruption. We conclude that the enzyme is located within the organelle. The possibility of an isopentenyl diphosphate/dimethylallyl diphosphate translocating system which would play a major role in the regulation of monoterpene metabolism is discussed.     

Mechanism of Arginine Transport in Vitis vinifera L. Protoplasts

Protoplasts were isolated from leaves of in vitro grown axenicshoots of grapevine (Vitis vinifera L. cv. Soultanina) and usedto study the characteristics of arginine transport. Uptake waslinear up to at least 60 min and the rate did not differ significantlybetween light and dark assaying conditions whereas incubationin darkness for 24 h caused a 70% reduction in uptake rate,which was probably not due to an energy dependent factor. Kineticsanalysis revealed a biphasic uptake curve. The high affinitycomponent had a K_m, of 2.2 mol m^–3. Optimum pH value was5.5. Two carrier systems, one for basic and neutral and onefor acidic amino acids were identified. Use of inhibitors revealedthat those associated with ATP metabolism inhibited arginineuptake; more specifically, the proton motive force appearedto be the predominant energy source. Metabolic products of labelledarginine were consistent with the operation of the Krebs-Henseleitcycle. Key words: Grapevine protoplast, grapevine tissue culture, arginine transport     

Immunocharacterization of NADH-Glutamate Dehydrogenase from Vitis vinifera L

Rabbit antiserum was raised against NADH-glutamate dehydrogenase (GDH) isoenzyme 1, purified from leaves of Vitis vinifera L. cv Soultanina and its specificity was tested. This antiserum was used for immunocharacterization of the GDH from leaf, shoot, and root tissues. The antiserum recognized the seven isoenzymes of NADH-GDH and precipitated all the enzyme activity from the three tissues tested. Western blot following SDS-PAGE revealed the same protein band for the three tissues, with a molecular mass of 42.5 kilodaltons corresponding to NADH-GDH subunit. Results, based on the immunological studies, revealed that NADH-GDH from leaf, shoot, and root tissues are closely related proteins. Furthermore, addition of ammonium ions to the culture medium of in vitro grown explants resulted in a significant increase in NADH-GDH activity in root, shoot, and leaf tissues.     

Indications for post-translational regulation of Vitis vinifera L. arginine decarboxylase

In order to study arginine decarboxylase regulation, we produced an antiserum against a hybrid of a 615 amino acid residue fragment of grapevine arginine decarboxylase cDNA with maltose-binding protein. The antiserum generated recognized mainly a protein band of ca. 80 kDa in extracts from grapevine tissues. Extracts from leaves and internodes in different developmental stages showed differences in the quantity of the 80 kDa band recognized by the antiserum. However, these differences did not correspond with changes in arginine decarboxylase specific activity. Furthermore, western blot analysis of extracts from cell cultures, where enzyme-specific activity was induced or repressed, did not reveal respective changes in the quantity of the 80 kDa protein band. Digestion of the hybrid by the specific protease factor Xa resulted in a polypeptide of 90 kDa instead of the expected two polypeptides of 43 and 66 kDa. Finally, western blot analysis of shoot extract incubated with factor Xa or the hybrid protein previously digested by factor Xa revealed that factor Xa-digested hybrid protein cleaved the 80 kDa band, resulting in two bands of ca. 38 and 40 kDa, whereas factor Xa alone did not affect it. These results suggest that arginine decarboxylase protein levels and/or activity is post-translationally regulated, as has been shown for other enzymes of polyamine biosynthesis.     

Microsatellite inferred genetic diversity and structure of Western Balkan grapevines (Vitis vinifera L.)

A collection of 196 grapevine samples from five countries of the Western Balkan region, representing local and traditional cultivars, was genotyped with 22 SSR markers. Identity analysis revealed 125 unique genotypes, which were further used for diversity assessment. The average number of alleles per locus detected was 11?±?3.53, ranging from 6 to 21. The low cumulative probability of identical genotypes (2.96?×?10^?20) shown in this study implies an even distribution of alleles within the analyzed set of grapevines and a sufficient number of loci. On the basis of the discriminatory power of each SSR, a set of five markers (VVMD5, VVMD7, VVMD28, VChr3a, and VChr8b) was determined as sufficient for high-throughput discrimination of the target cultivars. The maximum discriminating power was evidenced for loci VVMD28 and Vchr8b (0.96, 0.94, respectively). A core collection covering the entire genetic diversity resulted in a set of 60 genotypes representing approximately 50 % of the samples from each country. Structure clustering of Balkan and West European cultivars resulted in four well distinct groups identified according to the classification of Negrul (1946). The lowest level of admixed genotypes was assigned for grapevines from Bosnia and Herzegovina (61 %) and the highest for Serbian (87 %) grapevines. In terms of grape use, the wine cultivars were divided into three groups and the fourth group was intermixed, with half wine and half table grapes. The highest Nei’s genetic distance (0.22) was discovered between Slovenian and Macedonian cultivars, while the lowest (0.09) was between Slovenian and Serbian cultivars. Macedonian cultivars were genetically most distant from the others (0.17). A similar pattern of differentiation among populations is seen with distance-based clustering. Analysis of molecular variance revealed only 1 % of genetic variation among groups of different origin, while the variation among individuals within geographical groups and within individuals explained 13 and 86 % of the total variation, respectively.     

Localization of stilbene synthase in Vitis vinifera L. during berry development

The localization of stilbene synthase (STS) (EC 2.3.1.95) in grape berry (Vitis vinifera L.) was investigated during fruit development. The berries were collected at 2, 4, 7, 11, and 15 weeks postflowering from the cultivar Nebbiolo during the 2005 and 2006 growing seasons. High-performance liquid chromatography analysis showed that berries accumulated cis- and trans-isomers of resveratrol mainly in the exocarp throughout fruit development. Immunodetection of STS protein was performed on berry extracts and sections with an antibody specifically developed against recombinant grape STS1. In agreement with resveratrol presence, STS was found in berry exocarp tissues during all stages of fruit development. The labeled epidermal cells were few and were randomly distributed, whereas nearly all the outer hypodermis cells were STS-positive. The STS signal decreased gradually from exocarp to mesocarp, where the protein was detected only occasionally. At the subcellular level, STS was found predominantly within vesicles (of varying size), along the plasma membrane and in the cell wall, suggesting protein secretion in the apoplast compartment. Despite the differences in fruit size and structure, the STS localization was the same before and after veraison, the relatively short developmental period during which the firm green berries begin to soften and change color. Nevertheless, the amount of protein detected in both exocarp and mesocarp decreased significantly in ripe berries, in agreement with the lower resveratrol content measured in the same tissues. The location of STS in exocarp cell wall is consistent with its role in synthesizing defense compounds and supports the hypothesis that a differential localization of phenylpropanoid biosynthetic machinery regulates the deposition of specific secondary products at different action sites within cells.     

Linkage disequilibrium in cultivated grapevine, Vitis vinifera L

We present here the first study of linkage disequilibrium (LD) in cultivated grapevine, Vitis vinifera L. subsp. vinifera (sativa), an outcrossing highly heterozygous perennial species. Our goal was to characterize the amount and pattern of LD at the scale of a few centiMorgans (cM) between 38 microsatellite loci located on five linkage groups, in order to assess its origin and potential applications. We used a core collection of 141 cultivars representing the diversity of the cultivated compartment. LD was evaluated with both independence tests and multilocus r ² , both on raw genotypic and reconstructed haplotypic data. Significant genotypic LD was found only within linkage groups, extending up to 16.8 cM. It appeared not to be influenced by the weak structure of the sample and seemed to be mainly of haplotypic origin. Significant haplotypic LD was found over 30 cM. Both genotypic and haplotypic r ² values declined to around 0.1 within 5–10 cM, suggesting a rather narrow genetic base of the cultivated compartment and limited recombination since domestication events. These first results open up a few application opportunities for association mapping of QTLs and marker assisted selection. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Restriction fragment length polymorphism and molecular taxonomy in Vitis vinifera L.

Forty-six accessions of grapevine (V. vinifera L.) were compared by restriction fragment length polmorphism (RFLP) analysis, and 111 informative or unique restriction fragments were found that revealed an important level of polymorphism. RFLP patterns were compared in two ways: by calculating electrophoretic similarity degree values further analyzed by principal component analysis and by studying the distribution of rare restriction fragments. Six taxonomic groups could be defined, which partially confirmed relationships derived from ampelographical data. Our data support the existence of ecogeographical groups.     

Sugars and flowering in the grapevine (Vitis vinifera L.)

Sugars play an important role in grapevine flowering. This complex process from inflorescence initiation to fruit maturity takes two growing seasons. Currently, most of the available data concern the involvement of sugars as energy sources during the formation of reproductive structures from initiation of inflorescences during the summer of the first year, until flower opening during the following spring. Sugars devoted to the development of reproductive structures are supplied either by wood reserves or by photosynthesis in leaves or inflorescences, depending on the stage of development. Female meiosis appears to be a key point in the success of flower formation because (i) flowers are vulnerable at this stage and (ii) it corresponds in the whole plant to the transition between reserve mobilization from perennial organs (roots, trunk, and canes) towards efficient leaf photosynthesis. The perturbation of reserve replenishment during the previous year provokes perturbation in the development of inflorescences, whereas altering the photosynthetic sources affects the formation of flowers during the same year. In particular, a lack of sugar availability in flowers at female meiosis caused by various environmental or physiological fluctuations may lead to drastic flower abortion. Apart from energy, sugars also play roles as regulators of gene expression and as signal molecules that may be involved in stress responses. In the future, these two topics should be further investigated in the grapevine considering the sensitivity of flowers to environmental stresses at meiosis.     

Characterization of anthocyanic vacuolar inclusions in Vitis vinifera L. cell suspension cultures

Anthocyanic vacuolar inclusions (AVIs) are intra-vacuolar structures capable of concentrating anthocyanins and are present in over 50 of the highest anthocyanin-accumulating plant species. Presence of AVIs alters pigment intensity, total anthocyanin levels, pigment hue and causes bathochromic shifts in a spatio-temporal manner within various flowers, vegetables and fruits. A year-long study on Vitis vinifera cell suspension cultures found a strong correlation between AVI prevalence and anthocyanin content, but not the number of pigmented cells, growth rate or stilbene content. Furthermore, enhancement of the prevalence of AVIs and anthocyanins was achieved by treatment of V. vinifera cell suspension cultures with sucrose, jasmonic acid and white light. A unique autofluorescence of anthocyanins was used to demonstrate microscopically that AVIs proceed from the cytosol across the tonoplast and were able to coalesce intravacuolarly, with fewer, larger AVIs predominating as cells mature. Purification and characterisation of these bodies were performed, showing that they were dense, highly organic structures, with a lipid component indicative of membrane-encasement. These purified AVIs were also shown to comprise long-chain tannins and possessed an increased affinity for binding acylated anthocyanins, though no unique protein component was detected.     

Characterization of Vitis vinifera L. somatic variants exhibiting abnormal flower development patterns

Mutants have proven to be a key resource for functional genomic studies in model annual plant species. In perennial plant species where mutants are difficult to generate and to screen, spontaneous somatic variants represent a unique resource to understand the genetic control of complex developmental patterns. The morphological and histological characterization of six Vitis vinifera L. somatic variants that display four different abnormal phenotypes of flower development are described here. A phenotype of reiterated reproductive meristems (RRM), with both flower and petal reiteration, was observed in a somatic variant of the cultivar Carignan. An abnormal development of reproductive organs was displayed by the unfused carpels (UFC) somatic variant of cv. Bouchalès, while a somatic variant of cv. Mourvèdre named carpel-less (CLS) developed abnormal ovules in the absence of carpels. Finally, three independent somatic variants in cvs Gamay, Morrastel, and Pinot displayed a phenotype of multiple perianth whorls (MPW). Gene expression studies showed that the expression profiles of VvMADS-box 1, 2, and 3 (putative orthologues of Arabidopsis flowering genes AG, SEP, and AGL13), were altered during grapevine flower development in the somatic variants, whereas the corresponding original cultivars displayed similar VvMADS-box gene expression profiles. Phenotypic and molecular characterization of these variants allowed the development of hypotheses on genetic functions that might be altered in most of the variants in light of the current ABCDE flower model.     

Agrobacterium rhizogenes mediated transformation of grapevine (Vitis vinifera L.)

Genetically transformed grapevine (Vitis vinifera L.) roots were obtained after inocultation of in vitro grown whole plants (cv. Grenache) with Agrobacterium rhizogenes. The strain used contains two plasmids: the wild-type Ri plasmid pRi 15834 and a Ti-derived plasmid which carries a chimaeric neomycin phosphotrans-ferase gene (NPT II) and the nopaline synthase gene. Expression of the NPT II gene can confer kanamycin resistance to transformed plant cells. Slowly growing axenic root cultures derived from single root tips were obtained. Opine analysis indicated the presence of agropine and/or nopaline in established root cultures. For one culture, the presence of T-DNA was confirmed by dot-blot hybridization with pRi 15834 TL-DNA. Callogenesis was induced by subculturing root fragments on medium supplemented with benzylaminopurine and indoleacetic acid.Transformation of in vitro cultured grapevine cells has recently been reported (baribault T.J. et al., Plant Cell Rep (1989) 8: 137–140). In contrast with the results presented here, expession of the NPT II gene Conferred kanamycin resistance to Vitis vinifera calli that was sufficient for selection of trasformed cells.Abbreviations BAP benzylaminopurine - IAA indoleacetic acid - NAA naphtaleneacetic acid - NPT II neomycin phosphostransferase II - EDTA ethylenediaminetetraacetic acid     

A circulatory system useful both for long-term somatic embryogenesis and genetic transformation in Vitis vinifera L. cv. Thompson Seedless

To establish an efficient regeneration protocol for functional validation and variety resistance improvement, a long-term system that useful for embryogenic culture maintenance and transformation was developed through recurrent cycles of secondary embryogenesis from Vitis vinifera L. cv. Thompson Seedless. Three media and five types of somatic embryo in secondary embryogenesis were evaluated. Somatic embryos (SE) in the torpedo and mid-cotyledonary stages gave the best embryogenic responses with re-induction rates of about 80 %. Embryogenic callus, proembryonic masses and SE produced in the system, could be propagated for over 3 years and all proved competent for Agrobacterium-mediated transformation. Based on this system, different transgenic selection regimes were compared. Addition of kanamycin at 4 weeks after co-cultivation was optimal for embryo recovery. Plant conversion was improved by alternating culture on two media: one containing 0.2 mg l^?1 BA and the other 0.25 mg l^?1 kinetin. To further test the efficiency of the system, a ubiquitin ligase gene (VpPUB23) from Chinese wild Vitis pseudoreticulata was transferred into Thompson Seedless for functional evaluation. Of the 351 transgenic plants obtained, those overexpressing VpPUB23 exhibited decreased resistance to powdery mildew compared with non-transgenic plants.     

Italian wild grapevine (Vitis vinifera L. subsp. sylvestris) population: insights into eco-geographical aspects and genetic structure

An extended survey showed that the Italian wild grapevine (Vitis vinifera L. subsp. sylvestris (Gmelin) Hegi) population is still numerous, in spite of a range of threats. In this study, one hundred and sixty-one sites distributed in 10 Italian regions were explored and 814 wild samples were collected. The individuals were unequally distributed on all the national territory and they were mainly concentrated in some areas of Central and Southern Italy. The highest percentage of wild individuals was sampled in sites below 300 m of altitude with a connection to a hydrographic network, marginal from dense vegetation and with a high-quality environment conservation state. A possible correlation between the presence of wild grapevine individuals and carbonatic soil substratum was found. On 668 individuals, a genetic analysis was carried out by the 20 microsatellite loci commonly utilized for V. vinifera genotyping, to infer information on the population genetic diversity and structure; 645 individuals resulted as unique genetic profiles. According to Simple Sequence Repeat (SSR) analysis, the Italian wild population still conserves a high genetic diversity and a low inbreeding level. Moreover, any clear genetic structure was not observed inside the population, although a likely splitting between the two subpopulations from Central and Southern Italy was identified. Two alternative hypotheses were proposed to explain this result: (i) the beginning of a progressive differentiation process inside the population and (ii) the product of recolonization events occurred after the last glaciation.     

Morphogenetic Effects of Roots and of Some Synthetic Cytokinins in Vitis vinifera L.

In woody cuttings of the grape vine, inflorescences of fertilebuds usually fail to develop and atrophy soon after bud burst.Results presented here indicate that this effect is relatedto absence of roots at planting. Inflorescences were retainedin pre-rooted cuttings which were propagated by holding thetemperature of the medium at 25° C and the air temperatureat 4° C. Similar effects on inflorescence growth were obtainedby applying synthetic cytokinins to the bases of unrooted cuttingsin solution cultures, and by applications to emergent inflorescences.Promotion of inflorescence growth was found with 6-benzyl-aminopurine(BAP) and 6-(benzylamino)-9-(2-tetrahydropyranyl)-9H purine(‘SD 8₃₃₉’). Stimulation of inflorescence growthby BAP was accompanied by reduction in vegetative growth, andby development of red pigments in inflorescences and leaves.Results of cincturing experiments indicate that BAP is transportedacropetally in the xylem of vines. Effects of roots, and effectsof synthetic cytokinins, are discussed in relation to recentdiscoveries of endogenous cytokinins in the ascending sap ofvines.