Grapevine Genotypes Research Articles

Grapevine downy mildew dual epidemics: a leaf and inflorescence transcriptomics study

Grapevine downy mildew (DM) is one of the most important plagues affecting viticulture, especially in humid climates. The pathogen responsible of the disease, Plasmopara viticola, is able to differentially attack leaves and grapes and is currently controlled with a massive use of fungicides. Economical costs, adverse impact on environment and negative effects on human health have led to use resistant grapevine varieties. Nevertheless, cases of divergent dual epidemics underline that, in the same genotype, organs can vary in their susceptibility to DM. Grapevine genotypes exhibit a wide spectrum of resistance to DM, but little is known about the transcriptional basis behind dual epidemics. Based on a previous phenotypic screening under field and controlled conditions, we selected 'Cabernet Cortis' which displays a different pattern of susceptibility to DM: mid-resistant leaves and mid-susceptible inflorescences. So far, we identified genes constitutively more highly or lowly expressed in 'Cabernet Cortis' compared to 'Cabernet Sauvignon' leaf and inflorescence.

Molecular variants of Grapevine rupestris stem pitting-associated virus infecting grapevines (Vitis spp.) in Brazil

ABSTRACT: Grapevine rupestris stem pitting-associated virus (GRSPaV) is one of the most common viruses of grapevine. It is involved in the graft-transmissible disease rupestris stem pitting of the rugose wood complex. The objective of the research was to perform the molecular characterization of the coat protein (CP) gene of sixteen Brazilian GRSPaV isolates aiming to determine the occurrence of molecular variants (strains) of this virus. Nine grapevine samples were evaluated, from which dsRNA was extracted. Nucleotide sequences were generated by Next generation sequencing (NGS). Fifteen complete sequences of the GRSPaV CP gene were obtained and phylogenetically analyzed. Multiple alignments of the sequences showed identities of nucleotides ranging from 82% to 99%, suggesting high variability among the CPs of Brazilian isolates. The study revealed that genetic variability of GRSPaV comprising three molecular variants is also present in Brazilian grapevine genotypes.

Secreted proteins produced by fungi associated with Botryosphaeria dieback trigger distinct defense responses in Vitis vinifera and Vitis rupestris cells.

Grapevine trunk diseases (Eutypa dieback, esca and Botryosphaeria dieback) are caused by a complex of xylem-inhabiting fungi, which severely reduce yields in vineyards. Botryosphaeria dieback is associated with Botryosphaeriaceae. In order to develop effective strategies against Botryosphaeria dieback, we investigated the molecular basis of grapevine interactions with a virulent species, Neofusicoccum parvum, and a weak pathogen, Diplodia seriata. We investigated defenses induced by purified secreted fungal proteins within suspension cells of Vitis (Vitis rupestris and Vitis vinifera cv. Gewurztraminer) with putative different susceptibility to Botryosphaeria dieback. Our results show that Vitis cells are able to detect secreted proteins produced by Botryosphaeriaceae, resulting in a rapid alkalinization of the extracellular medium and the production of reactive oxygen species. Concerning early defense responses, N. parvum proteins induced a more intense response compared to D. seriata. Early and late defense responses, i.e., extracellular medium alkalinization, cell death, and expression of PR defense genes were stronger in V. rupestris compared to V. vinifera, except for stilbene production. Secreted Botryosphaeriaceae proteins triggered a high accumulation of δ-viniferin in V. vinifera suspension cells. Artificial inoculation assays on detached canes with N. parvum and D. seriata showed that the development of necrosis is reduced in V. rupestris compared to V. vinifera cv. Gewurztraminer. This may be related to a more efficient induction of defense responses in V. rupestris, although not sufficient to completely inhibit fungal colonization. Overall, our work shows a specific signature of defense responses depending on the grapevine genotype and the fungal species.

Investigations on the role of cuticular wax in resistance to powdery mildew in grapevine

Cuticular wax on the plant epidermis inhibits or enhances prepenetration events of powdery mildew (Erysiphe necator Schwein). We examined the role of cuticular leaf and berry waxes as a resistance mechanism in four grapevine genotypes (Italia × Mercan-174, Gurcu, Isabella, Ozer Karasi) resistant to powdery mildew after natural infection and inoculation. To understand cuticular wax properties, we determined the amount of wax and antifungal effects of thin layer chromatography (TLC) fractions from cuticular leaf and berry waxes, then assessed the chemical composition of fractions with different antifungal activities using gas chromatography/mass spectrometry (GC/MS). Susceptible genotypes Cabernet Sauvignon and Italia were used for comparison. Resistant and sensitive genotypes did not differ significantly in the total amount of wax on leaves and berries; however, 27 fatty acids, 26 alkanes, 6 terpenes, 4 indole derivatives and 4 ketones, and 3 amides, 3 phenols and 3 steroids were detected in fractions with high antifungal activity (≥75% inhibition of germination) in leaf and/or berry cuticular waxes of resistant genotypes only. These compounds may be evaluated as markers for powdery mildew resistance during genotype selection in a grapevine breeding program.

Transcriptional Responses to Pre-flowering Leaf Defoliation in Grapevine Berry from Different Growing Sites, Years, and Genotypes.

Leaf removal is a grapevine canopy management technique widely used to modify the source–sink balance and/or microclimate around berry clusters to optimize fruit composition. In general, the removal of basal leaves before flowering reduces fruit set, hence achieving looser clusters, and improves grape composition since yield is generally curtailed more than proportionally to leaf area itself. Albeit responses to this practice seem quite consistent, overall vine performance is affected by genotype, environmental conditions, and severity of treatment. The physiological responses of grape varieties to defoliation practices have been widely investigated, and just recently a whole genome transcriptomic approach was exploited showing an extensive transcriptome rearrangement in berries defoliated before flowering. Nevertheless, the extent to which these transcriptomic reactions could be manifested by different genotypes and growing environments is entirely unexplored. To highlight general responses to defoliation vs. different locations, we analyzed the transcriptome of cv. Sangiovese berries sampled at four development stages from pre-flowering defoliated vines in two different geographical areas of Italy. We obtained and validated five markers of the early defoliation treatment in Sangiovese, an ATP-binding cassette transporter, an auxin response factor, a cinnamyl alcohol dehydrogenase, a flavonoid 3-O-glucosyltransferase and an indole-3-acetate beta-glucosyltransferase. Candidate molecular markers were also obtained in another three grapevine genotypes (Nero d’Avola, Ortrugo, and Ciliegiolo), subjected to the same level of selective pre-flowering defoliation (PFD) over two consecutive years in their different areas of cultivation. The flavonol synthase was identified as a marker in the pre-veraison phase, the jasmonate methyltransferase during the transition phase and the abscisic acid receptor PYL4 in the ripening phase. The characterization of transcriptome changes in Sangiovese berry after PFD highlights, on one hand, the stronger effect of environment than treatment on the whole berry transcriptome rearrangement during development and, on the other, expands existing knowledge of the main molecular and biochemical modifications occurring in defoliated vines. Moreover, the identification of candidate genes associated with PFD in different genotypes and environments provides new insights into the applicability and repeatability of this crop practice, as well as its possible agricultural and qualitative outcomes across genetic and environmental variability.

Evaluation of Greek grapevine cultivars for resistance to Phaeomoniella chlamydospora

In the present study, four Greek (Agiorgitiko, Asyrtiko, Roditis and Xinomavro) and one international (Soultanina) grapevine cultivars (Vitis vinifera L.) were screened for their resistance to Phaeomoniella chlamydospora. Artificial inoculation was carried out by drilling a hole into the trunk and injecting a concentrated conidial suspension into the vessels. Disease reactions were evaluated in an 87-day assessment period, on the basis of external symptoms (disease incidence, disease severity and mortality) and by calculating the relative areas under disease progress curves (relative AUDPC). The extension of vascular browning as well as the isolation ratio along the inoculated vine trunks were also taken into account as additional parameters for evaluating resistance. The results indicated that the resistance of grapevine cultivars to P. chlamydospora varied significantly. ‘Agiorgitiko’ and ‘Soultanina’ were susceptible, whereas ‘Asyrtiko’ and ‘Xinomavro’ were resistant; ‘Roditis’ showed an intermediate level of resistance. Cultivars’ resistance was mostly distinguished in terms of the extension of vascular browning and pathogen isolation ratio. On the contrary, the disease incidence, final disease severity, mortality and relative AUDPC provided less distinctive efficiency in resistance evaluation. The robust methodology presented here could be useful in rapid evaluation experiments for future screening programs to search and recognize natural resistant sources within grapevine genotypes against P. chlamydospora.

Characterization of Wild North American Grapevine Cold Hardiness Using Differential Thermal Analysis

The cold hardiness of 33 different grapevine genotypes, representing six wild North American grapevine species, one wild Asian grapevine species, and six hybrid grapevines, was evaluated by measuring lethal temperatures for dormant buds using low temperature exotherms. Studies were conducted over three different winters to characterize the relative level of cold hardiness and responsiveness to changing weather patterns of each species. Major differences in the winter conditions demonstrated that wild grapevine has a great capacity for responding to both warm and cold temperature events during the dormant season. Results indicated that wild grapevine species with northern distributions tended to exhibit greater cold hardiness and responsiveness to temperature fluctuations than their southern-dwelling counterparts. Statistical modeling of low temperature exotherm results across the three winters indicated that each grapevine species had a distinct and innate capacity for responding to temperature. These results may be important for future grapevine breeding in areas with expected increases in winter temperature variation. Additionally, these results demonstrate the potential for genetic determinants of temperature responsiveness that may be investigated and mapped for future grapevine improvement.

Genetic Diversity of Wild Grapevine [ Vitis vinifera L. subsp. sylvestris (Gmel.) Hegi] in the Eastern Adriatic Region

The genetic diversity of wild grapevine [Vitis vinifera L. subsp. sylvestris (Gmel.) Hegi] in the eastern Adriatic region, which includes Croatia and Bosnia and Herzegovina, has previously not been documented. Natural populations of wild grapevine were identified in their natural habitats, and their genetic diversity was studied and compared with both local and widespread European cultivars. Ninety-two grapevine genotypes were determined at 21 nuclear microsatellite loci, including 53 wild grapevine individuals, 32 diverse local Croatian and west European cultivars, and seven commonly used rootstocks. Two hundred thirty-nine alleles were detected, with a mean of 11.4 alleles per locus and high heterozygosity, ranging from 0.461 to 0.897 for observed heterozygosity and from 0.391 to 0.837 for expected heterozygosity. The genetic diversity of the wild grapevines was slightly lower than that observed in cultivars. Distance- and model-based cluster analysis differentiated three main groups, indicating clear separation between wild, cultivated, and rootstock accessions. This study greatly contributes to knowledge of genetic diversity in local wild grapevine populations in Croatia and Bosnia and Herzegovina, and provides necessary information for their conservation and further characterization.

Specific adjustments in grapevine leaf proteome discriminating resistant and susceptible grapevine genotypes to Plasmopara viticola

Grapevine downy mildew is an important disease affecting crop production leading to severe yield losses. This study aims to identify the grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0h) and in after inoculation (6, 12 and 24h). Leaf proteome analysis was performed using 2D difference gel electrophoresis followed by protein identification via mass spectrometry. In addition, we analysed ROS production, antioxidant capacity, lipid peroxidation and gene expression. Proteins related to photosynthesis and metabolism allowed the discrimination of resistant and susceptible grapevine cultivars prior to P. viticola inoculation. Following inoculation increase of hydrogen peroxide levels, cellular redox regulation, establishment of ROS signalling and plant cell death seem to be key points differentiating the resistant genotype. Lipid associated signalling events, particularly related to jasmonates appear also to play a major role in the establishment of resistance. The findings from this study contribute to a better understanding of genotype-specific differences that account for a successful establishment of a defence response to the downy mildew pathogen. Biological significanceHere, we present for the first time grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0h) and in after inoculation (6, 12 and 24h). We have highlighted that, following inoculation, the major factors differentiating the resistant from the susceptible grapevine cultivars are the establishment of effective ROS signalling together with lipid-associated signalling events, particularly related to jasmonates. It is believed that plants infected with biotrophic pathogens suppress JA-mediated responses, however recent evidences shown that jasmonic acid signalling pathway in grapevine resistance against Plasmopara viticola. Our results corroborate those evidences and highlight the importance of lipid- signalling for an effective resistance response against the downy mildew pathogen.

Life traits of four Botryosphaeriaceae species and molecular responses of different grapevine cultivars or hybrids

Botryosphaeriaceae is a fungal family comprising many species involved in botryosphaeria dieback, a worldwide grapevine trunk disease. Currently, the interactions between Botryosphaeriaceae species and various grapevine cultivars are poorly understood and little data is available. This study investigated various life traits of five isolates belonging to four species of Botryosphaeriaceae found in French vineyards (Diplodia mutila, Diplodia seriata, Lasiodiplodia viticola and Neofusicoccum parvum). The two species N. parvum and L. viticola exhibited the highest optimal growth temperature and the best growth rates. They were also responsible for the most extensive necrosis and cankers in three Vitis vinifera cultivars (Cabernet Sauvignon, Merlot and Ugni‐Blanc) that differed in susceptibility to botryosphaeria dieback, and in two genotypes resistant to downy and powdery mildew (RV4 and RV5). Identification of the extracellular toxins produced by isolates in culture media showed that the N. parvum isolate had a different metabolite profile from the others, producing terremutin and salicylic acid derivatives, which are known to be compounds associated with virulence. In a second step, life traits were associated with nondestructive monitoring of gene expression involved in the defence mechanisms of five grapevine cultivars and genotypes after inoculation of wood cuttings with Botryosphaeriaceae. The transcript analyses were carried out at different times and were associated with principal component analysis (PCA). Each cultivar presented a specific transcript signature and several transcripts were correlated either with the size of necrosis/cankers or with symptom reduction, thus offering useful markers for breeding or estimating the defence status of plants.

Disclosing the Molecular Basis of the Postharvest Life of Berry in Different Grapevine Genotypes.

The molecular events that characterize postripening grapevine berries have rarely been investigated and are poorly defined. In particular, a detailed definition of changes occurring during the postharvest dehydration, a process undertaken to make some particularly special wine styles, would be of great interest for both winemakers and plant biologists. We report an exhaustive survey of transcriptomic and metabolomic responses in berries representing six grapevine genotypes subjected to postharvest dehydration under identical controlled conditions. The modulation of phenylpropanoid metabolism clearly distinguished the behavior of genotypes, with stilbene accumulation as the major metabolic event, although the transient accumulation/depletion of anthocyanins and flavonols was the prevalent variation in genotypes that do not accumulate stilbenes. The modulation of genes related to phenylpropanoid/stilbene metabolism highlighted the distinct metabolomic plasticity of genotypes, allowing for the identification of candidate structural and regulatory genes. In addition to genotype-specific responses, a core set of genes was consistently modulated in all genotypes, representing the common features of berries undergoing dehydration and/or commencing senescence. This included genes controlling ethylene and auxin metabolism as well as genes involved in oxidative and osmotic stress, defense responses, anaerobic respiration, and cell wall and carbohydrate metabolism. Several transcription factors were identified that may control these shared processes in the postharvest berry. Changes representing both common and genotype-specific responses to postharvest conditions shed light on the cellular processes taking place in harvested berries stored under dehydrating conditions for several months.

Evaluation of Grapevines for Resistance to Downy Mildew (Plasmopara viticola) under Laboratory and Field Conditions

Downy mildew of grapevine (Vitis vinifera L.), caused by the oomycete Plasmopara viticola, can seriously devastate grapevine production in tropical countries, such as Thailand. Four susceptible grapevine cultivars, four potentially resistant lines and 18 F1 hybrids, propagated by air layering and chip budding, were evaluated for resistance to downy mildew at laboratory (using a detached leaf assay) and field (natural infection in 2011 and 2013) levels. Significant differences in the disease scores among grapevine genotypes, ranging from 0.54 (resistant) to 4.83 (susceptible) and 3.30 (resistant) to 7.70 (susceptible), were observed under the laboratory and field conditions respectively. No significant difference in disease severity was observed between the two propagation methods or between the two different years of field evaluations. Resistance evaluations under both conditions consistently classified ‘NY88.0517.01’ and ‘NY65.0550.04’ as resistant lines what would be useful as parents for future breeding programmes. Moreover, one F1 hybrid, ‘SUT0403.09’, was reported to have considerable resistance to downy mildew under both laboratory and field conditions for the first time. The field resistance level of this hybrid was almost comparable to its highly resistant parent ‘Wilcox 321’, suggesting its potential for the future development of resistant cultivars in Thailand. Although the ranking of genotypes varied between screening methods, the resistance levels of the 26 grapevine genotypes evaluated under laboratory and field conditions were comparable based on the Spearman’s rank correlation coefficients of 0.73 (p ≤ 0.01). These results suggest that the laboratory screening assay is efficient for the rapid, reliable and economical identification of resistant hybrids in grapevine breeding programmes.

Hyperspectral phenotyping of the reaction of grapevine genotypes to Plasmopara viticola.

A major aim in grapevine breeding is the provision of cultivars resistant to downy mildew. As Plasmopara viticola produces sporangia on the abaxial surface of susceptible cultivars, disease symptoms on both leaf sides may be detected and quantified by technical sensors. The response of cultivars 'Mueller-Thurgau', 'Regent', and 'Solaris', which differ in resistance to P. viticola, was characterized under controlled conditions by using hyperspectral sensors. Spectral reflectance was suitable to differentiate between non-infected cultivars and leaf sides of the bicolored grapevine. Brown discoloration of tissue became visible on both leaf sides of resistant cultivars 2 days before downy mildew symptoms appeared on the susceptible 'Mueller-Thurgau' cultivar. Infection of this cultivar resulted in significant (P<0.05) reflectance changes 1-2 days prior to abaxial sporulation induced by high relative humidity, or the formation of adaxial oil spots. Hyperspectral imaging was more sensitive in disease detection than non-imaging and provided spatial information on the leaf level. Spectral indices provided information on the variability of chlorophyll content, photosynthetic activity, and relative water content of leaf tissue in time and space. On 'Mueller-Thurgau' downy mildew translated reflectance to higher values as detectable by the index DMI_3=(R470+R682+R800)/(R800/R682) and affected reflectance at 1450nm. Tissue discoloration on 'Regent' and 'Solaris' cultivars was associated with lower reflectance between 750 and 900nm; blue and red reflectance demonstrated differences from leaf necroses. With high inoculum densities, P. viticola sporulated on even resistant cultivars. Hyperspectral characterization at the tissue level proved suitable for phenotyping plant resistance to pathogens and provided information on resistance mechanisms.

Transcriptome pathways in leaf and root of grapevine genotypes with contrasting drought tolerance

Most of the world’s wine-producing regions are subjected to seasonal drought,and,in the light of the dramatic climate-change events occurring in recent years, the selection of resistant rootstocks is becoming a crucial factor for the development of sustainable agricultural models to ensure optimal grape berry development and ripening. In this study, roots and leaves of 101.14 (drought-susceptible) and M4 (drought-tolerant) rootstocks were sampled in progressive drought and mRNA-seq profiles were evaluated. Physiological characterization indicated that only M4 was able to maintain high leaf transpiration and net assimilation rates under severe stress conditions. Statistical analyses, carried out on mRNA-seq data, highlighted that “treatment” (water stress) and “genotype” (rootstock-genotype) seem to be the main variables explaining differential gene expression in roots and leaves tissues, respectively. Upon water-stress, roots and leaves of the tolerant genotype M4 exhibit a higher induction of stilbenes (i.e., STS) and flavonoids (e.g., CHS, F3H, FLS) biosynthetic genes. Moreover, the higher expression of STS genes in M4 is coupled with an up-regulation of WRKYs transcription factors. STS genes promoter regions, extracted from whole genome of M4 and 101.14, highlighted a higher number of WBOX cis elements (binding site for WRKYs) in the tolerant genotype.

ABA-mediated responses to water deficit separate grapevine genotypes by their genetic background.

BackgroundABA-mediated processes are involved in plant responses to water deficit, especially the control of stomatal opening. However in grapevine it is not known if these processes participate in the phenotypic variation in drought adaptation existing between genotypes. To elucidate this question, the response to short-term water-deficit was analysed in roots and shoots of nine Vitis genotypes differing in their drought adaptation in the field. The transcript abundance of 12 genes involved in ABA biosynthesis, catabolism, and signalling were monitored, together with physiological and metabolic parameters related to ABA and its role in controlling plant transpiration.ResultsAlthough transpiration and ABA responses were well-conserved among the genotypes, multifactorial analyses separated Vitis vinifera varieties and V. berlandieri x V. rupestris hybrids (all considered drought tolerant) from the other genotypes studied. Generally, V. vinifera varieties, followed by V. berlandieri x V. rupestris hybrids, displayed more pronounced responses to water-deficit in comparison to the other genotypes. However, changes in transcript abundance in roots were more pronounced for Vitis hybrids than V. vinifera genotypes. Changes in the expression of the cornerstone ABA biosynthetic gene VviNCED1, and the ABA transcriptional regulator VviABF1, were associated with the response of V. vinifera genotypes, while changes in VviNCED2 abundance were associated with the response of other Vitis genotypes. In contrast, the ABA RCAR receptors were not identified as key components of the genotypic variability of water-deficit responses. Interestingly, the expression of VviSnRK2.6 (an AtOST1 ortholog) was constitutively lower in roots and leaves of V. vinifera genotypes and higher in roots of V. berlandieri x V. rupestris hybrids.ConclusionsThis study highlights that Vitis genotypes exhibiting different levels of drought adaptation differ in key steps involved in ABA metabolism and signalling; both under well-watered conditions and in response to water-deficit. In addition, it supports that adaptation may be related to various mechanisms related or not to ABA responses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0778-4) contains supplementary material, which is available to authorized users.

Comparative effects of water deficit and enhanced UV-B radiation on photosynthetic capacity and leaf anatomy traits of two grapevine (Vitis vinifera L.) cultivars

The effects of drought and enhanced UV-B radiation on photosynthetic and leaf anatomical characteristics were studied in two different grapevine genotypes (Vitis vinifera L. cvs ‘Romeiko’ and ‘Soultanina’), which were grown outdoors in 25 L pots containing a peat:perlite:sand (3:1:1, v/v/v) potting mix. The plants were subjected to three levels of UV-B radiation (i) ambient, (ii) intermediate (ambient plus 15 % UV-B radiation), (iii) high (ambient plus 30 % UV-B radiation) and two irrigation regimes (i) well-watered (irrigated on daily basis to substrate capacity) and (ii) drought-stressed (irrigated daily with the 50 % of the amount of water provided to well-watered plants). Both abiotic stressors induced a significant decrease in net photosynthesis (Pn) while the combination of drought and elevated UV-B radiation appeared to have synergistic effects on photosynthetic rate. At leaf anatomical level, drought resulted in the development of idioblastic cells in leaves of both grapevine varieties as well as in the reduction of leaf expansion rate, total lamina thickness and total biomass per leaf area whereas leaf tissue density considerably increased. On the other hand, enhanced UV-B radiation did not affect leaf anatomical characteristics of both genotypes in a similar way. In particular, different responses, closely depended to UV-B levels applied, were observed concerning the chlorenchymatic tissue and total lamina thickness. Moreover, the stimulation of UV-B absorbing compounds biosynthesis under enhanced UV-B radiation, could not manage to preserve photosynthetic performance. The implication of the watering regime and the UV-B in the induction of leaf anatomical and morphological alterations in net photosynthesis is discussed.

Flavonols in skins of wild grapes (Vitis viniferaL., subsp. sylvestris (Gmelin) Hegi)

Flavonols are a group of grape phenolics that play an important role in young red wines, as they are involved in copigmentation of the flavylium form of anthocyanins. A study on the flavonol composition of grape skins in several wild grapevine genotypes from different Iberian natural populations, preserved at El Encin Germoplasm Bank, has been carried out in 2012. Flavonol glycosides contained in grape skins were determined by HPLC-DAD and HPLC-MS, through a previous phase of purification, using ion exchange chromatographic columns to retain anthocyanins and other phenolic compounds, so that flavonols do not suffer co-elution with other components, improving HPLC analysis. Thus, it was possible to separate 12 flavonol glycosides, and eight of them were successfully identified. The major flavonols were quercetin-3-O -glucoside, quercetin-3-O -glucoronide and myricetin-3-O -glucoside. The diversity and number of flavonols differed for each genotype. The total content of flavonols ranged from 25 to 350 mg/kg grapes; the richest genotype was three times richer than Tempranillo grapes, used as a reference. The most significant difference between wild genotypes and reference cultivars was that, in many cases, myricetin-3-O -glucoside or quercetin-3-O -glucuronide predominated in wild genotypes.

Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits

Seedlessness, one of the most desired traits in fleshy fruits, can be obtained altering solely AGL11 gene, a D -class MADS-box. Opposite to overlapping functions described for ovule identity. AGAMOUS like-11 (AGL11) is a D-class MADS-box gene that determines ovule identity in model species. In grapevine, VviAGL11 has been proposed as the main candidate gene responsible for seedlessness because ovules develop into seeds after fertilization. Here, we demonstrate that AGL11 has a direct role in the determination of the seedless phenotype. In grapevine, broad expression analysis revealed very low expression levels of the seedless allele compared to the seeded allele at the pea-size berry stage. Heterozygous genotypes have lower transcript accumulation than expected considering the diploid nature of grapevine, thereby revealing that the dominant phenotype previously described for seedlessness is based on its expression level. In a seeded somatic variant of Sultanina (Thompson Seedless) that has well-developed seeds, Sultanine Monococco, structural differences were identified in the regulatory region of VviAGL11. These differences affect transcript accumulation levels and explain the phenotypic differences between the two varieties. Functional experiments in tomato demonstrated that SlyAGL11 gene silencing produces seedless fruits and that the degree of seed development is proportional to transcript accumulation levels. Furthermore, the genes involved in seed coat development, SlyVPE1 and SlyVPE2 in tomato and VviVPE in grapevine, that are putatively controlled by SlyAGL11 and VviAGL11, respectively, are expressed at lower levels in silenced tomato lines and in seedless grapevine genotypes. In conclusion, this work provides evidence that the D-class MADS-box AGL11 plays a major and direct role in seed development in fleshy fruits, providing a valuable tool for further analysis of fruit development.

Short day transcriptomic programming during induction of dormancy in grapevine.

Bud dormancy in grapevine is an adaptive strategy for the survival of drought, high and low temperatures and freeze dehydration stress that limit the range of cultivar adaptation. Therefore, development of a comprehensive understanding of the biological mechanisms involved in bud dormancy is needed to promote advances in selection and breeding, and to develop improved cultural practices for existing grape cultivars. The seasonally indeterminate grapevine, which continuously develops compound axillary buds during the growing season, provides an excellent system for dissecting dormancy, because the grapevine does not transition through terminal bud development prior to dormancy. This study used gene expression patterns and targeted metabolite analysis of two grapevine genotypes that are short photoperiod responsive (Vitis riparia) and non-responsive (V. hybrid, Seyval) for dormancy development to determine differences between bud maturation and dormancy commitment. Grapevine gene expression and metabolites were monitored at seven time points under long (LD, 15 h) and short (SD, 13 h) day treatments. The use of age-matched buds and a small (2 h) photoperiod difference minimized developmental differences and allowed us to separate general photoperiod from dormancy specific gene responses. Gene expression profiles indicated three distinct phases (perception, induction and dormancy) in SD-induced dormancy development in V. riparia. Different genes from the NAC DOMAIN CONTAINING PROTEIN 19 and WRKY families of transcription factors were differentially expressed in each phase of dormancy. Metabolite and transcriptome analyses indicated ABA, trehalose, raffinose and resveratrol compounds have a potential role in dormancy commitment. Finally, a comparison between V. riparia compound axillary bud dormancy and dormancy responses in other species emphasized the relationship between dormancy and the expression of RESVERATROL SYNTHASE and genes associated with C3HC4-TYPE RING FINGER and NAC DOMAIN CONTAINING PROTEIN 19 transcription factors.

Cultivar Identity, Intravarietal Variation, and Health Status of Native Grapevine Varieties in Croatia and Montenegro

Croatia and Montenegro (located in southeast Europe) host numerous native (autochthonous) minor grapevine (Vitis vinifera L.) varieties that might irreversibly disappear in the near future because of limited conservation efforts and lack of virus-free propagation material. To prevent their disappearance and help sustain these native populations, a collaborative project was launched to gather and evaluate extremely rare and neglected native grapevine genotypes by establishing true-to-type and virus-tested stock material, ready to use for nurseries. During several field expeditions, 284 grapevine accessions collected in 22 different locations in Croatia and Montenegro were subjected to ampelographic in situ description, genetic identification by nine simple sequence repeat (SSR) markers, serological testing for the presence of common grape viruses, and intravarietal variability assessment by sequence-specific amplification polymorphism (S-SAP) markers. We observed 53 unique SSR profiles, of which 25 genotypes have not been previously reported and are thus worthy of further research. Comparison of these genetic profiles with those from some very rare and poorly described accessions within the European Vitis Database and other published studies revealed several previously unknown synonyms and homonyms, which helped properly name some of the profiles and provided novel information on the history of these cultivars and their distribution in the region. Virus testing revealed high frequencies of infection with Grapevine leafroll-associated viruses 1 and 3 and indicated the urgent need for sanitary selection. Assessment of intravarietal genetic variation indicated the presence of accumulated mutations, which strongly suggests a need for clonal selection.