Plavac Mali Research Articles

Single nucleotide polymorphism (SNP) analysis reveals ancestry and genetic diversity of cultivated and wild grapevines in Croatia

BackgroundCroatia is a geographically small country with a remarkable diversity of cultivated and spontaneous grapevines. Local germplasm has been characterised by microsatellite markers, but a detailed analysis based on single nucleotide polymorphisms (SNPs) is still lacking. Here we characterize the genetic diversity of 149 accessions from three germplasm repositories and four natural sites using 516,101 SNPs to identify complete parent-offspring trios and their relations with spontaneous populations, offering a proof-of-concept for the use of reduced-representation genome sequencing in population genetics and genome-wide association studies (GWAS).ResultsPrincipal component analysis revealed a clear discontinuity between cultivated (V. vinifera subsp. sativa) and spontaneous grapevines, supporting the notion that the latter represent local populations of the wild progenitor (V. vinifera subsp. sylvestris). ADMIXTURE identified three ancestry components. Two sativa components are alternatively predominant in cultivars grown either in northern Adriatic Croatia and Continental Croatia or in Dalmatia (i.e. central and southern Adriatic Croatia). A sylvestris component, which is predominant in accessions from spontaneous populations, is a minor ancestry component in cultivated accessions. TREEMIX provided evidence of unidirectional migration from the vineyards to natural sites, suggesting that gene flow has gone preferentially from the introduced domesticated germplasm into local wild populations rather than vice versa. Identity-by-descent analysis indicated an extensive kinship network, including 14 complete parent-offspring trios, involving only cultivated accessions, six full-sibling relationships and invalidated a presumed pedigree of one of the most important varieties in Croatia, ‘Plavac Mali’. Despite this strong population structure, significant association was found between 143 SNPs and berry skin colour and between 2 SNPs and leaf hairiness, across two previously known genomic regions.ConclusionsThe clear genetic separation between Croatian cultivars and sylvestris ruled out the hypothesis that those cultivars originated from local domestication events. On the other hand, the evidence of a crop-to-wild gene flow signals the need for an urgent adoption of conservation strategies that preserve the residual genetic integrity of wild relatives. The use of this reduced-representation genome sequencing protocol in grapevine enables an accurate pedigree reconstruction and can be recommended for GWAS experiments.

Positive Impact of Late Harvest Date on Polyphenolic Composition of Plavac Mali (Vitis vinifera L.) Wine Depends on Location.

Asynchronous ripening is a significant challenge in winemaking. Green berries reduce alcohol and pH while increasing acidity. Green berries are rich in bitter and astringent compounds, with an unknown impact on wine quality. The aim of this study was to evaluate the impact of harvest date and vineyard location on the polyphenolic composition of Plavac Mali wines in Dalmatia, Croatia. Experiments were conducted in two locations, Split and Zadar, producing fifteen wines per location from four harvest dates (H1-H4), including green berry wines from H1. The first harvest date occurred 27 days after véraison (DAV) and the last at 69 DAV, corresponding to overripeness. Green berry wines of H1 had low alcohol content up to 4.4% (v/v) in Split. Epigallocatechin was the main flavonoid in those wines, followed by dimer B1 in Split and catechin in Zadar. Green wines from Split had a higher concentration of phenolic acids, flavan-3-ol monomers and dimers. Wines of H3 had the highest concentration of malvidin-3-O-glucoside. With a later harvest date, a dramatic decrease in catechin and dimers was observed in wines from Split, and a decrease in epicatechin, epigallocatechin and dimer B1 in those from Zadar. The final expression of the physiochemical and polyphenolic composition of Plavac Mali wine is determined by the dynamics of harvest date, location and their interactions.

X-ray Microanalysis of Elemental Composition of Vitis sylvestris Pollen Grains.

The flowering and fruit set of grapevines are determined by many morphological, physiological, and environmental factors. Although the elemental composition of pollen grains plays a crucial role in the fruit set, there is still a considerable gap in our knowledge. To date, no study has been conducted on the elemental composition of Vitis vinifera subsp. sylvestris (hereafter V. sylvestris) pollen grains. The aim of this work was to investigate the elemental composition of pollen grains of V. sylvestris using scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX). The pollen grains of ten V. sylvestris individuals (eight male and two female) and one hermaphrodite cultivar 'Plavac mali crni' were analyzed. SEM-EDX analysis revealed the presence of eight elements (carbon-C, oxygen-O, magnesium-Mg, phosphorus-P, potassium-K, calcium-Ca, molybdenum-Mo, and aluminum-Al) in the pollen grains. Interestingly, aluminum was detected exclusively in the pollen of the cultivated grape cultivar 'Plavac mali crni', while it was not present in the genotypes of V. sylvestris. No significant differences between genotypes were found for oxygen and phosphorus, while significant differences were found for other elements. Pollen dimorphism was not associated with differences in element composition, although principal component analysis separated the genotypes into two distinct groups, with two female individuals (Pak10 and Pak12) and one male (Im19) tending to form separate clusters. This study is the first report on the elemental composition of pollen grains of V. sylvestris genotypes and provides valuable insights for further studies on pollen functionality.

Biochemical Response and Gene Expression to Water Deficit of Croatian Grapevine Cultivars (Vitis vinifera L.) and a Specimen of Vitis sylvestris.

The biochemical response and gene expression in different grapevine cultivars to water deficit are still not well understood. In this study, we investigated the performance of four traditional Croatian Vitis vinifera L. cultivars ('Plavac mali crni', 'Istrian Malvasia', 'Graševina', and 'Tribidrag'), and one wild (Vitis vinifera subsp. sylvestris) genotype exposed to water deficit (WD) for nine days under semi-controlled conditions in the greenhouse. Sampling for biochemical and gene expression analyses was performed at days six and nine from the beginning of WD treatment. The WD affected the accumulation of metabolites with a significant increase in abscisic acid (ABA), salicylic acid (SA), and proline in the leaves of the stressed genotypes when the WD continued for nine days. Lipid peroxidation (MDA) was not significantly different from that of the control plants after six days of WD, whereas it was significantly lower (297.40 nmol/g dw) in the stressed plants after nine days. The cultivar 'Istrian Malvasia' responded rapidly to the WD and showed the highest and earliest increase in ABA levels (1.16 ng mg-1 dw, i.e., 3.4-fold increase compared to control). 'Graševina' differed significantly from the other genotypes in SA content at both time points analyzed (six and nine days, 47.26 and 49.63 ng mg-1 dw, respectively). Proline level increased significantly under WD (up to 5-fold at day nine), and proline variation was not genotype driven. The expression of aquaporin genes (TIP2;1 and PIP2;1) was down-regulated in all genotypes, coinciding with the accumulation of ABA. The gene NCED1 (9-cis-epoxycarotenoid dioxygenase) related to ABA was up-regulated in all genotypes under stress conditions and served as a reliable marker of drought stress. This work suggests that the stress response in metabolite synthesis and accumulation is complex, treatment- and genotype-dependent.

Application of statistical models in the detection of grapevine phenology changes

In recent decades, there have been significant changes in temperature and precipitation, as well as in the occurrence of phenological stages of the grapevine in most wine-growing regions around the world. These changes are not the same for each variety, nor in all locations. Due to the vulnerability of the viticulture sector, including the possible losses in production in the current winegrowing areas, as well as the planting of vineyards in new areas, it is of great importance to examine the trends in the occurrence of individual stages and to link them as successfully as possible with changes in meteorological parameters. The simplest approach to this is using agrometeorological indices (e.g., Growing degree day, GDD) which can determine the possibility of growing a certain variety. There is also the possibility of developing and testing simple statistical phenological models that serve to predict the occurrence of phenological stages. Four such models were tested for the prediction of four phenological stages (budburst, flowering, veraison, and harvest) for four grape varieties ('Graševina', 'Chardonnay', 'Merlot', and 'Plavac mali') in Croatia. The first two models are commonly used GDD models with a temperature base of 10 °C or 5 °C, and thresholds necessary for phenological stage to start depending on variety or variety and location. The other two models are based on the determination of the best multi-linear regression using as predictors monthly and multi-month averages of minimum temperature, maximum temperature, mean temperature, and total precipitation. The increase in temperature index values from the 1990s to today is particularly significant. Statistical phenological models also proved to be a good indicator of the occurrence of individual phenological stages. GDD models proved to be somewhat better in prediction, GDD models that use a temperature of 5 °C as a base proved to be better for predicting budburst, those that use a base of 10 °C proved to be better for the other stages and particularly for flowering (with agreement index d up to 0.8 and root mean square error of prediction RMSE from 5 to 10 days). Linear regression that uses temperature as a predictor and the same equation regardless of location proved to be very good in predicting the harvest of autochthonous varieties ('Graševina' and 'Plavac mali') with low RMSE (up to 10 days). The presented results indicate that these models could be applied to future scenarios and with that help to make decisions in the wine sector in Croatia and worldwide.

Effect of Sequential Fermentation with Lachancea thermotolerans/S. cerevisiae on Aromatic and Flavonoid Profiles of Plavac Mali Wine.

In this study, the effects of sequential fermentation of Lachancea thermotolerans/S. cerevisiae on the production of Plavac Mali wines were investigated in comparison with the commonly used inoculation of the commercial Saccharomyces cerevisiae strain and spontaneous fermentation. A total of 113 aroma compounds and 35 polyphenolic compounds were analyzed. Sequential inoculation resulted in a decrease in alcohol content and pH (up to 0.3% v/v and 0.12 units, respectively) and an increase in total acidity (0.6 g/L, expressed as tartaric acid). The wines produced by spontaneous fermentation exhibited the greatest diversity of volatile compounds and the highest concentration of C13 norisoprenoids, lactones, and other compounds. These wines exhibited maximum hydroxycinnamic acids, prodelphinidin monomer units, epigallocatechin, B1, B3, and B4 dimers, and total flavan-3-ols. Sequential inoculation decreased the content of the aromas and polyphenols in the wines. The practical significance of this procedure lies in the selective effect on aroma compounds, the decrease in green aromas, undetectable volatile phenols, and the decrease in bitter and astringent compounds such as gallic acid, flavan-3-ol monomers (catechin and epicatechin), and dimers (B1, B2, B3, and B4). This work demonstrates the potential of sequential and spontaneous fermentation to improve the aromatic characteristics and overall quality of Plavac Mali wines.

The Effect of Seed Removal and Extraction Time on the Phenolic Profile of Plavac Mali Wine

The maceration process is one of the indispensable winemaking steps in red wine production that has a marked impact on phenolic compound extraction and can strongly influence the color and gustatory quality. Seed removal can have a substantial impact on the intensity of bitterness and astringency which are mainly influenced by the presence of flavanols. The main aim of this study was to investigate the influence of seed removal and extraction time on the phenolic composition, color parameters, and organoleptic properties of Plavac mali wines produced from the grapes from the Pelješac Peninsula. The achieved results showed that the control wines differ in terms of their highest content of total anthocyanins in both years and total flavonols in one year of investigation, while prolonged maceration without seeds decreases the concentration of phenolic acids as well as that of flavan-3-ols. A prolonged extraction time influenced the color intensity and tonality, moving it towards more yellow pigments. These changes could possibly be explained by the pronounced interaction between phenolic compounds and the formation of the new ones. Interestingly, seed removal probably diminished that change because the potential absorption of the anthocyanins by grape seeds and tannins were excluded. The highest quality was sensory defined in wines produced by prolonged maceration without seeds.

Characterization of Berry Skin Phenolic Profiles in Dalmatian Grapevine Varieties

Dalmatian vineyards host many autochthonous varieties. The phenolic profile, defined by the relative proportions of different phenolic compounds, is specific for each grape variety. The aim of this study was to determine and analyze the flavonoid compounds of twenty rare red grape varieties. Nineteen phenolic compounds, represented by anthocyanins, flavanols, and flavonols, were detected and quantified using HPLC in three consecutive vintages. The content of grape skin anthocyanins (10414.06 (Plavac mali crni)-19.58 (Trišnjavac) mg kg−1 d.w. of grape skin), flavonols (1742.08 (Pošip crni)-215.56 (Crljenak viški) mg kg−1 d.w. of grape skin), and flavan-3-ols (448.04 (Pošip crni)-87.88 (Glavinuša) mg kg−1 d.w. of grape skin) showed significant differences in investigated varieties. According to the investigated phenolic compounds, Pošip crni, Ljutun, Zadarka, Dobričić, Plavac mali crni, and Trnjak differed from other investigated varieties. These local varieties can be perceived as an alternative to widespread varieties in Dalmatia. This was shown by one of the first studies on phenolic profiles of (mostly) rare autochthonous varieties.

Prevalence of Virus Infections and GLRaV-3 Genetic Diversity in Selected Clones of Croatian Indigenous Grapevine Cultivar Plavac Mali.

The cultivar Plavac Mali (Vitis vinifera L.), the most important indigenous red grapevine cultivar in Croatia, was tested for the presence of 16 grapevine viruses. Thirty-five samples from the collection vineyard were tested for the presence of grapevine leafroll-associated viruses-1, -2, and -3 (GLRaV-1, GLRaV-2 and GLRaV-3, respectively), grapevine fanleaf virus (GFLV), arabis mosaic virus (ArMV), grapevine virus-A (GVA), -B (GVB), -G (GVG), -H (GVH), -I (GVI), -J (GVJ), grapevine fleck virus (GFkV), grapevine rupestris stem pitting associated virus (GRSPaV), and grapevine pinot gris virus (GPGV) by reverse transcription–polymerase chain reaction (RT-PCR). Furthermore, standard PCR was conducted for grapevine badnavirus 1 (GBV-1) and grapevine red blotch virus (GRBV). Mixed infections were most common and GLRaV-3, the most abundant virus found in 85.71% of the vines tested, was further molecularly characterised. Different genomic variants of the heat shock protein homologue (HSP70h) were separated by cloning, detected by single-strand conformation polymorphism (SSCP) analysis, sequenced, and phylogenetically analysed. The presence of phylogenetic groups I and II was only confirmed. This study demonstrates the high virus infection rate of Plavac Mali vines and the heterogeneity of GLRaV-3 present nowadays in a collection vineyard.

Impact of Commercial Yeasts on Phenolic Profile of Plavac Mali Wines from Croatia

Wine quality is influenced by the presence of over 500 different chemical compounds, with polyphenols having a crucial role in color intensity and tonality, astringency, mouthfeel, and overall impression formation, especially in red wine production. Their concentrations in wine can vary notably depending on the grape variety, the temperature and the length of maceration process, aging duration, and yeast selection. Therefore, in this work, the main goal was to determine the influence of five commercially available Saccharomyces yeasts provided from Lallemand, France and AEB, Italy, on the phenolic compound composition and chromatic parameters of Plavac mali wines produced from the grapes from coastal Dalmatia, grown at two different micro-locations. The achieved results pointed out the marked difference in individual polyphenol compound adsorption between tested yeasts. Fermol Super 16 was the one with the lowest and Lalvin D21 the strongest adsorption ability, regardless of vine growing location. These differences can be explained by the content of some anthocyanins (delphinidin and petunidin-3-O-glucoside) and gallic acid, and some flavan-3-ols. Tested strains also influenced wine color intensity, pointing out the possibility of modulating the style of a Plavac mali by the use of commercial yeasts.

Extended Harvest Date Alter Flavonoid Composition and Chromatic Characteristics of Plavac Mali (Vitis vinifera L.) Grape Berries.

This study delivers a comprehensive flavonoid fingerprint profile, physiochemical and external color characterization of Plavac Mali grapes through four harvest dates at two distinct vineyards (Split and Zadar) in the Eastern Adriatic region. The experimental harvest lasted 56 days, at total soluble solids content from 18.4 to 22.4°Brix in Split and 16.8 to 20.4°Brix in Zadar. Patterns of 27 skin and seed flavonoid compounds at each location indicate unique flavonoid composition of berries at each harvest date. Extended harvest increased six compounds in skin with maximum values of main anthocyanin malvidin-3-O-glucoside in H3 (4406.6 and 6389.5 mg kg−1, Split and Zadar, respectively) followed by a decrease in October. Peak values of seed and skin catechins and galloylated flavan-3-ol subunits are seen in H1 and H2 at Split, while constantly high values are reported in the case of Zadar, with an incoherent pattern of those in skin extracts. Minimal values of epigallocatechin were detected with an extended harvest date at both locations. Berries of extended harvest dates underwent colorimetric improvements, trough decrease in L*, a*, b* and C characteristics and increase in skin color index for red grapes CIRG. The extended harvest date promotes flavonoid composition, and improves the quality of Plavac Mali grape berries.

Somatic embryogenesis as a tool for virus elimination in Croatian indigenous grapevine cultivars

Most Croatian indigenous grapevine cultivars and vineyards are infected with a few dominant viruses. The goal of this study was to establish somatic embryogenesis as an efficient method for virus elimination from valuable Croatian cultivars and creating a reliable source of healthy plants. Somatic embryogenesis was induced from immature anthers and somatic embryogenesis-derived plantlets for seven indigenous cultivars were successfully regenerated. This procedure led to the elimination of viruses GFLV, GLRaV-1, GLRaV-3 and GFkV that were initially detected in the field-grown cultivars ‘Plavac mali’ and ‘Babica’ with an elimination success of at least 30%. The described method has the potential for production of virus-free rooted plantlets for all economically important cultivars or superior cultivar clones and for the establishment of a steady source of certified virus-free planting material.

Climate change impacts on viticulture in Croatia: Viticultural zoning and future potential

AbstractSince changes in temperature and precipitation have different effects on (a) all developmental stages of grapevines in most of the wine regions worldwide (i.e., on their phenological characteristics) and (b) different varieties, a comprehensive database of bioclimatic indices has been calculated and analysed for Croatian wine producing regions. The database consists of the average growing season temperature, growing degree‐days, Huglin index, dryness index and cool night index that are based on all available meteorological measurements as well as the outputs of regional climate models (RCMs) from the EURO‐CORDEX database. The horizontal grid spacing of 0.11° from the RCM ensembles enabled a fine‐scale determination of bioclimatic indices for the present and future climate in Croatia. In addition, statistical analyses (standard statistical parameters and Bayesian method) were carried out to examine trends in sugar content, total acidity and date of harvest. Calculations were performed for the present and future climate on the basis of data from seven selected vineyards/wineries and four varieties (‘Graševina’, ‘Plavac mali’, ‘Chardonnay’ and ‘Merlot’). The results show whether the part of Croatia that is suitable for grape cultivation in the present climate will continue to be favourable in the future within the Mediterranean area. In general, projections suggest further warming and drying of the climate in Croatia and an earlier harvest, with some variations among varieties that show latitude dependence. Projections for the future climate also suggest that the existing viticultural zoning will be much less adequate for the Croatian territory because it reduces the economically sustainable production of wine in certain areas.

Total phenols, stilbene and antioxidative activity in Babić and Plavac mali wines; Eficiency of pectolytic enzymes

Special importance of wine polyphenols have been associated with the bioactive compounds because of their benefits on human health. Research of acceptable and safe technological processes that would enable the production of wines with a higher content of such compounds is a need and a challenge. For this reason, the aim of this study was to investigate the influence of commercial pectolytic enzymes to total phenols (UF), stilbene and antioxidative activity (AA) of Babić and Plavac mali red wines, originated from middle quality localities. Two enzymatic product with different properties were used: pectinase with celulase and hemicelulase additional activity (E1) and pectinase with inactive yeast cells (E2), in comparison to maceration without egzogenous enzymes (control, K). The quality parameters were analysed in different production stages; from the end of maceration and fermentation to wine after the second decanting and bottling. Both products affected to those quality parameters. Maceration with pectolytic enzymes (E1) provided the best extraction and production of wines with the significant highest concentrations of total phenols of both varieties, in both years. The highest values of AA were obtained in wines of treatmant E1, in both years of research and for both varieties. Significant differences were obtained for both varieties, but not in both years. Maceration with egzogenous enzymes effected enrichment of wines with stilbenes, resveratrol and piceid particularly, however the significant differences did not obtained in both years of research. The differences between varieties were noted: while the resveratrol dominated in Plavac mali wines, higher proportion were obtained for piceid in Babić wines. Despite relatively better results in comparison to control, the use of product E2 was not found to be beneficial; significant differences were not confirmed for any variety during both years of the experiment. Significant positive correlation were found between the total phenols and antioksidative activity. The changes in parameters after 6 months aging were obtained in wines of both varieties, but some positive effect were reduced. The obtained differences in parameter values were not as significant as at the end of fermentation. The use of exogenous enzyme can be desirable in case of Babić and Plavac mali young wine production.

Prolonged ripening on the vine affects the polyphenolic profile of grapes and wine of ‘Plavac Mali’ (Vitis viniferaL.)

Compositional differences in the polyphenolic profile and physicochemical parameters of grapes and corresponding wines of 'Plavac Mali' at five different harvest dates were evaluated by standard chemical analysis and high-performance liquid chromatography. Prolonged ripening enhanced the anthocyanin and flavonol concentration in skin tissue, with the exception of 3-O-glucosides of delphinidin, petunidin and quercetin, and 3-O-rutinoside of quercetin. Various trends in skin low-molecular-weight phenolic compounds were detected: increases in (+)-gallocatechin and (-)-epicatechin, and decreases in the others including (+)-catechin, (‑)‑epigallocatechin, and proanthocyanidins (B1, B2, B3, B4). Grape seeds reached stable concentration of low-molecular-weight phenolic compounds at the third harvest date, while prolonged ripening had a significant influence on (-)-epicatechin and (-)-epigallocatechin. In contrast, in the corresponding wines, the concentrations of anthocyanins had two peaks NDASH one at the third harvest date and one at the fifth harvest date NDASH with no significant difference between the two, except in peonidin-3-O-glucoside. Prolonged ripening caused a production of wines with high concentration of (+)-gallocatechin and (-)-epicatechin, and the lowest concentration of (‑)‑epigallocatechin. The results indicate the potential of 'Plavac Mali' for prolonged grape ripening, but negative aspects of the prolonged ripening practice should also be considered.

Croatian Wines from Native Grape Varieties Have Higher Distinct Phenolic (Nutraceutic) Profiles than Wines from Non-Native Varieties with the Same Geographic Origin.

Croatian wines made from native (Plavac mali and Teran) and non-native grape varieties (Cabernet Sauvignon and Merlot), all grown in Croatian coastal regions, were investigated. Analyses included measurements of antioxidant activities, total phenolic contents and concentrations of non-colored phenolic compounds, chosen based on their known nutraceutical properties. Plavac mali wines were distinguished by higher antioxidant activity, total phenolic content and catechin concentrations but lower flavonol concentrations. Teran wines had higher hydroxytyrosol, myricetin and resveratrol concentrations. Merlot and Cabernet Sauvignon wines had higher flavonol concentrations (except myricetin). Canonical analysis was successful in discriminating Plavac mali from Teran wines, and both varieties were separated from non-native varieties. The results indicate distinct genetic potentials of studied varieties and enable wine authentication based on the investigated bioactive compounds.

Targeted UPLC-QqQ-MS/MS profiling of phenolic compounds for differentiation of monovarietal wines and corroboration of particular varietal typicity concepts

Targeted ultra-performance liquid chromatography with triple quadrupole mass spectrometric (UPLC-QqQ-MS/MS) profiling of phenolic compounds was utilised for varietal differentiation of 173 wines made from four red and six white grape varieties. Among 58 identified phenols many were found relevant as exclusive or partial discriminators between wines. Successful differentiation models were built by linear discriminant analysis with the percentage of correct classification higher than 95% in all cases, with peonidin 3-(6″-acetyl)-glucoside and taxifolin as the most potent differentiators between red, and cis-piceid between white monovarietal wines. Diverse typical colour attributes among the monovarietal wines were tentatively ascribed to the variations in the composition of monomeric anthocyanins. Plavac mali red wine exhibited the most specific composition, and its most typical samples were distinguished by the abundance in trans-fertaric acid, isorhapontin, phlorizin, quercetin 3-rhamnoside, and myricitrin. Despite positive correlations with particular astringent flavonols, the typical astringency of Plavac mali wine remained unresolved.

Reproductive potential of the functionally female native Croatian grapevine 'Grk bijeli'

A native Croatian grapevine 'Grk bijeli', sharing a parent-offspring relationships with 'Tribidrag' (aka 'Zinfandel'), is grown exclusively on the Adriatic island of Korcula. It is one of the grape cultivars with female-only functional flowers, causing reduced fertilisation and leads to problems in grape production and wine quality. A typical cluster of 'Grk bijeli' at maturity contains fully developed seeded berries, in addition to a highly variable proportion of undersized seedless berries. The aim of this study was to identify the cause of the reduced reproduction potential of 'Grk bijeli' in order to establish a better growing environment for improved yield and grape quality. 'Grk bijeli' female gametophyte develops normaly and at maturity contains both egg and central cell, together with two synergid cells and three antipodal cells. On the other hand, 'Grk bijeli' pollen grains show developmental deviations. Specifically, most of the pollen grains undergo the first pollen mitosis and contain one vegetative cell and one generative cell, while 20 % of ungerminating grains also accomplish the second pollen mitosis, giving rise to two sperm cells and one vegetative cell. Morover, 'Grk bijeli' pollen has acolporate morphology, which prevents germination and contributes to reduced reproduction. Furthermore, fertilisation after pollination with other varieties results in various degrees of ovule abortion depending on the pollinator, revealing Croatian cultivars 'Plavac mali crni' and 'Posip bijeli' as favourable varieties. Although this study describes a highly valuable cultivar of local importance, it also contributes to fundamental knowledge of grapevine reproductive biology and offers a strategy for improvement of wine production and oenological performance of semi-fertile varieties in general.

Anthocyanin profile of wild grape Vitis vinifera in the eastern Adriatic region

Wild grapevine (Vitis vinifera L., subsp. sylvestris) was found recently along the rivers in the karst landscape of the eastern Adriatic region, which includes Croatia and Bosnia and Herzegovina. The wild grapevine is almost extinct and grows in a restricted habitat in Europe. The anthocyanin profile of red grape berries from naturally growing populations of female wild var. sylvestris were characterized in two consecutive years and compared to the domesticated cultivars ‘Plavac mali’, ‘Merlot’ and ‘Xinomavro’. Five anthocyanidin-3-monoglucosides and their corresponding five 3-(6-O-acetyl) and five 3-(6-O-coumaryl) derivatives were identified and quantified using HPLC-DAD. Most wild grape samples had both acylated and non-acylated anthocyanin monoglucosides, although two individuals lacked acylated forms. DpGl were a greater percentage of the total in wild grape than in ‘Plavac mali’, ‘Merlot’ or ‘Xinomavro’. Principal component analysis showed differentiation of sylvestris from vinifera by anthocyanin profile. The anthocyanin profiles of sylvestris genotypes from the eastern Adriatic region, here presented for the first time, suggest genetic diversity within sylvestris in anthocyanin synthesis.

Differences in chemical composition of 'Plavac mali' grape berries

Evaluation of differences in yield and quality of grapevine is often based on the random samples of berries harvested on the same date. Due to differences in ripeness of berries from the same cluster, and among berries from different clusters of the same vine, the determined differences among examined treatments (clones, agricultural practices etc.) based on random berry samples may be over- or underestimated. The aims of this study were to determine: (1) differences among three 'Plavac mali' clones in the proportion of berries of different density classes (sugar concentration level) using the flotation method; (2) differences in berry weight and chemical composition among density classes of the same clone, and (3) differences between clones of the same density class. Significant differences were determined for all observed characteristics, among different berries density classes and clones. The results obtained indicate that berry samples used to compare different clones should be, to the greatest possible extent, at the same level of ripeness, as this significantly effects chemical composition. This can be achieved using the density separation of berries with the simple flotation method presented in this study. The presented results could be useful in the future improvement of clonal selection methodology as well as in improvement of sampling strategy for other types of researches.