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Numerous grapevine breeding programs are carried out in the world with the aim of getting varieties resistant to fungal diseases. The basics are often native varieties in order to preserve the authenticity of a certain area. Croatia counts more than a hundred native varieties but there is no research on their downy mildew susceptibility, which is essential for further breeding. The aim of this study is to compare the two most widely used methods for estimating the susceptibility of grapevine varieties: in vivo (field trial) and in vitro using the leaf disk assay. The research was conducted on nine genotypes: seven native varieties, Cabernet Sauvignon and Vitis riparia. In vivo disease symptoms were monitored after flowering four times in two-week intervals, and the leaf disks were evaluated once on the leaves of different developmental stages (from the 1st to 7th leaf from the shoot tip). Significant differences in susceptibility between the examined genotypes were found using both methods. By using the in vivo method significant differences were noticed during the initial terms, while the differences were reduced later on suggesting that the rate of disease development is peculiar to the genotype. In vitro method showed, together with differences between the genotypes, significant differences between the leaves of different developmental stages. The highest level of correlation between the results of these two methods (r>0, 5) was determined between the third term in vivo and the 3rd to the 6th leaf (from the shoot tip) in in vitro conditions.

Context and purpose of the study – Croatia has rich grapevine genetic resources with more than 125 autochthonous varieties preserved. Coastal region of Croatia, Dalmatia, is well known for wine production based on autochthonous grapevine varieties. Nevertheless, only couple of these are widely cultivated and have greater economic importance. Grape seeds are sources of polyphenols which play an important role in organoleptic and nutritional value of grape and wine. Hence, the aim of this study was to evaluate the simple polyphenols from grape seeds in 20 rare autochthonous grapevine varieties. Material and methods – Samples were collected during two consecutive years (2011. and 2012.) from germplasm collection in Split (Dalmatia). Grape samples were constituted of five bunches of fully ripe grapes. Seeds were manually separated, freeze-dried, grounded and stored at a low temperature until analyses. Polyphenolic compounds were analysed using HPLC analysis. Results – Eight polyphenolic compounds, galic acid, monomeric flavan-3-ols (catechin, epicatechin, gallocatechin and epicatechin 3-O-gallate) and procyanidin dimers (B1, B2 and B4) were detected. According to the investigated polyphenolic compounds significant differences between investigated varieties were found. Gallic acid content ranged from 91.0 to 245.08 total monomeric flavan-3-ols from 619.2 to 13539.6 mg kg-1 and total procyanidin dimers from 975.3 to 4140.2 mg kg-1 of seed. Catechin (263.2 to 8124.2 mg kg-1 seed) was found as main monomeric flavan-3-ol, epicatechin 3-O-gallate, gallocatchin and epicatechin varied between 0-164.31, 37.19-155.07 and 277.5-5224.4 mg kg-1 seed, respectively. Procyanidin B2 (420.2 to 2207.8 mg kg-1 seed) was found as a main procyanidin dimer. Procyanidin B1 and B4 amount varied between 401.80-165.19 and 276, 7-1539.4 mg kg-1 seed, respectively. Gegić had lowest and varieties Plavac mali and Babić highest amount of all investigated polyphenolic compounds. This study presents the first evaluation of Croatian grapevine varieties by characterization of seed polyphenolic compounds and it shows huge variability among them. More detailed analysis of polyphenolic compounds in selected varieties are carry out in our further research activities.

INTRODUCTION – Cryopreservation is a method for long-term conservation in which explants are stored at the ultra-low temperature of liquid nitrogen. In the case of grapevine, cryopreservation protocols have not been defined enough for wide application to numerous cultivars. Cryotherapy is a novel technique, which eliminates plant pathogens during exposure to ultra low temperatures. Plant cryopreservation is a complex procedure during which explants are exposed to potentially stressing events. Genetic stability of regenenerated plants is a part of cryopreservation protocol establishment. AIMS AND SCOPES – The main goals of this research are: to determine the efficient cryopreservation protocol for Croatian native grapevine cultivars and international cultivars, to test efficiency of cryotherapy in virus eradication and to check genetic stability of regenerated plants. This research was performed aiming to introduce this technique in general viticulture. MATERIALS AND METHODS – In vitro explants of Croatian (Plavac mali, Maraština-(plus their infected genotypes), Pošip and Škrlet) and international cultivars (Chardonnay, Cabernet Sauvignon, Merlot and Pinot Noir, within their infected genotypes) were taken for cryopreservation experiment, which had been established previously on cultivar Portan. Droplet- vitrification protocol was performed. Regenerated two-months in vitro plants from infected genotypes (control and cryopreserved apices) were subjected to ELISA testing. AFLP markers were employed to test genetic stability on cultivar Portan. Eight AFLP primer combinations were used on cryopreserved and non-cryopreserved shoot tips (sampled after sucrose preculture, loading and treatment with half-strength PVS2 solution) for a total of 43 plants tested. RESULTS AND DISCUSSIONS –The basal cryopreservation protocol, performed on international cultivars revealed: intermediate regrowth was achieved with Chardonnay (30%) and high with Merlot (70%), before and after cryopreservation, which was comparable to previously published results on 10 Vitis cultivars, which produced an average recovery of 64%. Similar results were obtained with healthy and virus infected cultivars. The same protocol, performed on Croatian cultivars, showed a low survival and recovery with only one (Maraština) of the four cultivars tested. This low success might be due to the fact that cryopreservation experiments were performed using cultures which had been introduced in vitro very recently and that the in vitro culture conditions employed were not adapted to the multiplication of these Croatian cultivars. The results of the cryotherapy experiments revealed that a high proportion of Chardonnay (100%) and Cabernet Sauvignon (78%) plants were virus-free after being subjected to cryopreservation. Control plants showed a high proportion of healthy plants, almost the same as with LN exposed ones for both viruses. AFLP markers revealed no significant differences in AFLP profiles after sucrose preculture, treatment with the loading solution and with half-strength PVS2, whereas an increase in polymorphic fragments was observed in non- cryopreserved and cryopreserved samples treated with PVS2 solution, the number of polymorphic fragments increasing with longer durations of exposure to PVS2 solution. CONCLUSIONS AND POSSIBLE APPLICATIONS – Cryotherapy as a method can be efficient for virus eradication even in the preconditioning steps of a cryopreservation protocol. A genetic stability study should be included in post- cryopreservation experiments.

INTRODUCTION – Before Philloxera crisis in Croatia were more than 400 grape varieties under cultivation, but today their number is drastically decreased. Recently, many efforts in the preservation of grapevine biodiversity have been undertaken: detailed inventory of wine-growing regions, establishing of National and regional collections and their systematic ampelographic and genetic characterisation. Recent genetic research resulted in revealing synonyms and homonyms, as well as genetic relationship within Croatian native cultivars, as well as with cultivars from other regions. Evaluation of quality traits of native cultivars has also started. AIMS AND SCOPES – The main goals of this research are: to determine the number of Croatian native grapevine cultivars based on inventarisation of viticultural areas, ampelographic and genetic characterization ; to perform detailed ampelographic characterization using OIV descriptors ; to perform the genetic characterization of Croatian native grapevine cultivars using SSR markers and to determine the level of genetic variability within Croatian native grapevine cultivars. MATERIALS AND METHODS – Vines of a potentially native cultivars ware selected and marked and used as a source of propagating material for establishment of a National collection of Croatian grapevine cultivars located at experimental station Jazbina, at Faculty of Agriculture, University of Zagreb. In this process more than 140 accessions were introduced to this collection. Phenotyping of collected accessions was performed using 47 OIV descriptors for grapevine cultivars and Vitis species that were selected within the GrapeGen 06 and used for cultivars description in European Vitis database. SSR genotyping was performed using a total of nine markers. Six SSR markers (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, VrZAG79) recommended by OIV and three additional ones (VVMD25, VVMD28, VVMD32) selected during the Grapegen06 project. RESULTS AND DISCUSSIONS – Based on results of inventarisation, ampelographic characterization and genetic characterization the list of 103 Croatian native grapevine cultivars is defined together with their synonyms and population status/vulnerability evaluation. Based on presented information on population status of Croatian native grapevine cultivars we can conclude that more than 50% of cultivars are critically endangered, endangered or vulnerable. The results of ampelographic characterization of Croatian native cultivars are showing the high level of variability. Comparison of Croatian cultivars SSR profiles with the results of the other cultivars in EU- vitis database confirmed that majority of them are unique and exist only in Croatia. Since most of them are duplicated in grapevine germplasm collections throughout Croatia they are considered as “saved”. Ongoing genetic studies with increased number of SSR loci (>30) as well as use of SNP markers will allow us more detail analysis of genetic structure and relatedness of Croatian native cultivars. CONCLUSIONS AND POSSIBLE APPLICATIONS – The preserved native cultivars are presenting a good base for future development of Croatian viticulture and because of high genetic variability, as well as some traits that are detected within Croatian native cultivars they could be of interest to other countries for production as well as for breeding.

Genotyping old grapevine samples, in particular variety identification via microsatellite profiling is still far from being a routine. The successful identification depends on several factors, the age of the investigated material being the most obvious one. In addition, the amount and integrity of DNA depends on conditions of samples storing. Contamination and fragmentation processes make the isolated ancient DNA (aDNA) a rather difficult template for PCR amplification. Three old grapevine samples were investigated in this study. Two samples were taken from approximately 90-years old herbarium of cv. Tribidrag, a Croatian autochthonous grapevine variety not anymore present in the germplasm collections and one was approximately 2000 years old underwater archeological grapevine specimen (woody parts). Several different approaches of aDNA isolation were tested. However, the use of a commercial plant DNA isolation kit showed to be the best choice in terms of yield and prevention of possible contamination with temporary grapevine DNA. The initial attempts to amplify standard grapevine microsatellite loci by standard PCR protocol failed, most likely due to low copy number of template DNA. Therefore, the whole genome amplification (WGA) kit was successfully applied to overcome this problem. The WGA approach worked in case of one herbarium sample and the archeological specimen. DNA fragments ranging between 100 and 500 bp were cloned and sequenced. While the isolated DNA from the underwater archeological sample was proved to be from different origins (not from grapevine), indicating contamination of the specimen, the six sequenced clones from the herbarium specimen corresponded to six different grapevine chromosomes. Therefore, we used the WGA-processed herbarium sample and managed to PCR-amplify the VVS2 locus, thereby demonstrating the proof of principle. Eventually, we successfully genotyped the same template using six standard microsatellite loci (VVS2, VVMD5, VVMD7, VVMD27, VvZAG62 and VvZAG79). The obtained SSR profile was identical to Zinfandel/Primitivo/Crljenak Kastelanski, except for the VVS2 locus which was homozygous (141/141) instead of heterozygous (131/141). In the light of these results we hypothesize on the origin of Zinfandel as an old Croatian variety under the historical name Tribidrag, which was being used in documents dating as late as 15th century.

At the beginning of the 20(th) century more than 400 grape varieties were under cultivation in Croatia. But because of introduced pests and pathogens, modern economic pressure and the changeover to internationally well-known varieties, a drastic germplasm erosion has occurred. During the last decade, a detailed inventory of wine-growing regions has shown that a lot of autochthonous varieties still can be found, but many of them are extremely rare and endangered. For their conservation collecting expeditions have been organized and in 2001 a national ex situ collection was established at the Faculty of Agriculture in Zagreb consisting nowadays of more than 140 native accessions. In addition, several regional (back-up) collections have been established to keep duplicates and enable economic evaluation under appropriate climatic conditions. Along with conservation efforts, a thorough amphelographic description and genetic fingerprinting of the varieties were carried out. So far, more than 100 varieties have been positively identified and genetically profiled according to the protocols of the EU project GrapeGen06. Consequently, their photos, descriptions and genetic profiles have been entered into the EU Vitis Database. Comparison of their microsatellite genotypes with those of international varieties showed most of them having unique genotypes supporting the hypothesis of their Croatian origin. Microsatellite analysis enabled clarification of synonyms and homonyms as well as analyses of parent-progeny relationships. In case of few neglected varieties a high quality potential was observed, and their economic revitalization has been started ('Malvasija dubrovacka', 'Crljenak kastelanski', 'Sansigot'). Clonal and sanitary selections of major native varieties ('Plavac mali', 'Posip', 'Zlahtina', 'Kraljevina', 'Skrlet', 'Plavina', 'Debit', 'Marstina', 'Vugava', 'Grk') also have been launched.'

Traditional grapevine varieties have been arisen by spontaneous crosses centuries ago and their present populations are result of many generations of clonal propagation. Due to great multiplication over time and space the genotype of individual vines was exposed to spontaneous mutations. Consequently, varieties are not uniform but rather mixture of similar clones of different agronomic value. Conventional clonal selection is a long lasting procedure that aims to detect and propagate seldom vines that are from the perspective of winegrowing and market superior over the population. The problem: many of individual vines under selection have uniform genotype in spite of different phenotype. Several types of molecular markers have potential to detect genetic intravarietal variability and thus, enable breeders to detect unique mutant genotypes what can provide more rational clonal selection and clone identification tool. The clones resulting from individual clonal selection of two grape varieties (Plavac mali and Skrlet) have been analyzed in replicated trial for several agronomic traits. The contrast clones that showed significant differences in bunch size and yield per vine have been assessed by transposon-based markers that have been reported as very polymorphic and repeatable. Unexpectedly, in this study the level of genetic variability was rather low.

There is a large number of grapevine cultivars grown in Croatia. Most of them are considered to be autochthonous. Unfortunately, clonal selection of these cultivars has never been done and there is no planting material of adequate quality standards. As a first step in clonal selection, mass positive selection together with sanitary selection of 15 economically important cultivars has been started. In rpoceeding of genetic selection visual symptoms of the viruses have also been observed. Samples of dormant canes have been taken from vines with positive agronomic traits and no visible symptoms of virus infection. The presence of four economically important viruses was evaluated using ELISA (enzyme linked immunosorbent assay): two nepoviruses, Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV) and two closteroviruses Grapevine leafroll-associated virus 1 (GLRaV-1) and Grapevine leafroll-associated virus 3 (GLRaV-3). Survey of virus deseases in production vineyards of different vine-growing region of Croatia has disclosed the widespread presence of viruses and the number of non infected vines was rather low. This is true especially for the coastal region where it was not possible to find any healthy plant of certain cultivars. Although no visible symptoms were observed , it was not rear to detect multiple infections with two or even three viruses. Mostly spread virus in Dalmatia was GFLaV-3 with approximately 80% of vines infected. The situation is much better in northern region, where almost half analysed plants were healthy and most represented virus is GLRaV-1. The presence of both nepoviruses is rather low, especially ArMV (only 1% of vines infected) and somewhat higher of GFLV in Dalmatia.

At the end of 19th and beginning of 20th century in Croatia were, according some literature data, more than 400 grape varieties. Unfortunately, because of many reasons (introduced pests and pathogens, modern economic pressure and introduction of world well known cultivars), drastic erosion of autochthonous cultivars has occurred. In last decade, many efforts in the preservation of grapevine biodiversity in Croatia have undertaken. Detail inventarisation of vine-growing regions in Croatia has shown that lot of autochthonous cultivars still can be found, but many of them are endangered and for a long time neglected. To preserve them against the extinguishing, we have established since 2001 national ex situ collection at the Faculty of Agriculture in Zagreb, where we have collected about 100 autochthonous accessions so far. Besides, we started to build few regional collections, to save duplicates and enable their economic evaluation in appropriate climatic conditions. Besides multiplication and planting into collection, amphelographic description (OIV descriptors) and genetic analyses (at least 6 core SSR loci) of cultivars have been carrying out. So far, about 60 cultivars have known genetic profile, and all others are in the progress. Comparison of the microsatellite genotypes of the analyzed cultivars with international varieties shows that most of them have unique genotype which supports hypothesis they are Croatian autochthonous cultivars. In few cases microsatelites were also useful in clarification of synonym and homonym questions as well as parent/progeny relationships. All this data, along with photos from different plant parts, are supposed to be entered in the electronic database. We have observed a high quality potential in case of some neglected cultivars, and we started economic revitalization for few most promising (Malvasija dubrovačka, Crljenak kaštelanski). Beside that, we started with clonal and sanitary selection in case of most important native varieties (Plavac mali, Pošip, Babić, Žlahtina, Kraljevina, Škrlet, Plavina, Debit, Maraština, Vugava).

Among 48 cultivars holding the „ Malvasia“ name listed in the Vitis International Variety Catalogue (http://www.genres.de/idb/vitis/vitis.htm) there are also two cultivated in Croatia – „ Malvazija istarska“ and „ Malvasia dubrovačka“ . Since recently it is known that Malvasia dubrovacka is not a unique local cultivar (genotype) but the member of the synonym group which is scattered across the Mediterranean and even at the Atlantic islands (Malvasia delle Lipari, Malvasia di Sardegna, Malvasia de Sitges, Malvasia de Tenerife, ...) (Crespan et al., 2006). So far, Malvazija istarska seems to be a unique genotype spread only in the area of peninsula Istria and north-east of Italy. According to Boursiquot et al. (2007), the real number of Malvasia cultivars is not so large as it used to be believed, i.e. they found that several so called Malvasia cultivars are synonyms of already well known cultivars. Taking into account the historical fact that „ Malvasia wine“ used to be exported from Greece to Venice for centuries and an assumption that the name „ Malvasia“ has Greek origin (according to medieval city Monemvasia), as well as the fact that by vegetative propagation cultivars can be easily disseminated and preserved over the space and time we have hypothesized that Croatian Malvasias might still be grown (under different names) in Greece or some other places along the historical naval routs. We have used an opportunity to participate in the sailing expedition „ Malvasia Mediterranea 2005“ (http://www.malvasia-mediterranea.com/) to explore and sample the tissue for DNA of the cultivars along the eastern coast of Adriatic and Ionian Sea, and south of Peloponnesus. Twenty-eight grapevine samples from Greece (islands and hinterland), more than 30 from Croatia, and several from Italian coastal regions that used to be on the ancient naval routs from Monemvasia to Venice, have been assessed by genomic DNA SSR markers and analyzed in term of genetic similarity. Similarly as in several previous studies we have found a weak relationship of actual Greek cultivars with Croatian and Italian ones. We have found only one original genotype that is nowadays present in Greece, Italy and Croatia under three different names (synonyms), Pavlos, Malvasia lunga (M. del Chianti) and Maraština, respectively.

Borgonja crna is grapevine cultivar (variety) grown in Croatia only on peninsula Istria (north Croatian coast) and has regional importance. Borgonja crna has superior quality in comparison to the other black grapevine cultivars grown in that region. All the available literature data suggests that Borgonja and Gamay noir (Gamay Beaujoilais) could be synonyms for the same variety. This assumption is based on the similarity of phenotype. For that reason even producers started to plant Gamay noir due to the lack of planting material of Borgonja crna. The aim of this work was to examine whether the Borgonja crna and Gamay noir are identical or two different cultivars. In the year 2004 various ampelographic and genetic analyzes were conducted. As a part of ampelographic analysis selected OIV descriptors, uvometry and philometry were applied. Jung leaves from the selected vines were collected, liophilised and DNA extraction was performed according to Doyle and Doyle (Focus, 1990.). Microsatellite markers (SSR) were chosen for plant genotyping. According to the project GENRES 081, 6 core SSR loci and three additional were used. The results shown that, although morphological similar, Borgonja crna and Gamay Beaujolais have different genetic profiles and cannot be considered as synonyms.

Due to clonal propagation, longevity and conservative attitude of wine makers and consumers in term of cultivar importance for wine uniqueness and quality, some of the present cultivars can be very old. Place of origin and dissemination routes for many cultivars are still mistery, especially in the Mediterranean. It is commonly believed that viticulture on the Eastern Adriatic coast is founded by ancient Greeks. Some of the present cultivars (e.g. 'Grk', 'Malvasia', 'Cipar') have names pointing to Greece as the possible place of their origin. Centuries of very alive trade along naval routes from Greece, via Dubrovnik, Dalmatian islands towards Venice and vice versa, make cultivar exchange as very likely. By the aid of SSR markers it is possible to study genetic relatedness among cultivars from different areas and to test the hypotheses on cultivar origin, i.e. to discover synonyms, closely related cultivars and in some cases even parentage. Approximately 30 Croatian and 30 Greek cultivars so far ungenotyped, being cultivated in the area along the ancient and middle age naval routes (islands and coast line), were screened by 10 SSR markers in order to check their genetic relatedness. Genotypes, genetic similarity indices and allele frequencies do not reveal any close genetic relative among two analyzed sets of cultivars.