Chapter 7 Genetic and Phenotypic Diversity and Relations Between Grapevine Varieties : Slovenian Germplasm

Slovenia is a small central European country situated between the Alps and the Adriatic Sea, at the crossing between Italy (to the West), Austria (to the North), Hungary (to the East) and Croatia (to the South), a historical place of significant grape and wine trading. Grapevines have been cultivated in Slovenia since ancient times, although the first literal proofs on grape growing and winemaking date back merely to the Austro-Hungarian Empire [1,2]. However, the oldest Vitis vinifera ssp. L. (grape) pips (seeds) found during the archaeologi‐ cal excavation of the late Neolithic (Copper Age) pile-dwelling settlement of Hočevarica at the Ljubljansko barje moor and date back to the 37th/36th century B.C. [3].


Introduction
Slovenia is a small central European country situated between the Alps and the Adriatic Sea, at the crossing between Italy (to the West), Austria (to the North), Hungary (to the East) and Croatia (to the South), a historical place of significant grape and wine trading.Grapevines have been cultivated in Slovenia since ancient times, although the first literal proofs on grape growing and winemaking date back merely to the Austro-Hungarian Empire [1,2].However, the oldest Vitis vinifera ssp.L. (grape) pips (seeds) found during the archaeological excavation of the late Neolithic (Copper Age) pile-dwelling settlement of Hočevarica at the Ljubljansko barje moor and date back to the 37th/36th century B.C. [3].
The geographical position of the country as well as the regarding climate conditions, and the socio-political development throughout the nation's history, have contributed to a diverse assortment of grapevine [1,2].At least around 100 old and less known varieties are enumerated especially in the western part of Slovenia (Sub Mediterranean) and accompanied by the widespread European, allochthon, autochthon/landrace and local varieties, a large number of grapevines is cultivated in Slovenia nowadays [4,5].
The intense trade in grapes and wine, especially due to the Venetians (Venetian Republic from the end of 7 th to the end of 18 th Century; Figure 1), and the varying climatic and geological conditions contributed to a great diversity of Vitis vinifera L. varieties.Additionally, the multiculturalism and multilingualism of the area as well as the turbulent historical events have contributed to identical genotypes having different names [1,2].Nowadays, Slovenian winegrowers (27.802) produce on 15.973 ha an annual of around 54.3 mio litres of wine; 62% whites and 38% reds.Fifty grapevine varieties (Vitis vinifera L.), international, allochthon, local and autochthon as well, are registered in the official Slovenian varietal list, but numerous accessions are still observed and non-descripted in many parts of Slovenia [1,6].Although some of them form the basis of the renown regional varieties ('Rebula' in Goriška brda, 'Zelen' and 'Pinela' in Vipavska dolina, 'Šipon' in Ljutomer-Ormož etc.), many face extinction, since in some areas only a few plants are reported.However, Slovenia is facing the threat of the rapid erosion of the native/local germplasm on account of the introduction of foreign varieties, such as 'Merlot', 'Cabernet', 'Chardonnay' and 'Pinot'.Very often, however, the problems with the identification of these local, unknown varieties, occur, because of the presence of various synonyms of the same variety throughout the country [2,6,7].The gene pool of the varieties cultivated in Slovenia is composed of old allochthon, autochthonous, domestic/local varieties, which can all be additionally divided into two groups.The first group consists of commercially used varieties which are planted on more than 100 ha each, such as 'Rebula', 'Žametovka', 'Zelen', 'Pinela', 'Šipon', 'Radgonska Ranina', and 'Refošk'.The second group of varieties, such as 'Vitovska grganja' and 'Belina', are cultivated on less than 100 ha each and are known as local varieties.Moreover, around 50 well-known and rare varieties/accessions exist in Slovenia today; the majority of which have not yet been listed in the International List of Vine Varieties and Their Synonyms of the O.I.V. (International Organisation of Vine and Wine).Even though some morphological and agronomical descriptions related to these accessions exist, we are talking about mostly unexplored plant material and some of these varieties had survived only in less productive vineyards or owing to germplasm collections.The systematically collection of these endangered accessions started in 1980.Nowadays, there are five grapevine collections in the whole country: Slap at Vipava, Ampelografski vrt in Kromberk, the grapevine collection at Dobrovo in Goriška brda, Meranovo near Maribor and one near Ormož that include old varieties, clonal candidates and clones (Table1).Nowadays, varieties can be characterized by several methods: (i) by means of a morphological description of plant parts at different phenological stages; (ii) morphometry based on the measurements of the parameters of the plant organs and (iii) quantitative or qualitative analysis of biochemical compounds.Furthermore, traditional methods of varietal description based on vegetative and reproductive (ampelography) parts of plants, contributed greatly to the full description of the identities and relationships among V. vinifera L. varieties; what also suggests that ampelographic characterization according to the characters put forward by the O.I.V. is the first step in the examination of grapevine varieties/accessions. Several authors described various analyses of primary and secondary metabolites and methods based on DNA polymorphism as outstandingly useful methods to complete the morphological identification of grapevine varieties [8,9].

Slovenian germplasm
One of the problems in the management of these germplasm collections is the use of synonymic and homonymic designations.The lack of order caused by synonyms and homonyms is caused by inadequate documentation and poor preservation of historical facts related to grape growing and trade.The identification and comparison of plant material by ampelographic methods often results in misinterpretations [10].In contrast, DNA-based markers are independent of environmental factors and are therefore more appropriate for varietal identification [9,11].In the last decade, more than 60 SSR primers from the genomic libraries of Vitis vinifera L. have been developed and used for identification purposes [12,13].The genetic characterization of grapevine varieties (Vitis vinifera L.) using microsatellite markers and ampelometric methods has been done by many countries and regions already.The integration of the resulting molecular analyses and detection of synonymous grapevine varieties has already been performed among countries which share a common grapevine assortment, such as Croatia, North Italy, Austria, Germany, France, Spain, Portugal, Greece, etc. [5,9,14,15].In the last decade many genetic studies of local varieties have been conducted in Slovenia [4,5,[15][16][17][18] as well, and have consequently revealed the genetic biodiversity of grapevine varieties grown and cultivated in the country (Table 2).

Genetic diversity and relations among varieties
SSR markers offer some advantages over other molecular markers, including their co-dominant inheritance, hypervariability, and high cross-species transferability [8,13].Consequently a large number of markers has been developed for characterisation of grapevine by many research groups [8,12,19] -these markers have provided a very useful and convenient tool for analysing genetic diversity of grapevine.In order to efficiently manage these conserved local germplasm resources and to understand the genetic relationships among them, it is necessary to characterize the genetic diversity existing in the Slovenian collection and production vineyards.However, a majority of cultivated and grown grapevine accessions in the Sub-Mediterranean part of Slovenia and a subset of 6 widespread European varieties taken as reference have been genotyped with microsatellite loci in order to: (1) identify and/or differentiate varieties, especially those of similar morphological characteristics; (2) assess genetic diversity and relationships among them; (3) and compare these varieties to their synonyms [5,[15][16][17][18] (Figures 2 and 3).The Mediterranean Genetic Code -Grapevine and Olive Figure 3. Dendrograms of genotyped grapevine accessions grown in Slovenia [17].
SSR profiles of groups of varieties with similar names but some different morphological characteristics were compared in order to assess their relationships and resolve existing doubts on their identity.In order to illustrate the population structure among Slovenian varieties/accessions dendrograms were constructed, which classify the varieties according to the proportion of shared alleles (Figures 2 and 3).The average similarity of all varieties is 34% of shared alleles (Figure 2, left), which is close to the average similarity observed for mid-European and Portuguese accessions [5].Overall, two distinct clusters were obtained, with many sub-clusters.The accessions from the first cluster are related to 'Laški rizling' ('Welschriesling'), 'Rdeča žlahtnina' ('Roter Gutedel', 'Chasselas red') from the West European gene pool (Proles occidentalis), but also 'Rebula' ('Ribolla') known as Proles pontica.Another two varieties, 'Guštana' and 'Danijela', grouped with the first cluster, are both characterised by an early grape ripening.The name 'Guštana' may describe the ripening time of this variety, which is in August.The variety 'Danijela' is planted only in gene bank vineyards and its provenance is still uncertain.The variety 'Picolit' is cultivated in the western part of Slovenia (Northern Primorska) and in the north-east part of Italy -in Slovenia two distinct, morphologically different types of this variety/accession, 'Picolit Italia' and 'Picolit Vienna', are known.Microsatellite markers revealed no differences at 21 loci; matching also the same allelic profiles at all 7 SSR loci with a variety 'Picolit' from Italy [25].In the second cluster, which includes 25 accessions, three groups of synonyms were discovered [5]: 'Vitovska grganja' = 'Racuk', 'Poljšakica Drnovk' = 'Istrska Malvazija' and 'Prosecco'= 'Briška Glera' = 'Števerjana'.'Vitovska grganja' is an old grape variety cultivated in Slovenia in the winegrowing districts of the Vipavska dolina and Kras and also in the north-east part of Italy, where it played an important role in the past [1].The synonymy found between 'Vitovska grganja' and 'Racuk' could not yet be confirmed despite obtaining identical allelic patterns, because an accurate morphological characterization of 'Racuk' is still lacking.The allelic profiles of our 'Vitovska grganja' have been further compared with the Italian variety 'Vitouska', recently published by [26] -dissimilarity was revealed at 14 out of 16 loci, and indicating that there is a compatible parent/progeny.The varieties 'Prosecco', 'Briška Glera' and 'Števerjana' form another group of synonyms.'Prosecco' and 'Glera' have already been proved to be synonyms on the basis of morphological descriptors and isoenzyme analyses, while 'Števerjana' has not previously been considered to be a synonym.The variety 'Prosecco' takes its name from the village of Prosecco, in the Province of Trieste, where this variety is also known as 'Glera' [26].The name for the variety 'Števerjana' may originate in Števerjan, a small village in North-East Italy (Collio), near the Slovenian border.The comparison of 16 SSR loci of our 'Prosecco' with Italian 'Prosecco tondo', which was recently analysed [26], revealed no differences -on the basis of this comparison 'Prosecco' = 'Prosecco tondo' = 'Glera' but according to [26] 'Glera' is related mainly to 'Prosecco lungo' and less frequently to 'Prosecco tondo' [5].
Subsequent, more detailed study was done in 2011 [17], which included all accessions founded in the Sub-Mediterranean part of Slovenia.The observations are reported as follows [17] (Figure 3): The pairs/groups of vines denominated 'Duranja' and 'Duranja-Pregara', furthermore 'Belina' and 'Belina1', 'Zelen Rup2', Zelen66' and 'Zelen3' and 'Malvasia', 'Istrska Malvasia' and 'Malvasia Istriana' revealed identical genotypes in all SSR microsatellites analysed therefore they are regarded as synonyms.The designations related to 'Malvasia' very often comprise several types of grapevine varieties which are also morphologicaly heterogenous [20].Very closely related to 'Malvasia Istra' was 'Borgonja bela', sharing 19 out of 20 alleles, what was also expected according to known ampelographic characteristics.Unexpectedly, vine denominated 'Laščina 1' also showed identical SSR profiles as 'Malvasia Istra' but completely different than accession 'Laščina 2', what is considered as a designation error or homonym.In the group of variety 'Plavina', the vines signed as 'Plavina maločrn' and 'Maločrn' showed same genotyping results and they are closely related to vine denominated 'Plavina Pregara', what cannot be affirmed for accession signed as 'Plavina-Pčavina'.'Plavina-Pregara' in contrast to 'Plavina maločrn' and 'Maločrn' resulted in homozygous state (or heterozygous with a null allele) at locus VVMD7, but 'Plavina-Pčavina' was different from them in 8 loci out of 10 [17].The difference in the stage of homozygosity/heterozygosity at locus VVMD7 was also obtained between accessions 'Glera' and 'Briška Glera' and these kind of mutations at locus VVMD7 have previously often been reported [5,9,21].These results of close relatedness suggested that local varieties can be phenotypically partly different types or under-types and that one could have originated from the other through somatic mutations.Low relatedness (20% of similarity) was also found between Slovene 'Plavina' and Croatian 'Plavina' genotyped by [20].'Plavina' was reported the most common grape variety along the Dalmatian coast and was known as 'Pagadebiti' in 'Curzola', however in our study the vine denominated 'Pagadebiti' is more similar to variety 'Rebula' ('Ribolla') and 'Kanarjola' than to 'Plavina'; as pairwaise comparison revealed only 6% of genetic similarity.The obtained allelic profiles of 'Pagadebiti' are also different from the profiles of two 'Pagadebiti' accessions reported by [21] -'Pagadebiti' translates as "pay the debt" and so the name may had been used for several very productive grapevine varieties, resulting in many homonyms.
To the group of synonyms related to the designation 'Glera' ('Glera', 'Prosecco', 'Briška Glera', 'Števerjana', 'Beli teran') the Croatian variety 'Teran bijeli' genotyped by [14] was compared.The comparison of data of 7 SSR loci resulted in identical SSR profile confirming their synonymy.A high genetic difference was revealed between the accessions denominated as 'Glera'/'Briška glera' and 'Bela glera' and could be explained with the fact that the term "Glera" was quite frequently used for white grapevine varieties in the Sub-Mediterranean part of Slovenia in the past [1,24], therefore the obtained homonymy is not a surprise.By comparing allele sizes of 7 SSR loci of the Croatian 'Muškat ruža Porečki' [14] with the Slovene 'Cipro', identical genotypes were found and therefore synonymy between these two varieties was confirmed.On the other hand, 'Beli muškat', which is clustered together with 'Cipro' in the dendrogram, is related to them with a similarity of 60%.
Moreover, the genetic identity and relationship among 'Ribolla gialla', 'Rebula' and 'Robola' that have been traditionally cultivated in other European Countries (Goriška brda in Slovenia and Kefalonia Island in Greece) grown in private vineyards and in grape collections were additionally studied (Table 3) [29].For this purpose, 35 SSR loci were analysed to fingerprint 19 accessions uniformly grown in the three cultivation areas (Friulian Collio, Goriška brda and Ionian Islands).'Ribolla Gialla' and 'Rebula' accessions revealed identical genotype in all 35 analysed SSR markers, therefore are regarded as synonymous.Data proved the existence of full-sibling relationships between 'Ribolla gialla' and 'Robola' (Figure 5) [29].'Robola 7' Table 3. List of analyzed accessions and origin area of plant materials related to the designations 'Rebula', 'Ribolla' [29].

Accessions related to designations 'Vitovska' and 'Grganja'
The accessions related to 'Vitovska', 'Vitouska', 'Grganja' and 'Garganja' are cultivated in different winegrowing areas in Slovenia, Italy and Croatia; therefore their names (synonyms) still cause disorder, because they are often considered indigenous and ancient varieties in the area of cultivation.According to the accurate study [26], which included as much as 37 nuclear microsatellite locii confirmed the variety 'Vitouska' to be the progeny of 'Malvasia del Chianti' (syn.'Malvasia bianca lunga', 'Malvasia lunga') and 'Prosecco tondo'.Following this, [30] found an original genotype of 'Malvasia del Chianti' with the varieties 'Pavlos' present in Greece and 'Maraština' cultivated in the south coastal region of Croatia (Dalmatia).Already in 1949 it was affirmed that 'Maraština' and 'Malvasia lunga' are one and the same variety [31].In 2012 [18] reported genotyping and phenotyping of accessions (Vitis vinifera L.) cultivated in Slovenia, Croatia and Italia, mostly known as 'Vito(u/v)ska' and 'G(a)rgan(i)ja', while also referred as indigenous or landrace varieties [1,7,28].The studied varieties, with an additional focus on variety 'Vitovska grganja', were mostly taken from germplasm collections: 'Vitovska grganja' from Slovenia, 'Vitouska', 'Ribolla gialla' and 'Prosecco' from Italy, 'Garganja' and 'Maraština' from Croatia.The Mediterranean Genetic Code -Grapevine and Olive Vines designated as 'Vitovska grganja 1' and 'Vitovska grganja 2', which have been grown for more than 100 years in the Slovenian Karst (Kras) revealed identical genotypes in all 11 analysed SSR microsatellites.The variety 'Maraština', nowadays known as an indigenous grapevine variety from Central and South Dalmatia (Croatia) surprisingly showed highly related SSR profile as both 'Vitovska grganja' cultivated in Slovenia.According to [30,31] the variety 'Maraština' coincides to the varieties 'Pavlos' and 'Malvasia del Chianti' (Malvasia lunga).In our study 'Maraština' coincided in seven of eleven SSR loci compared to 'Malvasia del Chianti' reported by [26], therefore we can conclude only that they are in strong relationship.Variety 'Vitouska' received from VCR Rauscedo (Italy), compared to 'Maraština' and 'Vitovska grganja' 1 and 2, differed in almost all used microsatellites, except at locus VVMD 5 and VVMD36, what additionally confirm the already reported affirmation of null (weak) relationship between both varieties [5,15,26].Furthermore, considering that [26] affirmed the progeny of 'Vitouska' with 'Malvasia del Chianti' and 'Prosecco tondo', a quite weak match in their locii was found among them in our study.

Phyllometrical tolls for phenotyping
Morphological descriptions of different parts of the plant at different phenological stages, but also morphometry based on the measurements of parameters of plant organs are the oldest methods used in ampelography.Moreover, quantitative and qualitative analyses of biochemical compounds were also suggested at varietal description [33].Despite the technical advances in biochemical and molecular approaches over the last decades, the descriptions of morphological characteristics remain an important methodology in the description and research of the diversity among species, varieties and clones.The most commune and used methodologies are descriptions according to the OIV codes [34] and ampelometry, especially phyllometry.The advances of morphological descriptions can be characterised as simple, cheap and applicable in the laboratory or directly in the field.Moreover, the presentday studies focus on finding new parameters in order to differentiate the varieties of vine precociously, quickly and efficiently [35].The average vine leaves were reconstructed by measuring the proposed morphometrical parameters [36], furthermore a system for direct digitizing of phyllometric parameters from leaves was developed [37].In grapevine collections approximately 5-10% of existing varieties are misnamed and even in commercial viticulture, misnaming and confusion related to synonyms and homonyms [38] is a frequent phenomena.We started to collect our allochthon, autochthone, local and endangered varieties systematically in 1980 and today we have few germplasm collections around Slovenia.
Additional attention has also been given to varietal phenotyping, especially at those varieties where some misunderstandings in nomination still cause disorders, for example at the varieties 'Vitovska grganja', 'Refošk', 'Rebula', 'Glera', 'Vitouska', 'Garganja' and others, which are frequently treated as an indigenous, autochthonous or landrace varieties in different countries [18].Some new approaches to accelerate the phenotyping of leaves at different varieties were suggested; for example automatic measurements, as well as the possibility of typical-varietal mature leaf reconstruction [39,40].

Phyllometric measurements
Many studies in the last decades demonstrated the importance and contribution of phyllometry in grapevine description and distinction.The method provides around 82 criteria measurements of morphometrical characteristics on an individual leaf.According to a high variation in these characteristics among leaves of the same variety at least 10 mature, typically varietal leaves should be picked up, scanned or photocopied, placed in herbarium before being analysed.In our study we followed that procedure: Herbarium leaves were scanned (with the HP LaserJet M1005scanner) and analysed using AnalySIS image analysis program (Soft Imagining System GmbH).The quantitative base variables required the reconstruction of an average typical leaf of different varieties measured following the suggestions from [36] with some modifications.The following morphometrical parameters were measured in all leaves sampled per variety: leaf area (LA), length of nerves (L1, L2, L3, L4), distance from the petiole insertion to upper sinuses (OS), distance from the petiole insertion to lower sinuses (OI), total leaf height (H), total leaf width (W1), distance between the ends of left and right L2 (W2), distance between the basal tooth of the upper lobe (W3), angles between nerves measured from the petiole sinus to the first ramifications of vein (α, β, γ), angles between nerves from petiole sinus to apex of vein (α', β', τ), angle at the apex of upper lobe (δ1), angle between the apex of L1 and the apexes of L2 (δ2), angle between apex of L1 and apexes of L5 (δ3), distance between apex and basal tooth of upper lobes (D1), distance between the ends of L1 and L2 (D2), petiole length (lp), distance from the base of petiole sinus to the intersection of L3 vein to L4 vein (LO), tooth width at the end of L2 and L4 (b1, b2), tooth length at the end of L2 and L4 (h1, h2), distance between the ends of L5 (l), distance between the beginning of L5 (l') suggested by [41].Moreover, [42]   The aim of the study was to investigate the efficiency of ampelographic and morphologic methods to evaluate the significance of O.I.V. code lists (Paragraph 2.2.2.2) and phyllometry regarding the varietal diversity.In addition, a graphic reconstruction method was applied and improved to form a typical varietal mature leaf (Figure 9).We evaluated 38 morpholog-ic and 22 morphometric parameters per leaf.Statistical analysis was carried out, PCA (principal component analysis) was performed taking into account equations calculated from different leaf variables, and the qualitative variables proposed by the O.I.V. were preceded to Cluster Analysis.The combination of morphologic descriptors and phyllometric measurements proved to be complementary more than comparable methods.The results of the evaluation of O.I.V. descriptors showed a high level of varietal diversity, whereas accession 'Barbera type Bovcon' and 'Pokalca' as well as 'Barbera' and 'Syrah' showed some similarities.The most different variety in the group studied according to O.I.V. descriptors was 'Touriga national'.The PCA analysis showed the first three components responsible for more than 82% of the discriminating power.The reconstruction determinants were relations between the depth of the lateral sinuses and lateral veins (PC1), relation between first left lateral vein and central vein, relation between first right lateral vein and central vein (PC2), sum of angles between veins, relation between length of petiole and central vein and depth of the lateral sinuses (PC3).The enumerated relations enable and additionally improve the leaf reconstruction.At the varieties 'Sladkočrn' and 'Refošk' the shallowest lateral sinuses and at 'Tinta pinheira' the deepest ones were observed whereas 'Plovdina' and 'Touriga national'showed shorter lateral veins (L1d, L1g) in comparison with the main vein (L).The morphological descriptions and morphometric reconstructions of leaves gave a significant contribution to understanding the grapevine phenotypical biodiversity.Furthermore, [40] studied 28 morphometric characteristics of leaves in detail and their contribution to the similarity among accessions with designations linked to 'Glera' and 'Rebula'.Phyllometric measurements affirmed the genotyping of same varieties studied by [29].At the comparison of the groupings in Figure 5 and Figure 8, the measured 28 morphometric characteristics give suitable results what suggests that phyllometry grants an indispensable, cheap and credible methodology in the ampelography -varietal description and discrimination.

Morphological descriptions
The O.I.V. codes list still remains the most uses and suitable ampelographic tool in context of grapevine description [34].There are approx.147 descriptors; each regarding one significant characteristic, which offers a possibility of an accurate description of the main morphological parts of the vine, for example shoots tip, shoot, leaf, bunch, berry etc. [34].For basic and relatively fast description of the varieties, the so called the Primary Descriptor list of OIV codes for Grapevine Varieties and Species Vitis was formed [34], which directs a description of 14 grapevine characteristics regarding shoots, leaves, bunches and berries.The OIV descriptors, for some leaf characteristics, overlap with phyllometric parameters: OIV 601 = L1, OIV 602 = L2, OIV 603 = L3, OIV 604 = L4, OIV 605 = OS, OIV 606 = OI, OIV 607 = α, OIV 608 = β, OIV 609 = γ, OIV 612 = h1, OIV 613 = b1, OIV 614 = h2, OIV 615 = b2, OIV 618 = l (Figure 7), therefore a collective use of both methodologies upgrades the varietal description.In few studies we used a different number of OIV codes, what depends especially on the condition of the grapevine, environmental conditions and the scope of the study.
The aim of the first study was the evaluation of selected OIV codes for grapevine description and discrimination -if the selected OIV code descriptorsare enough informative for a precise varietal discrimination?We sampled from 10 to 12 fully developed leaves from the sixth to ninth node of the shoot per certain variety.For the ampelographic description, shoot tips were sampled in the earlier phenological stages and berries were observed during the whole ripening period.The description and measurements of the particular parts of the vine were conducted according to the instructions of the individual descriptive codes.The Mediterranean Genetic Code -Grapevine and Olive According to the obtained data (Figure 10) and to the already acquired knowledge of the studied varieties some expected results revealed -for example similar OIV codes between accessions 'Refošk' and 'Teran Istra', frequently used as synonyms; or 'Pokalca' and 'Syrah' especially because of shape of the leaves berries.On the other hand the applied OIV codes revealed some imprecisions as well, for example similarities between varieties 'Merlot' and 'Cabernet sauvignon'.Therefore we suggest that the grouping according to the OIV code descriptors should be done and interpreted carefully, as many misunderstandingscould be provoked.For a comparison and interpretation of the studied accessions we suggest that the reference grapevine varieties should be included always, and as many as possible (also with greater differences in characteristics).
The comparison of the ampelographic descriptions, morphometric and phyllometric characteristics among the 'Vitovska grganja' (VG), 'Vitovska grganija' (VGI), 'Vitouska' (V), 'Grganja' (G) and 'Maraština' (M) varieties is presented in Table 5.The ampelographic descriptions of the VGI, V and G varieties highly differed from the VG and M varieties [18].Similarities between VG and M varieties were observed in the characteristics of young and mature shoots, the opening shoot tips showed similar anthocyanin coloration and a similar intensity and coloration of prostrate hairs.Similarities between VGI and M varieties have to be underlined, especially in the size of young and mature leaves, dark green coloured upper side of the blade, both convex shaped sides of the teeth.The latter descriptor was in contrast to the report of [45], where both sides were described as straight.Moreover, [45] described the shape of the petiole base sinus of the M variety as 'U'-shaped whereas in [44] it is shown brace-shaped, what is also in accordance with our observations on VG variety.Both M and VG varieties were characterized by high density prostrate hairiness between the main veins on the lower side of the blade; however, hair density differed at other blades/vein parts.Our observations regarding the depth of the upper lateral sinuses of VG variety coincided with those mentioned by [45] for the M variety.In our study, M variety had a lower fertility of basal buds (1-3) compared to VG variety and also to the results reported by [45].
A comparison of bunch and berry characteristics among the studied varieties were also reported [18], what partly explains the reasons of the misuse of the variety nominations due to the rather similar phenotypes.All studied varieties have a cylindrical or conical shape of the bunches, obloid and globose berries, and a bunch length ranging between 160 and 200 mm with the exception of M and VG varieties, where berries are distinctively shorter and narrower.The description of single bunch and berry weight, as well as yield of the M variety reported by [45], coincided with our description for the VG variety.On the other hand, many significant differences were observed among the studied varieties, also between VG and M variety.Compared to M variety, the VG variety blooms later, has thinner berry skin, higher must yield, less firm flesh, shorter length of pedicle, lower sugar and total acidity content and lower specific pH level of the must.Moreover VG also has later bud bursting, but reaches full physiological maturity of the berry earlier.[18] found also some similarities among the studied varieties, specifically in morphometrical characteristics of mature leaves.Significant similarities were observed between our measurements of the VG variety and data cited by [45] for the M variety, which only differed in 'α' and 'β' angles.The comparison of phenotypic characteristics of the studied varieties showed many similarities, which suggests that a consistent attention to the ampelographic distinguishing among varieties, especially among those with the same designations or synonyms, has to be emphasized in the future.

Conclusions
The grapevine genepool is particularly vulnerable in the marginal areas of its distribution range.Many grapevine varieties have been already described and their genotypes determined; but many local grapevine accessions remain unidentified and their ampelographic characteristics overlooked.Accurate ampelographic description of variety should be done with combined methodologies which involve morphological description and DNA analyses.In the last decade a great effort was given to the DNA analyses -genotyping of the grapevine varieties, and quite soon revealed that DNA analyses is simply not enough to answer all questions about varietal discrimination and description.Parallel to the development of genetic techniques, the morphological and morphometric evaluations have fall into oblivion.Although, microsatellites are very powerful means of identifying synonyms in germplasm collections, they have to be supported with morphological descriptions, what was also demonstrated in our researches.Only such works will allow a characterization of a large number of varieties/accessions and will contribute to improve the organization of grapevine collections and the possibility of exchanging true-to-type material.The presented works are a first step towards true-to-type identification, which will facilitate the registration of varieties and allow growers to be sure of the value of their products.Starting true-to-type variety identification by comparing results with neighbouring, historically linked areas, will significantly clarify the confusion in nomenclature and help determine the origin and relationships among varieties over the whole area.In the future all the obtained results from different studies should be upgraded to the same database for grapevine varieties, to obtain a total overview of World germplasm, what may be the key to stop the erosion of the many varieties.

Figure 8 .
Figure 8. Dendrogram of grapevine accessions linked to the designations 'Glera' and 'Rebula' grouped on the basis of the 28 morphometric characteristics of mature leaves [40].

Figure 9 .
Figure 9. Graphical reconstruction of leaves of studied grapevine varieties according to determined phyllometric characteristics [39].

Table 1 .
List of varietal clones and clone candidates selected in Slovenia.