It was defined that, since ancient times people had engaged with cultivation of new varieties and forms possessed different biological-agricultural traits. This tradition is also being con‐ tinued today. Though the abundance of grape varieties of traditional breeding in Azerbaijan Republic, selection of new, of highly productive varieties, with big berries, with a high bio‐ logically active substances in berries, with a valuable economic characteristics, resistant to pests and diseases, as well as to stressful environmental factors were continued and some results were obtained in this field.

#### **4. Conclusions**

For the purpose of collecting ancient naturally selected varieties and their wild relatives of grape, a number of expeditions were organized in different regions of our Republic, their areal was determined, biological-agricultural traits of collected varieties and forms were evaluated, and for the first time ampelographic descriptions of newly threatened varieties were given and collected varieties were certificated and included in database. Some phyto‐ pathological, immunological and physiological parameters of grape varieties conserved in collection were evaluated and new resistant varieties were selected. Collected materials were included in gene pool, enriching the collections. Taking into account economic efficien‐ cy the growing of above mentioned grape varieties can have great perspectives, not only in Azerbaijan, but also in countries with similar climatic conditions. Therefore conservation of plant genetic diversity of grapes existed in Azerbaijan Republic, selection of productive samples, evaluation and protection are one of the most important problems in modern time.

#### **Author details**

Mirza Musayev\* and Zeynal Akparov

\*Address all correspondence to<EMAIL_ADDRESS>
Laboratory of Subtropical Plants and Grapevine, Genetic Resources Institute of the Azerbai‐ jan National Academy of Sciences, Baku, Azerbaijan

#### **References**

**Figure 9.** Changing the amount of chlorophyll in some grape varieties and forms of stress due to salinity and drought. 1.Gırmızı kishmish., 2.Zerefshan kishmishi., 3.Kishmish Yangiyer., 4.Ruşaki., 5.Polubessemyannıy., 6.İrtişar., 7.Bidane., 8.Vatkana., 9.Belqradskiy bessemyannıy., 10.Kishmish Xişrau., 11.Tezyetishen., 12.Kishmish Terakli., 13. Soqdiana., 14.Sarı kishmish., 15.Oktiyabiskiy., 16.Agh kishmish,. 17.Vishnyovıy., 18.Girde kishmish., 19.Form 21-18-36., 20.Gırmızı turkmen kishmishi., 22.Qara Qushureyi., 23.Kishmish Batır., 24. Samarkand kishmishi., 24.Vatkana-2., 25.Form 1.

It was defined that, since ancient times people had engaged with cultivation of new varieties and forms possessed different biological-agricultural traits. This tradition is also being con‐ tinued today. Though the abundance of grape varieties of traditional breeding in Azerbaijan Republic, selection of new, of highly productive varieties, with big berries, with a high bio‐ logically active substances in berries, with a valuable economic characteristics, resistant to pests and diseases, as well as to stressful environmental factors were continued and some

For the purpose of collecting ancient naturally selected varieties and their wild relatives of grape, a number of expeditions were organized in different regions of our Republic, their areal was determined, biological-agricultural traits of collected varieties and forms were evaluated, and for the first time ampelographic descriptions of newly threatened varieties were given and collected varieties were certificated and included in database. Some phyto‐ pathological, immunological and physiological parameters of grape varieties conserved in collection were evaluated and new resistant varieties were selected. Collected materials were included in gene pool, enriching the collections. Taking into account economic efficien‐ cy the growing of above mentioned grape varieties can have great perspectives, not only in Azerbaijan, but also in countries with similar climatic conditions. Therefore conservation of plant genetic diversity of grapes existed in Azerbaijan Republic, selection of productive samples, evaluation and protection are one of the most important problems in modern time.

results were obtained in this field.

120 The Mediterranean Genetic Code - Grapevine and Olive

**4. Conclusions**

Diversity in Wild Grape Samples (Vitis vinifera L. subsp. sylvestris) from the Cauca‐ sus Region". Materials of XXXIV World Congress of Vine and Wine, Porto, Portugal,.

[28] Akparov, Z, Musayev, M, Mammadov, A, & Salimov, V. (2010). Study of the genetic resources of grapevine in Azerbaijan. *Journal "Agricultural science in Azerbaijan* [1-2], *Baku* [1-2], 40-44, in Azeri.

[12] Makarov, S. N. (1964). Scientific basis of method of experimental work in viticulture.

[13] Morozova, G. (1987). Viticulture with the basics ampelography. M. Kolos, (in Russ),

[14] Prostoserdov, N. (1963). The study of grapes for determining its use. M., Food prom‐

[15] Smirnov, K, Kalmykova, T, Morozova, G, & 1987. Viticulture. M Agropromizdat in

[16] Chisinau, S. (1985). New methods of phytopathological and immunological studies

[17] Kushnirenko, M D, et al. (1976). Methods for assessing plant resistance to unfavora‐

[18] Udovenko, G. (1988). Diagnosis of plant resistance to stress. Leningrad, 22-46, in

[19] Lodhi, M. A, Ye, G, Weeden, N. F, Reisch, B. I, & 1994. A Simple and efficient method for DNA ex-traction from grapevine cultivars and Vitis species. *Plant Molecular Biolo‐*

[20] Schaal, B, Beck, J, Hsu, S, Beridze, C, Gamkrelidze, T, Gogniashvili, M, Pipia, M, Ta‐ bidze, I, This, V, Bacilieri, P, Gotsiridze, R, & Glonti, V. M. (2010). Plastid DNA se‐ quence diversity in a worldwide set of grapevine cultivars (Vitis vinifera L. subsp. vinifera). Abstracts of 10th International Conference on Grapevine Breeding and Ge‐

[21] Beridze, T, Pipia, I, Beck, J, Hsu, S-C, Gamkrelidze, M, Gogniashvili, M, Tabidze, V, This, P, Bacilieri, R, Gotsiridze, V, Glonti, M, & Schaal, B. (2011). Plastid DNA se‐ quence diversity in a worldwide set of grapevine cultivars (Vitis vinifera L. subsp.

[23] Sauer, J. D. (1993). Historical Geography of Crop Plants. CRC Press, Boca. Raton

[24] Jackson, R. S. (1994). Wine Science Principles and Application. Academic Press New

[25] Arroyo-garcia, R, Ruiz-garcia, L, Bolling, L, Ocete, R, Lopez, M. A, Arnold, C, & Er‐ gul, A. (2006). Genetic evidence for multiple centers of grapevine (Vitis vinifera L.)

[27] Pipia, I, Gogniashvili, M, Tabidze, V, Beridze, T, Gamkrelidze, M, Gotsiridze, V, Me‐ liyan, G, Musayev, M, Salimov, V, Beck, J, & Schaal, B. (2011). Plastid DNA Sequence

vinifera). *Bulletin of the Georgian National Academy of Sciences*, 5(1), 91-96.

[22] Negrul, A. (1946). Ampelography of USSR. v.I, Moscow. (in Russ).

[26] Phillips, R. (2000). A Short History of Wine. Harper Collins, New York.

domestication. *Molecular Ecology*, 15, 3707-3714.

*Proceedings, Volume IX. Chisinau: Map Moldovenyaske*, 280, in Russ.

ble environmental conditions. Leningrad: Kolos (in Russ), 87.

251.

izdat, 80, in Russ.

122 The Mediterranean Genetic Code - Grapevine and Olive

in viticulture.(in Russ). 138.

*gy Reporter*, 12, 6-13.

netics., 65, Geneva, New York.

Russ, 367.

Russ.

USA.

York.

### **Portuguese** *Vitis vinifera* **L. Germplasm: Accessing Its Diversity and Strategies for Conservation**

Jorge Cunha, Margarida Teixeira-Santos, João Brazão, Pedro Fevereiro and José Eduardo Eiras-Dias

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52639

#### **1. Introduction**

#### **1.1. Economical, cultural and historical importance of grapevine in Portugal**

Grapevine (*Vitis vinifera* L.) is the most widely cultivated and economically important fruit crop in the world. In the different Portuguese agro-ecosystems, grapevine plays an impor‐ tant role either as a border culture or as an extensive crop. The surface area used by vine‐ yards amounts to 4.9 % of the arable land [1], representing 240,000 ha, being the 7th largest area in the world and the 4th in the European Union [2]. In 2011 Portugal produced 5.9 mil‐ lion hectoliters of which 2.9 million hectoliters were exported, making the country the 12th world wine producer [2]. There are fourteen wine regions with Protected Geographical Indi‐ cation (Figure 1) and 31 wine areas with Designation of Origin status including Porto, estab‐ lished since 1756, the oldest legally established wine production region in the world. Each one of the wine regions has a particular set of grapevine cultivars adapted to its specific *ter‐ roirs*. Officially there are 343 cultivars allowed to be use in wine production in Portugal [3].

Grapes were eaten by Neolithic and Bronze Age populations of the Iberian Peninsula since the 3rd millennium BCE as proven by archaeological remains [4, 5, 6]. Consumption and production of wine is thought to have started by the Iberian populations in contact with the Phoenicians and Greeks trading ports. It further expanded during the Roman occupation and reach important religious prominence with the Christianization of population. It even continued during the Muslim caliphate since part of the population maintain the Christian faith. After the 10th century convents and monasteries spread again grapevine cultivation and implemented new tools for wine production. Since the 12th century, Portugal produces

wine not only for local consumption but also for export, especially to northern Europe. This remote history of grapevine cultivation allowed the building up of great diversity. The num‐ ber of cultivars increased until the tree waves of destruction from North American pest and diseases: powdery mildew (*Uncinula necator* Schweinf. Burrill ) in 1851, phylloxera (*Dactylos‐ phaera vitifoliae* Fitch) in 1863 and downy mildew [*Plasmopara viticola* (Berk. & M.A. Curtis) Berl & de Toni] in 1880. Until these severe pathological events grapevine was multiply sim‐ ply by self-rooting of cutting our seed germination. Since the introduction of phylloxera the use of rootstocks from hybrids of other *Vitis* species is mandatory, except in areas were the phylloxera cannot survive. Such a case occurs in the Designation of Origin Colares wine re‐ gion where the vineyards are settled in sandy soil and the roots are over tree meters deep. As early as the 19th century attempts to improve grape production result in a number of cul‐ tivars as Tinta do Aurélio (red cultivar selected by someone called "Aurélio"). However a truth breeding program to obtain new varieties was only started in the mid of the 20th cen‐ tury by José Leão Ferreira de Almeida and two of the obtain cultivars, Dona Maria (table grape) and Seara Nova (wine grape), occupy today a significant acreage [7]. The exact num‐ ber of cultivars in use is unknown but from the 340 allowed for wine production, 240 are thought to be autochthonous [ 8, 9].

**Figure 1.** Location of the Portuguese wine regions. (Source: Wines of Portugal - http://www.winesofportugal.info/ pagina.php?codNode=18012).

Traditionally morphological descriptors were used to characterize cultivars until the ad‐ vent of molecular markers. Presently these have been successfully used in a wide range of applications such as assessing genetic diversity [10], linkage mapping [11], cultivar identi‐ fication and pedigree studies [12], [13]. Microsatellites (SSR) are being used to character‐ ize grapevine cultivars and wild vines [10, 14] and to carry out genetic diversity analyses [15]. Usually six *loci* are sufficient for differentiating between genotypes [16], but closely related cultivars require a larger number of *loci* [17]. Sequence variation at the chloroplas‐ tidial *loci* has been extensively used to assess phylogenetic relationships among plant *taxa*, based on their low rate of sequence evolution, the almost absent recombination and sin‐ gle parent inheritance [18]. All this range of tools is useful to make decisions on the strat‐ egies for conservation.

#### **2. Diversity of the Portuguese grape germplasm**

wine not only for local consumption but also for export, especially to northern Europe. This remote history of grapevine cultivation allowed the building up of great diversity. The num‐ ber of cultivars increased until the tree waves of destruction from North American pest and diseases: powdery mildew (*Uncinula necator* Schweinf. Burrill ) in 1851, phylloxera (*Dactylos‐ phaera vitifoliae* Fitch) in 1863 and downy mildew [*Plasmopara viticola* (Berk. & M.A. Curtis) Berl & de Toni] in 1880. Until these severe pathological events grapevine was multiply sim‐ ply by self-rooting of cutting our seed germination. Since the introduction of phylloxera the use of rootstocks from hybrids of other *Vitis* species is mandatory, except in areas were the phylloxera cannot survive. Such a case occurs in the Designation of Origin Colares wine re‐ gion where the vineyards are settled in sandy soil and the roots are over tree meters deep. As early as the 19th century attempts to improve grape production result in a number of cul‐ tivars as Tinta do Aurélio (red cultivar selected by someone called "Aurélio"). However a truth breeding program to obtain new varieties was only started in the mid of the 20th cen‐ tury by José Leão Ferreira de Almeida and two of the obtain cultivars, Dona Maria (table grape) and Seara Nova (wine grape), occupy today a significant acreage [7]. The exact num‐ ber of cultivars in use is unknown but from the 340 allowed for wine production, 240 are

**Figure 1.** Location of the Portuguese wine regions. (Source: Wines of Portugal - http://www.winesofportugal.info/

thought to be autochthonous [ 8, 9].

126 The Mediterranean Genetic Code - Grapevine and Olive

pagina.php?codNode=18012).

#### **2.1. Wild vine populations: Geographical distribution, morphological and molecular characterization**

Wild vine populations of *Vitis vinifera* L. subspecies *sylvestris* [(Gmelin) Hegi)] is closely re‐ lated to the cultivated grapevine (*Vitis vinifera* subsp. *vinifera*), first domesticated 10,000 years BP around the Caspian Sea [19]. In Portugal these wild vine populations are distribut‐ ed along riparian woods and flooded river banks in the southern part of the country in what is the most western habitats of this subspecies. From the Atlantic coasts of southwest Europe and northwest Africa this subspecies is distributed in patches adjacent to rivers along the Mediterranean basin, Central Europe and in Asia between the Black Sea and the Hindu Kush [20]. Once this subspecies occupied a larger area as a result of the its expansion after the last Quaternary glaciations [21, 22] but today´s remaining areas are refuges from human pressure and North-American pest and diseases introduced during the 19th century. Hu‐ man populations since the early settlements in the Iberian Peninsula collected and con‐ sumed wild grapes [6] and this resource continued to be used until the late 20th century in folk medicine [20].

The wild vine populations found up to now in Portugal live in riparian woods along small streams (Figure 2) belonging to three large river basins – Tagus (Tejo in Portu‐ guese), Guadiana and Sado (Table 1). The first two rivers are common to Portugal and Spain and the populations along these basins, even if found in patches, could be consid‐ ered as a continuum [23, 24].

In these riparian woods the plants species most frequently found as tutors of V*itis vinifera* L. ssp. *sylvestris* are: *Adenocarpus complicatus*, *Alnus glutinosa*, *Fraxinus angustifolia*, *Nerium ole‐ ander*, *Olea europea*, *Quercus faginea* subsp. *Broteroi*, *Quercus suber*, *Rubus ulmifolius*, *Salix atroc‐ inerea*, *Salix neotricha* and *Salix salvifolia* subsp. *salvifolia* [23, 25] . The thirteen populations found until now (Table 1) thrive in a typically Mediterranean environment. Fifty three plants belonging to four of these populations were characterized morphologically using the OIV [26] and GENRES-081 [27] descriptors [23, 28, 29].

**Figure 2.** *Vitis vinifera* subspecies *sylvestris* male plant from the São José/ Toutalga population in its natural habitat, a riparian forest along a small stream from the Guadiana river basin.

**Table 1.** *Vitis vinifera* ssp. *sylvestris* Portuguese populations data: River basin; geographic coordinates, elevation (in meters) estimated size of the population, and risk of extinction.

2

The characterized wild vine plants featured the particularly morphological characteristics of the subspecies *sylvestris*: i) open young shoots, which is a characteristic allowing to dif‐ ferentiate between *Vitis vinifera* and the other *Vitis* species and hybrids; ii) the presence of male and female plants in each population (dioecious plants) (hermaphrodite plants are rare in wild vine populations and the rule in cultivated grapevines); iii) Stummer's Index (breadth/length ratio x 100) [30] of pips is equal or greater than 75 in wild vines. The morphological characteristics of the leaves, shoots and bunches were used to distinguish different phenotypes in the field. Until now only blue black berries were found and the ratio of male to female plants varies from population to population [28]. The 53 different wild vine accessions collected were genotyped using the six nuclear microsatellites sug‐ gested by the OIV [31, 32]. The diversity founded in wild vine genotypes (Table 2) re‐ veals that the observed Heterozigocity (Ho) was less than the expected Heterozigocity (He) in all *loci*, confirming the result obtain in a different group of accessions from the same populations using a set of 11 SSRs [33] .

**Table 2.** Diversity obtained in 53 Portuguese wild vines: *locus*, accessions number (*N*), number of alleles (*Na*), number of effective alleles (*Ne*), observed Heterozygosity (Ho), expected Heterozygosity (*He*) and Fixation Index (*F*).

**Figure 2.** *Vitis vinifera* subspecies *sylvestris* male plant from the São José/ Toutalga population in its natural habitat, a

Série, Nº 226.

**Code Latitude Longitude Elevation**

[3] Portaria nº 380/2012, de 22 de Novembro. Diário da Republica. 1a

**(meters)**

**Estimated size of the population**

**PopRisk**

Série, Nº

**Reference**

PopRisk (survival risk of the population ): 1= No Risk; 3= Some Risk; 5= Medium Risk; 7= At Risk; 9= Extinct

**Table 1.** *Vitis vinifera* ssp. *sylvestris* Portuguese populations data: River basin; geographic coordinates, elevation (in

Sta Sofia - Montemor-o-Novo Tagus 01\*a 38°36'41''N 08°05'24''W 306 [30-40] 3 Pônsul - Castelo Branco Tagus 02\*a 39°45'16''N 07°26'06''W 119 [30-40] 7 Guadiana - Mourão Guadiana 03a 38°24'10''N 07°22'36''W 128 0 9 Vale do Guiso - Alcácer do Sal Sado 04\*a 38°14'46''N 08°22'30''W 49 [10-20] 3 Portel Guadiana 05\* 38°16'46''N 07°38'07''W 197 [20-30] 7 Ardila - Barrancos Guadiana 06 38°07'56''N 06°57'41''W 208 [20-30] 5 Vendinha - Évora Guadiana 07 38°27'18''N 07°41'02''W 163 [10-20] 5 Pintada - Montemor-o-Novo Tagus 08 38°37'59''N 08°11'31''W 204 [10-20] 5 Fronteira Tagus 09 39°02'38''N 07°42'14''W 93 [10-20] 5 Anta do Silval - Évora Tagus 10 38°36'45''N 08°03'29''W 292 <10 5 Q. do Pinheiro - Montemor-o-Novo Tagus 11 38°37'58''N 08°10'31''W 234 [20-30] 5 S.José/Toutalga - Moura Guadiana 12 38°02'37''N 07°15'54''W 176 [20-30] 5 Enxota tordos - Grândola Sado 13 38°13'27''N 08°30'22''W 34 >50 3

2

riparian forest along a small stream from the Guadiana river basin.

**basin**

\* Wild populations studied by [28, 29]/ a Wild populations studied by [33]

meters) estimated size of the population, and risk of extinction.

**17**1 [3] Portaria nº, 428/2000 de 17 de Julho. Diário da Republica. 1a

128 The Mediterranean Genetic Code - Grapevine and Olive

163.

New Table 1 (see below)

**Population River**

The values of the Fixation Index (F) range from 0.005 to 0.28, showing the existence of in‐ breeding in some wild vine populations, since F is expected to be close to zero under ran‐ dom mating [34].

An Analysis of Molecular Variance (AMOVA) performed on the same molecular data showed that the genetic diversity was attributable to differences among individuals within populations (93.0%), but Fst values among populations are still significant (*Fst* = 0.071; P, 0.001), showing a low inter-population differentiation (Table 3). The morphological and mo‐ lecular data confirmed that some of the collected plants were clones due to vegetative prop‐ agation (asexual propagation), but that the majority were different genotypes arising from seeds (sexual propagation).

Chloroplastidial microsatellites (cpSSRs) have been used to study the genetic relationships among grapevine cultivars [35], wild vines [36] and relations between both subspecies [37, 38 ]. Analysis of chloropastidial microsatellites (Figure 3) revealed the expected situation for the Iberian Peninsula [37] with the presence of chlorotypes A and B, being chlorotype A the most frequent within the wild vine populations (66%) of Portugal.

**Table 3. A**MOVA analyses of six nuclear microsatellites data of 53 Portuguese wild vines on four distinct Southern Portuguese populations.

**Figure 3.** Chlorotypes identified in each Portuguese wild vine population. Chlorotype nomination according to [37].

Chlorotype A is the most frequent in Western Europe and absent in Near East where the do‐ mestication of *Vitis vinifera* occurred. The distribution of chlorotypes in four Southern Portu‐ guese populations is heterogeneous. Only chlorotype A was found in plants of the population of Sta Sofia – Montemor-o-Novo. In the populations of Vale do Guiso - Alcácer do Sal, Pônsul – Castelo Branco and Portel both A and B chlorotypes were found but with distributions of 91.6%, 18% and 62.5% of chlorotype B respectively (Figure 3).

#### **2.2. Cultivated grapevine: Morphological and molecular diversity**

**Variance component Degrees of**

Portuguese populations.

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

100

0

130 The Mediterranean Genetic Code - Grapevine and Olive

**freedom**

**Total** 105 215.1 2.09

82

18

**Among Populations** 3 17.0 0.15 7% **Within Populations** 102 198.1 1.94 93%

Fixation index (Fst) 0.071 (P<0.001)

**Table 3. A**MOVA analyses of six nuclear microsatellites data of 53 Portuguese wild vines on four distinct Southern

**Sum of Squares**

8.4

Pop 01 Pop 02 Pop 04 Pop 05 TOTAL Chlorotype A Chlorotype B

**Figure 3.** Chlorotypes identified in each Portuguese wild vine population. Chlorotype nomination according to [37].

Chlorotype A is the most frequent in Western Europe and absent in Near East where the do‐ mestication of *Vitis vinifera* occurred. The distribution of chlorotypes in four Southern Portu‐ guese populations is heterogeneous. Only chlorotype A was found in plants of the population of Sta Sofia – Montemor-o-Novo. In the populations of Vale do Guiso - Alcácer

91.6

37.5

62.5

66

34

**Variance components**

**Percentage of variation**

> Portugal, a small country on the outer edge of Europe, has nonetheless a very rich diversity of grapevine cultivars build up over the centuries and back to the 19th century, 1482 differ‐ ent cultivar names were known. To organize the disarray that the different names caused to the wine sector the Ministry of Agriculture promoted a program to sort out the synonyms and homonyms using morphological descriptions [39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49]. Before Portugal joined the EEC (European Economic Community) in 1986, the Ministry of Agriculture finally drew up a list of "authorized" and "recommended" grapevine cultivars for each and every wine production areas (Figure 1). These efforts lead to the establishment of the Portuguese National Ampelografic Collection (in Portuguese "Coleção Ampelográfica Nacional" – CAN; international code PRT051) in 1988 after an extensive survey and collec‐ tion of accessions all over the country. All CAN accessions were grafted into SO4 rootstock and each access is represented by seven plants from the same original mother plant. This collection holds 691 accessions of *Vitis vinifera* ssp. *vinifera*; 30 accessions of *Vitis vinifera* ssp. *sylvestris*; 24 accessions of rootstocks and nine of other *Vitis* species. The sanitary status of the collection was also assessed for the principal viruses of grapevine (*Arabis* mosaic virus (ArMV), grapevine fanleaf virus (GFLV), grapevine fleck virus (GFKV), grapevine leafroll associated viruses 1, 2, 3 and 7 (GLRaV 1, 2, 3 and 7) grapevine virus A (GVA) and grape‐ vine virus B (GVB) [50].

> The molecular characterization of the Portuguese grapevine cultivars was initiated in 1999 by Lopes and collaborators and a number of known synonyms and homonyms as well as pedigrees were confirmed [51, 52, 53]. A systematic characterization of all the 340 varieties admitted for wine production in Portugal, including 243 autochthonous grape cultivars (Ta‐ ble 4) was done with the six nuclear SSRs recommended by OIV [ 8, 9]. These studies come to prove the synonyms and homonyms that previous morphologic description had estab‐ lished in the past and also allowed the finding out of new ones.

> The diversity present in the 243 autochthonous grapevine cultivars analyzed based on the six nuclear SSRs genetic markers (Table 5) reveals that the observed Heterozigocity (*Ho*) was slightly higher than the expected Heterozigocity (*He*) in all *loci*. The Fixation Index (*F*) is negative for all *loci*, indicating an excess of Heterozigocity, probably due to the strong barri‐ er caused by the vegetative propagation commonly used in grapevine.

> Four chlorotypes (A, B, C and D) were found in the autochthonous grapevine cultivars so far genotyped (roughly one quarter of the 243) (Figure 4). Chlorotype A is the most frequent, and it is present in 75% of the cultivars, followed by chlorotype D with 19%. Chlorotypes B and C are each present in a very restricted number of cultivars [29, 32, 37]. These results support the presumption that most of the Portuguese cultivated grapevine germplasm may have derived from local domestication, but that some are the result of introgressed with for‐ eign material as exemplified by important wine cultivars like Touriga Franca and Trinca‐ deira that show the presence of the D chlorotype.

**Access**

*Continued*

**number Grape cultivar Origin**

The Mediterranean Genetic Code - Grapevine and Olive

**Access**

 Seara Nova E.A.N. 41703 Malvasia Preta Roxa Douro. Assaraky E.A.N. 41705 Roxo de Vila Flor R Douro. Promissão Douro. 41702 Gouveio Roxo Douro. 40502 Branco Valente B Douro. 41707 Deliciosa E.A.N. Sercial Madeira. 41708 Bastardo Roxo Douro. 40603 Malvasia Babosa B Madeira. 41709 Donzelinho Roxo Douro. Malvasia São Jorge Madeira. 41806 Campanário E.A.N. Granho Alentejo. 50104 Ferral unknown Tinta Aurélio Douro. 50201 Complexa E.A.N. 40701 Alvarinho Lilaz B E.A.N. 50216 Terrantez do Pico Pico - Açores. Castália E.A.N. 50218 Arintaçor Terceira - Açores.

 Naia E.A.N. 50309 Castelo Branco E.A.N. 40704 Malvasia de Oeiras B E.A.N. 50314 Branca de Anadia E.A.N. Cornichon Alentejo. 50317 Verdelho Açores. Generosa E.A.N. 50602 Tinta Martins Douro. Rio Grande E.A.N. 50604 Tinta Mesquita Douro. Pé Comprido Douro. 50605 Português Azul Douro. Esganinho Vinhos Verdes. 50607 Tinta Gorda N Douro. Branco Gouvães Douro. 50608 Tinta Malandra N Douro. Branco Desconhecido Douro. 50611 Lameiro Vinhos Verdes. Branjo Vinhos Verdes. 50615 Água Santa E.A.N. Galego Vinhos Verdes. 50616 Gouveio Real Douro. Labrusco Vinhos Verdes. 50617 Gouveio Estimado Douro. Melhorio Vinhos Verdes. 50702 Mondet Douro. Transâncora Vinhos Verdes. 50703 Tinta Aguiar Douro. Verdial Tinto Douro. 50705 Touriga Fêmea Douro. Alvarelhão Ceitão Douro. 50706 Tinta Miúda de Fontes N Douro. Moscatel Galego Tinto Douro. 50707 Tinta Roseira N Douro. 41302 Barreto de Semente T Douro. 50708 Lourela Douro. Casteloa Douro. 50802 Gonçalo Pires Douro. Farinheira Douro. 50806 Padeiro de Basto N Vinhos Verdes. Gouveio Preto Douro. 50807 Tinta Pomar Douro. Mourisco de Trevões Douro. 50808 Tinta Varejoa N Douro. 41309 Tinta Melra T Douro. 50901 Casculho Douro. 41502 Alentejana N E.A.N. 50902 Concieira Douro. Lusitano E.A.N. 50904 Doçal Vinhos Verdes. 41504 Tinta de Alcobaça N E.A.N. 50905 Doçal de Refoios N Douro. Agronómica E.A.N. 50907 Tinta Pereira Douro. 41508 Portalegre N E.A.N. 50909 Malvasia Trigueira R Douro. Triunfo E.A.N. 50912 Malvasia Branca Açores. 41601 Monvedro de Sines N Sines 50914 Caracol Madeira. Manteúdo Preto Alentejo. 50915 Esganoso Vinhos Verdes. Listrão Madeira. 50916 Mourisco Branco Douro. Mindelo E.A.N. 50917 Rabigato Moreno Douro.

**number Grape cultivar Origin**

**Table 4.** Autochthonous grapevine cultivars used in wine production in Portugal: Access number in the PRT051 collection, name of the grapevine cultivar, origin of grapevine accession.

Portuguese *Vitis vinifera* L. Germplasm: Accessing Its Diversity and Strategies for Conservation http://dx.doi.org/10.5772/52639 135

 Santareno Douro. 52908 Amaral Vinhos Verdes. Donzelinho Tinto Douro. 52913 Galego Dourado Estremadura Donzelinho Branco Douro. 53006 Trincadeira Douro. 52309 Boal Ratinho B Estremadura 53013 Malvasia Rei Douro. Avesso Vinhos Verdes. 53015 Moscatel Nunes Setúbal. Arinto Bucelas. 53102 Primavera E.A.N. Almafra Estremadura 53103 Cabinda E.A.N. Fonte Cal Beira Interior. 53106 Castelão Ribatejo. Antão Vaz Alentejo. 53204 Amostrinha Estremadura Camarate Estremadura 53205 Malvasia Preta Douro. Barcelo Dão. 53206 Valbom E.A.N. Cerceal Branco Douro. 53207 Alvarelhão Dão. Cercial Bairrada. 53307 Tinto Cão Douro. Tinta Francisca Douro. 53308 Malvarisco Setúbal. Jaen Dão. 53312 Marquinhas E.A.N. 52505 Benfica N E.A.N. 53407 Mulata E.A.N. Tinto Pegões E.A.N. 53806 Roal Setúbal. Batoca Vinhos Verdes. 53807 Teinturier Estremadura

134 The Mediterranean Genetic Code - Grapevine and Olive

 Malvasia Fina Douro. 54006 Almenhaca \* Diagalves Estremadura 54007 Alvar \* Jampal Estremadura 54008 Alvar Roxo \* Carrasquenho Estremadura 54009 Arinto Roxo \* Baga Bairrada. 54010 Boal Barreiro \* Malvasia Fina Roxa Dão. 54011 Branco João \* Vital Estremadura 54012 Cainho \* Castelão Branco Estremadura 54013 Calrão \* Parreira Matias Estremadura 54015 Corval \* Preto Cardana Ribatejo. 54016 Crato Espanhol \* Castelino Estremadura 54017 Esgana Cão Tinto \* Folgasão Roxo Beira Interior. 54018 Galego Rosado \* Folgasão Douro. 54019 Leira \* Trajadura Vinhos Verdes. 54020 Malvasia Romana \* Malvasia Estremadura 54021 Malvia \* Viosinho Douro. 54022 Perigó \* Bastardo Douro. 54023 Pero Pinhão \* Borraçal Vinhos Verdes. 54025 Pexem \* Azal Vinhos Verdes. 54026 Rabo de Lobo \* Fernão Pires Bairrada. 54027 Santoal \* Fernão Pires Rosado Ribatejo. 54028 Zé do Telheiro \* Carrega Burros Ribatejo. 54029 Tinta \* Rabo de Anho Vinhos Verdes. 54030 Tinto Sem Nome \* Espadeiro Vinhos Verdes. 54031 Valveirinho \* Tinta Barroca Douro. 54032 Verdial Branco \* Tinta Grossa Alentejo. 54033 Xara \*

**Table 4.** Autochthonous grapevine cultivars used in wine production in Portugal: Access number in the PRT051

collection, name of the grapevine cultivar, origin of grapevine accession.

\* Recent Introduction in PRT051

**Table 5.** Analyses of diversity in 243 Portuguese autochthonous cultivars: *locus*, accessions (*N*), number of alleles (*Na*), number of effective alleles (*Ne*), observed Heterozygosity (*Ho*), expected Heterozygosity (*He*) and Fixation Index (*F*).

**Figure 4.** Chlorotypes of the Portuguese autochthonous grapevine cultivars. Chlorotype nomination according to [37].

The obtained results reinforce the suggestion that the Iberian Peninsula was a secondary center for grapevine domestication [37] despite the initial contribution of the Eastern gene pool some 3000 years ago and the more recent introgression from materials coming from central Europe.

Since 1978 a network of public and private associations lead by Antero Martins carried out an extensive work aiming at quantifying the intravarietal genetic variability within each of 45 Portuguese grapevine cultivars [54]. The static methods used were recently reviewed in [55]. These studies lead to the selection of a number of clones from Portuguese cultivars. In parallel and using the Geisenheim method of grapevine selection, a private nursery leaded by Jorge Böhm also selected a number of clones. Both groups registered a total of 122 clones from 27 different cultivars in the national grapevine catalogue (Table 6).

 Plansel/ JBP - Plansel (Wine and Nursery Company) / Jorge Böhm Plansel UTL/ISA – Universidade Técnica de Lisboa / Instituto Superior de Agronomia INIAV/ EAN – Instituto Nacional de Investigação Agrária e Veterinária/ Estação Agronómica Nacional

**Table 6.** List of the certified Portuguese clones of grapevine cultivars.

#### **2.3. Overall diversity of the Portuguese grapevine germplasm**

Plansel UTL INIAV clones JBP clones ISA clones EAN

Obtainers

Aragonez T 106; 110; 111; 114; 117 54; 55; 56; 57; 58; 59; 60 Arinto B 34; 35; 107 36; 37; 38; 39; 40

Fernão Pires B 1 68; 69; 70; 71; 72; 73; 74

Síria B 75; 76; 77; 78; 79; 80

Trincadeira T 6; 7; 8; 109 10; 11; 12; 13; 14; 15

INIAV/ EAN – Instituto Nacional de Investigação Agrária e Veterinária/ Estação Agronómica

Castelão T 5; 25; 26 29; 30; 31; 32; 33

Alfrocheiro T 41

136 The Mediterranean Genetic Code - Grapevine and Olive

Variety

Antão Vaz B 50

Bastardo T 48 Bical B 119

Cerceal Branco B 120

Moreto T 51 Perrum B 128 Sercial B 49; 105

Tinta Barroca T 9; 129 Tinta Caiada T 115; 116; 118

Trincadeira das Pratas B 124; 125; 126

Touriga Franca T 24

Viosinho B 53 PE 1103 P 4 PE 110 R 2 PE 140 Ru 113 PE 99 R 3 3

Nacional

Gouveio B 121; 122; 123

Alvarinho B 42; 43 44; 45; 46; 47

Jaen T 91; 92; 93; 94; 95; 96; 97 Loureiro B 81; 82; 83; 84; 85

Touriga Nacional T 16; 108; 112 17; 18; 19; 20; 21; 22; 23 Trajadura B 86; 87; 88; 89; 90

Vinhão T 61; 62; 63; 64; 65; 66; 67

 Plansel/ JBP - Plansel (Wine and Nursery Company) / Jorge Böhm Plansel UTL/ISA – Universidade Técnica de Lisboa / Instituto Superior de Agronomia

**Table 6.** List of the certified Portuguese clones of grapevine cultivars.

Malvasia Fina B 127 98; 99; 100; 101; 102; 103; 104

Portuguese wild vine populations are in an apparent geographic fringe of the species distribu‐ tion but the country richness in cultivar diversity [8, 9] and the importance in allele contribu‐ tion to the overall diversity of grapevine [56] tell another story. Figure 5 represents a Principal Coordinate Analysis of the diversity computed with the six nuclear SSRs used to genotype the 243 autochthonous cultivars and 53 wild vines, calculated with the program GenAlex6 [57] The two first coordinates represent 44.12% (1st coordinate - 24.08% and 2nd coordinate - 20.04%) of the total variance. Both subspecies are spread between the four quadrants although most wild vines are in the right quadrants. Even the plausible occurrence of feral forms cannot explain the overall dotting of the four quadrants since the alleles found in the wild vines population in‐ clude private and particular alleles (data from [32]). When a Multiple Discriminant Analysis was used to assign the accessions to the different wild vine populations or to the cultivated group, most plants were correctly assigned and only three wild vines were assigned to the *vin‐ ifera* subspecies. On the other hand eight cultivars were assigned to the *sylvestris* subspecies [58]. This seems to corroborate the assumption that the part of the Portuguese germplasm was locally domesticated and contributes to the hypothesis that the Iberian Peninsula has been a secondary center for grapevine domestication [37].

**Figure 5.** Scatter plot of a Principal Coordinate Analysis of six microsatellite loci from 243 Portuguese grapevine culti‐ vars (GC, in green) and 53 wild vines (WV, in red) from four Portuguese populations.

#### **3. The present situation of germplasm conservation in Portugal**

Different strategies are needed to preserve the germplasm of the two grapevine subspecies. One obvious strategy is to maintain the natural habitats where the wild vines are present and keep them subjected to the selection pressures of the natural environment. For the culti‐ vated subspecies the ideal situations should be maintaining the agro-systems where its di‐ versity was buildup. However these *in situ* dynamic strategies must be accompanied by more static *ex situ* strategies, since natural habitats undergo a number of hazards and even the risk of disappearance, and today's commercial agro-systems tend to rely in a very small number of genotypes. Knowledge of the available diversity by multiple tools as reported above is the first step to decide on the strategies of conservation.

*In situ* conservation of wild vines populations is the leading choice to be considerate. There are a number of different problems that arise from this option: the land ownership where the plants subsist; the legal protection status of the subspecies; natural hazards, like fire; hazards caused by humans, like brutal cleaning of river banks; etc. Most of the populations are located in private owned land even when situated in areas where there is some kind of legal environment protection (populations 02 and 12). The first approach is to contact the land owner and explain the importance of wild vine populations and of the riparian habi‐ tats. In Portugal all contacted owners were willing to cooperate in the process of preserving the populations and some were even enthusiastic. Any major occurrence is usually reported like river bank cleaning or fire. Another important action is to contact the municipal authori‐ ties responsible for stream cleaning in order to adjust their actions to protect the riparian habitat. A good outcome of this policy was the case when the area where the population 04 inhabits was clean under the supervision of trained staff. Despite the positive results of these approaches some situations prove to be out of hand like the building of a dam, floods and fire. Population 03 was destroyed due to the construction of the Alqueva dam and part of population 12 was uprooted due to severe flooding. Populations 02 suffered a major fire in its habitat although with little loss in the total number of plants that recovered subse‐ quently. To prevent the loss of the existing diversity an *ex situ* collection was started in 2005 at the CAN location (PRT051) with thirty wild vine accessions from three populations. Plants from other populations have been added to this collection.

Even though some European countries like France and Germany have a legal protection sta‐ tus for the subspecies *sylvestris*, in Portugal no such protection exists. An formal require‐ ment was sent to the Portuguese agency for wildlife protection to establish a similar protected status for the Portuguese populations of *Vitis vinifera* subspecies *sylvestris* based on the information described in the previous sections.

Until the middle of the 20th century, most Portuguese farmers used to grow a mixture of vine cultivars as a way to overcome the effects of biotic and abiotic stresses but this situation was became increasingly rare and the vineyards are now mostly monovarietal. Nevertheless a recent report on *in farm* conservation, still found a considerable diversity in cultivated vineyards [59]. This is particularly observed when there is a weak relationship between the owner and the wine market, and a farm agro-ecological heterogeneity [59]. Today world‐ wide viticulture relies in a very restricted number of cultivars an even in a country like Por‐ tugal that has not abandoned its autochthonous cultivars, only 25 cultivars are planted in 80% of the new vineyards. The majority of the ancient cultivars is thus neglected and needs to be preserved *ex situ*.

*Ex situ* collection of grapevine cultivars were settled initially in the 19th century after the ar‐ rival in Europe of *Dactylosphaera vitifoli*ae in order to be post philoxera repositories of local cultivars. Today two types of collections exist in Portugal: typical ampelographic collections (Table 7) and collections with a large number of different accessions of the same cultivar. These later were established as a result of a grapevine selection group network leaded by Antero Martins and today managed by PORVID - a public/private consortium. The method‐ ology used to establish these collections was recently reviewed in [55].

and keep them subjected to the selection pressures of the natural environment. For the culti‐ vated subspecies the ideal situations should be maintaining the agro-systems where its di‐ versity was buildup. However these *in situ* dynamic strategies must be accompanied by more static *ex situ* strategies, since natural habitats undergo a number of hazards and even the risk of disappearance, and today's commercial agro-systems tend to rely in a very small number of genotypes. Knowledge of the available diversity by multiple tools as reported

*In situ* conservation of wild vines populations is the leading choice to be considerate. There are a number of different problems that arise from this option: the land ownership where the plants subsist; the legal protection status of the subspecies; natural hazards, like fire; hazards caused by humans, like brutal cleaning of river banks; etc. Most of the populations are located in private owned land even when situated in areas where there is some kind of legal environment protection (populations 02 and 12). The first approach is to contact the land owner and explain the importance of wild vine populations and of the riparian habi‐ tats. In Portugal all contacted owners were willing to cooperate in the process of preserving the populations and some were even enthusiastic. Any major occurrence is usually reported like river bank cleaning or fire. Another important action is to contact the municipal authori‐ ties responsible for stream cleaning in order to adjust their actions to protect the riparian habitat. A good outcome of this policy was the case when the area where the population 04 inhabits was clean under the supervision of trained staff. Despite the positive results of these approaches some situations prove to be out of hand like the building of a dam, floods and fire. Population 03 was destroyed due to the construction of the Alqueva dam and part of population 12 was uprooted due to severe flooding. Populations 02 suffered a major fire in its habitat although with little loss in the total number of plants that recovered subse‐ quently. To prevent the loss of the existing diversity an *ex situ* collection was started in 2005 at the CAN location (PRT051) with thirty wild vine accessions from three populations.

Even though some European countries like France and Germany have a legal protection sta‐ tus for the subspecies *sylvestris*, in Portugal no such protection exists. An formal require‐ ment was sent to the Portuguese agency for wildlife protection to establish a similar protected status for the Portuguese populations of *Vitis vinifera* subspecies *sylvestris* based

Until the middle of the 20th century, most Portuguese farmers used to grow a mixture of vine cultivars as a way to overcome the effects of biotic and abiotic stresses but this situation was became increasingly rare and the vineyards are now mostly monovarietal. Nevertheless a recent report on *in farm* conservation, still found a considerable diversity in cultivated vineyards [59]. This is particularly observed when there is a weak relationship between the owner and the wine market, and a farm agro-ecological heterogeneity [59]. Today world‐ wide viticulture relies in a very restricted number of cultivars an even in a country like Por‐ tugal that has not abandoned its autochthonous cultivars, only 25 cultivars are planted in 80% of the new vineyards. The majority of the ancient cultivars is thus neglected and needs

above is the first step to decide on the strategies of conservation.

138 The Mediterranean Genetic Code - Grapevine and Olive

Plants from other populations have been added to this collection.

on the information described in the previous sections.

to be preserved *ex situ*.

**Table 7.** National and regional public and private ampelographic collections existing today.

The existing collections continue to perform several functions. These functions were initially related to the characterization and identification of cultivars using classic ampelography in‐ cluding: i) standardization of the morphological descriptors of *Vitis*; ii) morphological de‐

3

scription of the cultivars iii) production of illustrate catalogues of cultivars iv) and sorting out synonyms and homonyms. These roles have evolved with the availability of new tools particularly the use of molecular markers that allowed the confirmation of suspected pedi‐ grees and finding unsuspected ones. It also allowed tracing the remote history of grapevine domestication including the existence of several secondary domestication centers. The avail‐ ability in one location of large number of genotypes of a highly heterozygous species also allow the development of genetic association studies like the one developed by Cardoso [60] that establish a candidate gene association with berry colour and anthocyanin content in 149 red and rose grapevine cultivars. Field performance of large numbers of cultivars in one spot as is the case of Esporão collection (Table 7) will help in the decision of what cultivar to plant and how to develop new wine types on the climate change scenario. Finnaly, morpho‐ logical, molecular and field performance data will be useful in establishing core collections aiming a better management of the germplasm available.

#### **Acknowledgements**

This work was funded by: "Fundação para a Ciência e Tecnologia" (SFRH/BPD/ 74895/2010) and "Ministério da Agricultura, do Mar, do Ambiente e do Ordenamento do Territór‐ io" (PRODER - Ação <IP_ADDRESS>. - PA 18621).

#### **Author details**

Jorge Cunha1,2, Margarida Teixeira-Santos3 , João Brazão1 , Pedro Fevereiro2,4 and José Eduardo Eiras-Dias1

3 INIAV, Quinta do Marquês, Oeiras, Portugal

4 Universidade de Lisboa, Faculdade de Ciências, Lisboa, Portugal

#### **References**

[3] Portaria nº, 428/2000 de 17 de Julho. Diário da Republica. 1ª Série, Nº 163.

scription of the cultivars iii) production of illustrate catalogues of cultivars iv) and sorting out synonyms and homonyms. These roles have evolved with the availability of new tools particularly the use of molecular markers that allowed the confirmation of suspected pedi‐ grees and finding unsuspected ones. It also allowed tracing the remote history of grapevine domestication including the existence of several secondary domestication centers. The avail‐ ability in one location of large number of genotypes of a highly heterozygous species also allow the development of genetic association studies like the one developed by Cardoso [60] that establish a candidate gene association with berry colour and anthocyanin content in 149 red and rose grapevine cultivars. Field performance of large numbers of cultivars in one spot as is the case of Esporão collection (Table 7) will help in the decision of what cultivar to plant and how to develop new wine types on the climate change scenario. Finnaly, morpho‐ logical, molecular and field performance data will be useful in establishing core collections

This work was funded by: "Fundação para a Ciência e Tecnologia" (SFRH/BPD/ 74895/2010) and "Ministério da Agricultura, do Mar, do Ambiente e do Ordenamento do Territór‐

, João Brazão1

[1] INE. Recenseamento Agrícola 2009. Análise dos principais resultados. Instituto Na‐

[2] OIV. Statistical report on world vitiviniculture. Paris: International Organisation of Vine and Wine; 2012. http://www.oiv.int/oiv/info/enizmiroivreport (accessed 2 July

, Pedro Fevereiro2,4 and

aiming a better management of the germplasm available.

**Acknowledgements**

**Author details**

**References**

2012).

José Eduardo Eiras-Dias1

io" (PRODER - Ação <IP_ADDRESS>. - PA 18621).

140 The Mediterranean Genetic Code - Grapevine and Olive

Jorge Cunha1,2, Margarida Teixeira-Santos3

1 INIAV, Quinta d'Almoinha, Dois Portos, Portugal

3 INIAV, Quinta do Marquês, Oeiras, Portugal

2 Universidade Nova de Lisboa, ITQB, Oeiras, Portugal

cional de Estatística. Lisboa: I.P. Ed.; 2011.

4 Universidade de Lisboa, Faculdade de Ciências, Lisboa, Portugal

[29] Cunha J, Santos MT, Carneiro LC, Fevereiro P, Eiras-Dias JE. Portuguese traditional grapevine cultivars and wild vines (Vitis vinifera L.) share morphological and genet‐ ic traits. Genetic Resources and Crop Evolution 2009; 56: 975-989.

[15] Aradhya MK, Dangl GS, Prins BH, Boursiquot JM, Walker MA, Meredith CP, Simon CJ. Genetic structure and differentiation in cultivated grape, Vitis vinifera L. Geneti‐

[16] This P, Jung A, Boccacci P, Borrego J, Botta R, Costantini L, Crespan M, Dangl GS, Eisenheld C, Ferreira-Monteiro F, Grando S, Ibañez J, Lacombe T, Laucou V, Magal‐ hães R, Meredith CP, Milani N, Peterlunger E, Regner F, Zulini L, Maul E. Develop‐ ment of a standard set of microsatellite reference alleles for identification of grape

[17] Meredith CP, Bowers JE, Riaz S, Handley V, Bandman EB, Dangl GS. The Identity and Parentage of the Variety Known in California as Petite Sirah. American Journal

[18] Vendramin GG, Lelli L, Rossi P, Morgante M. A set of primers for the amplification of 20 chloroplast microsatellites in Pinaceae. Molecular Ecology 1996; 5: 595–598. [19] McGovern PE. Ancient Wine: The Search for the Origins of Viticulture. New Jersey:

[20] Ocete RR, López Martínez MÁ, Izquierdo MÁP, Moreno TR, Lara BM. Las pobla‐ ciones españolas de vid silvestre. Características de un recurso fitogenético a conser‐

[21] Scossiroli RE. Origine ed evoluzione della vite. Atti dell'Istituto Botànico e del labo‐

[22] Arnold C, Schnitzler A, Douard A, Peter R, Gillet F. Is there a future for wild grape‐ vine (Vitis vinifera subsp silvestris) in the Rhine Valley?. Biodiversity and Conserva‐

[23] Cunha J, Cunha JP, Lousã M, Eiras-Dias JE. Os bosques ribeirinhos, fonte de diversi‐ dade genética de Vitis vinifera L. Ciência e Técnica Vitivinícola / Journal of Viticul‐

[24] Ocete RR. Vitis sylvestris en Iberia.  As Galerias Ribeirinhas na Paisagem Mediter‐ rânica. Reconhecimento na Bacia Hidrográfica do Rio Sado. Lisboa: ISA Press; 1999.

[26] Office International de la Vigne et du Vin. Code des caractères descriptifs des varié‐

[27] GENRES 081. Primary and Secundary descriptor list for grapvine cultivars and spe‐ cies (Vitis L.). Siebeldingen: Institut Fur Rebenzuchtung Geilweilerhof; 1999.

[28] Cunha J, Baleiras-Couto M, Cunha JP, Banza J, Soveral A, Carneiro LC, Eiras-Dias JE. Characterization of Portuguese populations of Vitis vinifera ssp. sylvestris (Gmelin)

Hegi. Genetic Resources and Crop Evolution 2007; 54: 981–988.

Ibérica: História, Terroir, Ampelografia. Lisboa: Dinalivro; 2011 p96-101.

ratorio Crittogámico dell'Universittà di Pavía. Pavia: 1988; 7: 35-55.

cultivars. Theoretical and Applied Genetics 2004; 109: 1448–1458.

of Enology and Viticulture 1999; 50(3): 236-242.

cal Research 2003; 81: 179–192.

142 The Mediterranean Genetic Code - Grapevine and Olive

Princeton University Press; 2003.

var. Madrid: INIA; 1999.

tion 2005; 14: 1507-1523.

ture and Enology 2004; 19: 51–59.

tés et espèces de Vitis. Paris: O.I.V.; 1983.

[53] Magalhães R, Faria MA, Maria dos Santos NM, Eiras-Dias JE, Magalhães N, Mere‐ dith CP, Ferreira Monteiro F. Verifying the Identity and Parentage of Cruzado de Ra‐ bo de Ovelha with Microsatellite Markers. American Journal of Enology and Viticulture 2003; 54: 56-58.

[40] Antunes AA, Costa JF. Catálogo das Castas. Região de Pinhel. Lisboa: Instituto de Gestão e Estruturação Fundiária, Direcção Regional de Agricultura da Beira Interior;

[41] Banza JP. Catálogo das Castas. Região do Alentejo. Lisboa: Instituto de Gestão e Es‐ truturação Fundiária, Direcção Regional de Agricultura do Alentejo; 1986.

[42] Faustino RA. Catálogo das Castas. Região Demarcada do Algarve. Lisboa: Instituto de Gestão e Estruturação Fundiária, Direcção Regional de Agricultura do Algarve;

[43] Mota MT, Silva MF. Catálogo das Castas. Região Demarcada dos Vinhos Verdes. Lis‐ boa: Instituto de Gestão e Estruturação Fundiária, Comissão de Viticultura da Região

[44] Pereira CD, Duarte AP. Catálogo das Castas. Região Demarcada do Dão. Lisboa: In‐

[45] Pereira CD, Sousa AC. Catálogo das Castas. Região Demarcada do Douro. Lisboa: In‐ stituto da Vinha e do Vinho, Centro de Estudos Vitivinicolas do Douro; 1986.

[46] Duarte MTT, Eiras-Dias JE. Catálogo de Porta-enxertos mais utilizados em Portugal. Instituto da Vinha e do Vinho, Centro Nacional de Produção Agricola, Estação Vitivi‐

[47] Vaz JT Catálogo de Castas. Uvas de Mesa cultivadas em Portugal. Instituto de Gestão e Estruturação Fundiária, Direcção-Geral do Planeamento e Agricultura; 1987.

[48] Eiras-Dias JE, Pereira CA, Cunha JP. Catálogo das Castas. Região do Ribatejo, Oeste e Península de Setúbal. Lisboa: Instituto da Vinha e do Vinho, Estacão Vitivinícola Na‐

[49] Rocha ML, Barão AG, Martins JM. Catálogo de Novas Castas de Uva de Mesa obti‐ das na Estação Agronómica Nacional. Lisboa: Instituto da Vinha e do Vinho, Estação

[50] Santos MT, Brazão J, Cunha J, Eiras-Dias JE. Renewing and enlarging and the Portu‐ guese Ampelographic Collection: screening for nine viruses by Elisa. Proceedings of the 17th Congress of the International Council for the Study of Virus and Virus-like Diseases of the Grapevine (ICVG), Davis, California, USA, October 7-14, 2012;

[51] Lopes MS, Sefc KM, Eiras Dias E, Steinkellner H, Laimer da Câmara Machado M, Câ‐ mara Machado A. The use of microsatellites for germplasm management in a Portu‐ guese grapevine collection. Theoretical and Applied Genetics 1999; 99: 733–739.

[52] Lopes MS, Santos MR, Eiras Dias JE, Mendonça D, Câmara Machado A. Discrimina‐ tion of Portuguese grapevines based on microsatellite markers. Journal of Biotechnol‐

1986.

1986.

dos Vinhos Verdes; 1986.

144 The Mediterranean Genetic Code - Grapevine and Olive

nicola Nacional; 1990.

Agronómica Nacional; 1990.

cional; 1988.

272-273.

ogy 2006; 127: 34–44.

stituto de Gestão e Estruturação Fundiária; 1986.

## **Genetic and Phenotypic Diversity and Relations Between Grapevine Varieties: Slovenian Germplasm**

Denis Rusjan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51773

#### **1. 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 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].

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 enumer‐ ated 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 7th to the end of 18th Century; Figure 1), and the varying climatic and geolog‐ ical 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].

© 2013 Rusjan; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Rusjan; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** Territories of the Republic of Venice (697–1797). (http://en.wikipedia.org/wiki/File:Repubblica\_di\_Vene‐ zia.png)

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 Sloven‐ ian 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 vari‐ eties ('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', 'Chardon‐ nay' 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 varie‐ ty 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 addi‐ tionally 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', 'Pine‐ la', 'Šipon', 'Radgonska Ranina', and 'Refošk'. The second group of varieties, such as 'Vitov‐ ska 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 ex‐ ist, 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 system‐ atically collection of these endangered accessions started in 1980. Nowadays, there are five grapevine collections in the whole country: Slap at Vipava, Ampelografski vrt in Krom‐ berk, the grapevine collection at Dobrovo in Goriška brda, Meranovo near Maribor and one near Ormož that include old varieties, clonal candidates and clones (Table1).

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

Nowadays, varieties can be characterized by several methods: (i) by means of a morphologi‐ cal 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 descrip‐ tion based on vegetative and reproductive (ampelography) parts of plants, contributed greatly to the full description of the identities and relationships among *V. vinifera* L. variet‐ ies; 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 meth‐ ods based on DNA polymorphism as outstandingly useful methods to complete the mor‐ phological identification of grapevine varieties [8,9].

#### **2. Slovenian germplasm**

**Figure 1.** Territories of the Republic of Venice (697–1797). (http://en.wikipedia.org/wiki/File:Repubblica\_di\_Vene‐

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 Sloven‐ ian 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 vari‐ eties ('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', 'Chardon‐ nay' 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 varie‐ ty 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 addi‐ tionally 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', 'Pine‐ la', 'Šipon', 'Radgonska Ranina', and 'Refošk'. The second group of varieties, such as 'Vitov‐ ska 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 ex‐ ist, 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 system‐ atically collection of these endangered accessions started in 1980. Nowadays, there are five grapevine collections in the whole country: Slap at Vipava, Ampelografski vrt in Krom‐ berk, the grapevine collection at Dobrovo in Goriška brda, Meranovo near Maribor and one

near Ormož that include old varieties, clonal candidates and clones (Table1).

zia.png)

148 The Mediterranean Genetic Code - Grapevine and Olive

One of the problems in the management of these germplasm collections is the use of syno‐ nymic 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 ampelo‐ graphic 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 ge‐ netic characterization of grapevine varieties (*Vitis vinifera* L.) using microsatellite markers and ampelometric methods has been done by many countries and regions already. The inte‐ gration 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 [4,5,15-18] as well, and have consequently revealed the genetic biodiversity of grapevine varieties grown and cultivated in the country (Table 2).

of *Vitis vinifera* L. have been developed and used for identification purposes [12,13]. The ge‐ netic characterization of grapevine varieties (*Vitis vinifera* L.) using microsatellite markers and ampelometric methods has been done by many countries and regions already. The inte‐ gration 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 [4,5,15-18] as well, and have consequently revealed the genetic biodiversity of

**Variety / Accession Synonym Homonym Origin**

Al

Cipro Al

Borgonja rdeča Al

/ L

Bela glera / Prosseco, Briška glera G Beli teran / Vitovska grganja, Vitouska G Belina Heunish L

Borgonja bela Istrska malvazija Al

Briška glera Glera Bela glera, Glera G

Cividin Cividino bianco Al Cohovka / A Danijela / G Dolga petlja / G Drenik / G Duranja / Al Glera Prosecco, Prosekar / Al Grganc G Guštana Auguštana G

Rusti, Erbalus, Erbalucente, Uva Rustia

omiški, Moscato rosa, Rosenmuskateller blauer, Likvor, Rdeča muškateljka

grapevine varieties grown and cultivated in the country (Table 2).

Bianchera Erbaluce, Albaluce, Albalucent, Bianco

150 The Mediterranean Genetic Code - Grapevine and Olive

Borgonja rdeča Burgonja istarska, Gamay Beaujolais, Borgogna

Cipro Muškat ruža Porečki, Muškat ruža

Istrska malvazija Malvazija, Malvasia d'Istria, Istarska

Borgonja bela

malvasia, Istrijanka, Malvasia Istra,

Kanarjola Canaiolo Al Klarnica Klarnca, Klarna A Laščina G Maločrn Piccola nera, Negra Tenera, Petite Raisin Al

Istrijanka

**Table 2.** Names, designations and classifications of the less known cultivated grapevine varieties/accessions (*Vitis vinifera* L.) in Slovenia. Legend: Al – allochthon; Au – autochthon (landrace); L – local (domestic); G – germplasm (grown only in certain gene banks).

#### **2.1. Genetic diversity and relations among varieties**

SSR markers offer some advantages over other molecular markers, including their co-domi‐ nant inheritance, hypervariability, and high cross-species transferability [8,13]. Consequent‐ ly 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 pro‐ duction 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 dif‐ ferentiate varieties, especially those of similar morphological characteristics; (2) assess genet‐ ic diversity and relationships among them; (3) and compare these varieties to their synonyms [5,15-18] (Figures 2 and 3).

**Figure 2.** Dendrograms of genotyped grapevine accessions grown in Slovenia [5].

**Figure 3.** Dendrograms of genotyped grapevine accessions grown in Slovenia [17].

**2.1. Genetic diversity and relations among varieties**

152 The Mediterranean Genetic Code - Grapevine and Olive

synonyms [5,15-18] (Figures 2 and 3).

**Figure 2.** Dendrograms of genotyped grapevine accessions grown in Slovenia [5].

SSR markers offer some advantages over other molecular markers, including their co-domi‐ nant inheritance, hypervariability, and high cross-species transferability [8,13]. Consequent‐ ly 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 pro‐ duction 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 dif‐ ferentiate varieties, especially those of similar morphological characteristics; (2) assess genet‐ ic diversity and relationships among them; (3) and compare these varieties to their

> 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 va‐ rieties/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 Euro‐ pean gene pool (*Proles occidentalis*), but also 'Rebula' ('Ribolla') known as *Proles pontica*. An‐ other 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 west‐ ern 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 'Pi‐ colit 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'= 'Briš‐ ka 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 'Vi‐ tovska 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 'Prosec‐ co' 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 Štever‐ jan, 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].

Comparison between the two accessions denominated 'Glera' ('Briška Glera' and 'Bela Glera') included in our analysis [5] revealed differences at 16 out of 21 loci, so they are con‐ sidered homonyms. Two varieties, 'Poljšakica Drnovk' and 'Poljšakica Lože', which were ex‐ pected to have the same genetic profile, were different at various analysed microsatellite loci. The synonymy of 'Heunisch' = 'Ranfol' = 'Belina', which was first mentioned by [2], was also analysed - discrepancies were discovered at 13 loci, but the two varieties share one common allele at all 13 loci [5]. Furthermore, the accession 'Belina Pleterje' was compared to the synonymic variety 'Ranfol Bijeli' from Croatia using microsatellite data [14] - 8 com‐ pared SSR loci data showed the same allelic profiles for all, except VVMD7, where a triallelic profile was observed. A mutation in the microsatellite sequence of locus VVMD7 was often responsible for identifying grapevine synonyms or related types [19].The SSR profiles of ac‐ cessions linked to the designation 'Zelen' ('Zelen Pokov', 'Zelen 66' and 'Zelen 2.4') were compared as well.  The differences were re‐ vealed also at the comparison of 'Zelen' to 'Verduzzo' and 'Verdicchio'andcan therefore not be considered/used synonyms.In pairwise comparison, excluding *Vitis rupestris* L., the great‐

est distances detected (86%) were between accesions 'Rebula' – 'Volovnik', 'Dolga petlja' – 'Guštana' and 'Klarnica' – 'Pikolit' (Figure 2).

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 west‐ ern 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 'Pi‐ colit 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'= 'Briš‐ ka 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 'Vi‐ tovska 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 'Prosec‐ co' 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 Štever‐ jan, 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

Comparison between the two accessions denominated 'Glera' ('Briška Glera' and 'Bela Glera') included in our analysis [5] revealed differences at 16 out of 21 loci, so they are con‐ sidered homonyms. Two varieties, 'Poljšakica Drnovk' and 'Poljšakica Lože', which were ex‐ pected to have the same genetic profile, were different at various analysed microsatellite loci. The synonymy of 'Heunisch' = 'Ranfol' = 'Belina', which was first mentioned by [2], was also analysed - discrepancies were discovered at 13 loci, but the two varieties share one common allele at all 13 loci [5]. Furthermore, the accession 'Belina Pleterje' was compared to the synonymic variety 'Ranfol Bijeli' from Croatia using microsatellite data [14] - 8 com‐ pared SSR loci data showed the same allelic profiles for all, except VVMD7, where a triallelic profile was observed. A mutation in the microsatellite sequence of locus VVMD7 was often responsible for identifying grapevine synonyms or related types [19].The SSR profiles of ac‐ cessions linked to the designation 'Zelen' ('Zelen Pokov', 'Zelen 66' and 'Zelen 2.4') were compared as well.  The differences were re‐ vealed also at the comparison of 'Zelen' to 'Verduzzo' and 'Verdicchio'andcan therefore not be considered/used synonyms.In pairwise comparison, excluding *Vitis rupestris* L., the great‐

to 'Prosecco tondo' [5].

154 The Mediterranean Genetic Code - Grapevine and Olive

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 fol‐ lows [17] (Figure 3): The pairs/groups of vines denominated 'Duranja' and 'Duranja-Pre‐ gara', furthermore 'Belina' and 'Belina1', 'Zelen Rup2', Zelen66' and 'Zelen3' and 'Malvasia', 'Istrska Malvasia' and 'Malvasia Istriana' revealed identical genotypes in all SSR microsatel‐ lites analysed therefore they are regarded as synonyms. The designations related to 'Malva‐ sia' 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 denomi‐ nated '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 differ‐ ent from them in 8 loci out of 10 [17]. The difference in the stage of homozygosity/hetero‐ zygosity 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 com‐ mon 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 ge‐ netic similarity. The obtained allelic profiles of 'Pagadebiti' are also different from the pro‐ files 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 denominat‐ ed 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-Mediterra‐ nean 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%.

Unexpected the varieties 'Rožca' and 'Zelenika' showed the same genotype, what can be explained by the dissemination of scions of the same varieties throughout the winegrowing regions during the past, where the influence of dialects has also put its mark on the denomi‐ nation. The variety 'Rožca' is found quite close to another white accession signed as 'Rebula stara'; they share 77% of alleles. Vines denominated 'Rebula stara' and 'Rebula-100' were expected to be closely related but share only 55% of alleles; in common at least one allele at each locus, which could imply their parent/offspring relation.A genotype of 'Rebula stara' (='Stara Rebula'='Rebula old') perfectly matched at 7 loci with variety 'Gouais blanc' ('Heu‐ nisch'). Another interesting finding was the similarity between the vines 'Grganc' and 'Rebu‐ la-100', which showed similar genotypes with exception at locus VVMD5 at which 'Rebula-100' revealed a triallelic pattern [17].

#### *2.1.1. Grapevine accessions designated 'Rebula' and 'Vitovska'*

#### *<IP_ADDRESS>. Accessions related to designations 'Rebula' and 'Ribolla'*

'Rebula' (*Vitis vinifera* L.) has been one of the most important white grapevine varieties from the ancient times down to the present days and has been mostly cultivated in the area of Goriška brda and Collio where it is still gaining in importance. The first mentions of the name 'Rebula' date back to 1299, later 1376,to a deed of sale »Notariorum Joppi« in the area of the Slovenian Collio [27], whereas the first ampelographic descriptions of the variety are found in Vinoreja [1] and in Ampelographie [2]. Six different types of the variety 'Rebula' are enumerated and described ("green rebula", "yellow rebula", "less fertile rebula", "unfer‐ tile rebula", "crazy rebula" and "rebula with smaller and dissected leaf") [1] what confirms its biodiversity which decreases with narrow clonal selection. For this variety many syno‐ nyms are used - 'Garganja', 'Glera', 'Ribolla gialla', 'Rebolla', 'Ribolla', 'Ribolla Bianca', 'Ri‐ buèle', 'Rabuèle', 'Rosazzo', 'Ribollat', 'Raibola', 'Ràbola', 'Ribuole' and 'Gargania' [6,7,28]. DNA analyses regarding accessions of 'Rebula' have been already reported [15]. The variety 'Rebula' showed a very low similarity (16 %) with other analysed varieties, also with vines designed 'Rebula-100 years' and 'Rebula-old'. The accessions named 'Prosecco', 'Števerjana', 'Beli teran' and 'Briška Glera', however, revealed identical genotypes in all 11 SSR microsa‐ tellites analysed, and are therefore regarded as synonyms. The varieties 'Rebula' and 'Ribolla gialla' revealed an identical SSR profile at 8 out of 9 SSR loci (Figure 4).

Moreover, the genetic identity and relationship among 'Ribolla gialla', 'Rebula' and 'Robola' that have been traditionally cultivated in other European Countries (Goriška brda in Sloven‐ ia 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 exis‐ tence of full-sibling relationships between 'Ribolla gialla' and 'Robola' (Figure 5) [29].

varieties was confirmed. On the other hand, 'Beli muškat', which is clustered together with

Unexpected the varieties 'Rožca' and 'Zelenika' showed the same genotype, what can be explained by the dissemination of scions of the same varieties throughout the winegrowing regions during the past, where the influence of dialects has also put its mark on the denomi‐ nation. The variety 'Rožca' is found quite close to another white accession signed as 'Rebula stara'; they share 77% of alleles. Vines denominated 'Rebula stara' and 'Rebula-100' were expected to be closely related but share only 55% of alleles; in common at least one allele at each locus, which could imply their parent/offspring relation.A genotype of 'Rebula stara' (='Stara Rebula'='Rebula old') perfectly matched at 7 loci with variety 'Gouais blanc' ('Heu‐ nisch'). Another interesting finding was the similarity between the vines 'Grganc' and 'Rebu‐ la-100', which showed similar genotypes with exception at locus VVMD5 at which 'Rebula-100'

'Rebula' (*Vitis vinifera* L.) has been one of the most important white grapevine varieties from the ancient times down to the present days and has been mostly cultivated in the area of Goriška brda and Collio where it is still gaining in importance. The first mentions of the name 'Rebula' date back to 1299, later 1376,to a deed of sale »Notariorum Joppi« in the area of the Slovenian Collio [27], whereas the first ampelographic descriptions of the variety are found in Vinoreja [1] and in Ampelographie [2]. Six different types of the variety 'Rebula' are enumerated and described ("green rebula", "yellow rebula", "less fertile rebula", "unfer‐ tile rebula", "crazy rebula" and "rebula with smaller and dissected leaf") [1] what confirms its biodiversity which decreases with narrow clonal selection. For this variety many syno‐ nyms are used - 'Garganja', 'Glera', 'Ribolla gialla', 'Rebolla', 'Ribolla', 'Ribolla Bianca', 'Ri‐ buèle', 'Rabuèle', 'Rosazzo', 'Ribollat', 'Raibola', 'Ràbola', 'Ribuole' and 'Gargania' [6,7,28]. DNA analyses regarding accessions of 'Rebula' have been already reported [15]. The variety 'Rebula' showed a very low similarity (16 %) with other analysed varieties, also with vines designed 'Rebula-100 years' and 'Rebula-old'. The accessions named 'Prosecco', 'Števerjana', 'Beli teran' and 'Briška Glera', however, revealed identical genotypes in all 11 SSR microsa‐ tellites analysed, and are therefore regarded as synonyms. The varieties 'Rebula' and 'Ribolla

Moreover, the genetic identity and relationship among 'Ribolla gialla', 'Rebula' and 'Robola' that have been traditionally cultivated in other European Countries (Goriška brda in Sloven‐ ia 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 exis‐

tence of full-sibling relationships between 'Ribolla gialla' and 'Robola' (Figure 5) [29].

'Cipro' in the dendrogram, is related to them with a similarity of 60%.

revealed a triallelic pattern [17].

156 The Mediterranean Genetic Code - Grapevine and Olive

*2.1.1. Grapevine accessions designated 'Rebula' and 'Vitovska'*

*<IP_ADDRESS>. Accessions related to designations 'Rebula' and 'Ribolla'*

gialla' revealed an identical SSR profile at 8 out of 9 SSR loci (Figure 4).

**Table 3.** List of analyzed accessions and origin area of plant materials related to the designations 'Rebula', 'Ribolla' [29].

At least 9 distinct genotypes resulted from the 19 analysed accessions [29], due to several cases of detected synonyms (Figure 5) – for example the same genotype was founded among vines designated 'Robola 1 and 'Robola 2', than between 'Robola 6' and 'Robola 7', more‐ over among five Italian vines designated 'Ribolla' accessions (1-5) and three Slovenian acces‐ sions ('Glera 2', 'Rebula 1' and 'Rebula 2'), 'Bela Glera 5/6' with 'Glera 1'. Besides, several cases of homonyms revealed, two Slovenian accessions 'Bela Glera 5/6' and 'Bela Glera 8/1' showed different allelic profiles, sharing 48 % of alleles, so did the accessions 'Glera 1' and 'Glera 2'(34% of shared alleles). Five cases of homonyms of the group of Greek accessions were revealed [29]: 'Robola 1', 'Robola 2' and 'Robola 6', 'Robola 7' accessions showed allelic profiles different from other 'Robola' accessions. Vine designated 'Robola 1' and 'Robola 2' shared 81 % of alleles with 'Ribolla Gialla', 'Rebula' and 'Glera 2' accessions. In addition[29] report the SSR profiles of five accessions resulted to be unique: 'Bela Glera 8/1', 'Glera Mede‐ na', 'Robola 3', 'Robola 4' and 'Robola 5'. After the accurate consideration of the obtained results, the studied accessions are clustered into three major groups, but 'Glera Medena' and 'Robola 3' are presented as isolated samples. The cluster 2 presents a major number of ana‐ lysed vines, what reveals high homogeneity of the vines cultivated in Italy, as well as in the comparison with vines cultivated in Slovenia under the name 'Rebula' and those from Greece named 'Robola'. All these designations from cluster 2 can be perceived as synonyms; therefore the original name of this variety is still uncertain. It is demonstrated, that the groups of Italian and Slovenian vine from cluster 2 share 90% of alleles, suggesting the exis‐ tence of a common ancestor[29]. In the case of the other Greek accessions the situation seems to be more complicated and variability is higher – the largest number of accessions is group‐ ed in cluster 1 ('Robola 4', 'Robola 5', 'Robola 6'/'Robola 7' accessions) as they share about 75% of alleles, what suggests very close relationships. Grouped in cluster 3, we find the ac‐ cessions' Bela Glera 8/1', 'Glera 1' and 'Bela Glera 5/6', which show a similarity of 68% in comparison with cluster 2 (Figure 5). No case of parent-offspring (PO) relationships was re‐ vealed among analysed accessions. True-to-type 'Rebula' and 'Ribolla Gialla' (cluster 2) showed possible full-sibling (FS) relationships with 'Robola 1'/'Robola 2' accessions (same cluster), but shared only 55/70 alleles. Among the Greek accessions, 'Robola 4', 'Robola 5' and 'Robola 6'/'Robola 7', belonging to a different cluster are related by a FS relationship; 'Robola 4' and 'Robola 5' accessions sharing 68 % of alleles, 'Robola 4' and 'Robola 6'/'Robo‐ la 7' sharing 80 % of alleles and 'Robola 5' and 'Robola 6'/'Robola 7' sharing 78 % of alleles. Accessions 'Bela Glera 5/6', 'Bela Glera 8/1' and 'Glera 1' shared 48 % of alleles and show a probable HS relationship [29].

**Figure 4.** Dendrograms of grapevine accessions related to designations 'Rebula' and 'Ribolla' using D = 1-(proportion of shared alleles) as coefficient of distance and UPGMA as grouping method [15].

Genetic and Phenotypic Diversity and Relations Between Grapevine Varieties: Slovenian Germplasm http://dx.doi.org/10.5772/51773 159

**Figure 5.** Dendrograms of grapevine accessions related to designations 'Rebula' and 'Ribolla' using D = 1-(proportion of shared alleles) as coefficient of distance and UPGMA as grouping method [29].

#### *<IP_ADDRESS>. Accessions related to designations 'Vitovska' and 'Grganja'*

therefore the original name of this variety is still uncertain. It is demonstrated, that the groups of Italian and Slovenian vine from cluster 2 share 90% of alleles, suggesting the exis‐ tence of a common ancestor[29]. In the case of the other Greek accessions the situation seems to be more complicated and variability is higher – the largest number of accessions is group‐ ed in cluster 1 ('Robola 4', 'Robola 5', 'Robola 6'/'Robola 7' accessions) as they share about 75% of alleles, what suggests very close relationships. Grouped in cluster 3, we find the ac‐ cessions' Bela Glera 8/1', 'Glera 1' and 'Bela Glera 5/6', which show a similarity of 68% in comparison with cluster 2 (Figure 5). No case of parent-offspring (PO) relationships was re‐ vealed among analysed accessions. True-to-type 'Rebula' and 'Ribolla Gialla' (cluster 2) showed possible full-sibling (FS) relationships with 'Robola 1'/'Robola 2' accessions (same cluster), but shared only 55/70 alleles. Among the Greek accessions, 'Robola 4', 'Robola 5' and 'Robola 6'/'Robola 7', belonging to a different cluster are related by a FS relationship; 'Robola 4' and 'Robola 5' accessions sharing 68 % of alleles, 'Robola 4' and 'Robola 6'/'Robo‐ la 7' sharing 80 % of alleles and 'Robola 5' and 'Robola 6'/'Robola 7' sharing 78 % of alleles. Accessions 'Bela Glera 5/6', 'Bela Glera 8/1' and 'Glera 1' shared 48 % of alleles and show a

**Figure 4.** Dendrograms of grapevine accessions related to designations 'Rebula' and 'Ribolla' using D = 1-(proportion

of shared alleles) as coefficient of distance and UPGMA as grouping method [15].

probable HS relationship [29].

158 The Mediterranean Genetic Code - Grapevine and Olive

The accessions related to 'Vitovska', 'Vitouska', 'Grganja' and 'Garganja' are cultivated in different winegrowing areas in Slovenia, Italy and Croatia; therefore their names (syno‐ nyms) still cause disorder, because they are often considered indigenous and ancient variet‐ ies 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 'Mal‐ vasia del Chianti' (syn. 'Malvasia bianca lunga', 'Malvasia lunga') and 'Prosecco tondo'. Fol‐ lowing 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 stud‐ ied 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.

**Table 4.** List of the studied accessions related to the terms 'Vitovska', and 'Garganja' describing their already used synonyms, homonyms and cultivation areas[18]. Indexes: 1-[1]; 2-[28]; 3-[32]; 4-[7]; 5-[30]; 6-[31]

**Variety / Type / Vine / Abbreviation**

160 The Mediterranean Genetic Code - Grapevine and Olive

'Vitovska grganja' 1 (VG) Gerganja1, Vitouska2, Gargania2,

'Vitovska grganja' 2 (VG) Gerganja1, Vitouska2, Gargania2,

'Vitouska' (V) Vitovska grganja2, Vitouska2,

'Prosecco' (P) Gljera6, Prosekar6, Prosecco tondo2,

'Maraština' (M) Pavlos6, Malvasia del Chianti6,

'Ribolla gialla' (RG) Garganja2, Glera2, Refosco bianco2,

**Synonyms and homonyms (Translation)**

Ribolla gialla2, Grganja3, Garganja3, Vitovška3, Vitevška3, Vitovka3, Gorjanska3, Malvazija s piko3, Beli refošk3, Vrbina3, Vrbovna3, Črna pika3

Ribolla gialla2, Grganja3, Garganja3, Vitovška3, Vitevška3, Vitovka3, Gorjanska3, Malvazija s piko3, Beli refošk3, Vrbina3, Vrbovna3, Črna pika3

Gargania2, Ribolla gialla2

Prosecco bianco2, Gargana2, Brešanka1,2 (Brescia)

Malvasia lunga6, Rukatac6, Marinkuša mala6, Maraškin6, Đerđevina6, Kukuruz6, Rukac6, Krizol6, Višana6

Teran bijeli2, Ribola bijela2, Erbula2, Jerbula2, Gorička ribola2, Rebolla2, Ribolla2, Ribolla bianca2, Ribuèle2, Rabuèle2, Rosazzo2, Ribollat2, Raibola2, Ràbola2, Ribuole2, Gargania2

**Table 4.** List of the studied accessions related to the terms 'Vitovska', and 'Garganja' describing their already used

synonyms, homonyms and cultivation areas[18]. Indexes: 1-[1]; 2-[28]; 3-[32]; 4-[7]; 5-[30]; 6-[31]

'Garganja' (G) Rebula6, Ribolla gialla6 Sampled in the germplasm collection of

**Cultivation area and notes**

The vine is approx. 100 years old and grows in the village Sveto v (address Sveto 4), Kras (Slovenia) as an individual

The vine is over 110 years old and grows in the village Briščki (address Briščki 5), Carso, Italy cultivated as a pergola-

Sampled in the VCR (Rauscedo) selection of vines from vineyards planted near the Prosecco village, Italy. The variety is usually planted and cultivated in Carso region, north-east part of Italy.

Sampled in the VCR (Rauscedo) clone VCR101, Italy. The variety is usually planted and cultivated in Carso region,

Sampled in the germplasm collection of the University of Zagreb, near Split (Dalmatia, Croatia). The variety is usually planted and cultivated in Dalmatia,

the Faculty of agriculture and tourism Poreč, near Poreč (Croatia). The variety is usually planted and cultivated in Croatian part of the Istria peninsula, near

Sampled in the VCR (Rauscedo) clone VCR01, Italy. The variety is planted in Collio (Italy) presumably also in Goriška brda (Slovenian Collio), and Istria (Croatia). Sampled in the collection of

the University of Ljubljana.

pergola-trained vine.

trained individual vine.

north-east part of Italy.

coastal part of Croatia.

the village Buzet.

**Figure 6.** Dendrogram of studied grapevine (*Vitis vinifera* L.) accessions and of *Vitis rupestris* based on standard genet‐ ic distance [18].

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 'Malva‐ sia del Chianti' reported by [26], therefore we can conclude only that they are in strong rela‐ tionship. 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] af‐ firmed 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.

#### **2.2. Phenotyping experiences at grapevine varieties cultivated in Slovenia**

#### *2.2.1. 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 techni‐ cal advances in biochemical and molecular approaches over the last decades, the descrip‐ tions 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, es‐ pecially 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 collec‐ tions approximately 5-10% of existing varieties are misnamed and even in commercial viti‐ culture, misnaming and confusion related to synonyms and homonyms [38] is a frequent phenomena. We started to collect our allochthon, autochthone, local and endangered variet‐ ies systematically in 1980 and today we have few germplasm collections around Slovenia.

Additional attention has also been given to varietal phenotyping, especially at those variet‐ ies 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 differ‐ ent 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].

#### *<IP_ADDRESS>. Phyllometric measurements*

Many studies in the last decades demonstrated the importance and contribution of phyllom‐ etry in grapevine description and distinction. The method provides around 82 criteria meas‐ urements 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, typi‐ cally varietal leaves should be picked up, scanned or photocopied, placed in herbarium be‐ fore 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 re‐ construction of an average typical leaf of different varieties measured following the sugges‐ tions 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 up‐ per 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), dis‐ tance between the ends of L1 and L2 (D2), petiole length (lp), distance from the base of pe‐ tiole 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] suggested the following characterizing equations, in which both parts of the same leaf are used separately: Rel.1 = Lp/L, Rel.2 (left side) = L1/L, Rel.3 (right side) = L1/L, Rel.4 (left side) = α+β+γ, Rel.5 (right side) = α+β+γ, Rel.6 (left side) = α'+β'+τ, Rel.7 (right side) = α'+β'+τ, Rel.8 (left side) = (OS+OI)/(L1+L2), Rel.9 (right side) = (OS+OI)/(L1+L2), Rel.10 (left side) = OS/L1, Rel.11 (right side)= OS/L1, Rel.12 (left side) = OI/L2, Rel.13 (right side) = OI/L2 (Figure 7).

**2.2. Phenotyping experiences at grapevine varieties cultivated in Slovenia**

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 techni‐ cal advances in biochemical and molecular approaches over the last decades, the descrip‐ tions 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, es‐ pecially 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 collec‐ tions approximately 5-10% of existing varieties are misnamed and even in commercial viti‐ culture, misnaming and confusion related to synonyms and homonyms [38] is a frequent phenomena. We started to collect our allochthon, autochthone, local and endangered variet‐ ies systematically in 1980 and today we have few germplasm collections around Slovenia.

Additional attention has also been given to varietal phenotyping, especially at those variet‐ ies 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 differ‐ ent 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

Many studies in the last decades demonstrated the importance and contribution of phyllom‐ etry in grapevine description and distinction. The method provides around 82 criteria meas‐ urements 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, typi‐ cally varietal leaves should be picked up, scanned or photocopied, placed in herbarium be‐ fore 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 re‐ construction of an average typical leaf of different varieties measured following the sugges‐ tions 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

*2.2.1. Phyllometrical tolls for phenotyping*

162 The Mediterranean Genetic Code - Grapevine and Olive

typical-varietal mature leaf reconstruction [39,40].

*<IP_ADDRESS>. Phyllometric measurements*

**Figure 7.** The morphometrical characteristics of mature leaves suggested by different authors [36,41,42].

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 <IP_ADDRESS>) 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 (prin‐ cipal 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 measure‐ ments proved to be complementary more than comparable methods. The results of the eval‐ uation 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 similari‐ ties. 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 na‐ tional'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.

**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].

Furthermore, [40] studied 28 morphometric characteristics of leaves in detail and their contri‐ bution 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 char‐ acteristics give suitable results what suggests that phyllometry grants an indispensable, cheap and credible methodology in the ampelography – varietal description and discrimination.

ic and 22 morphometric parameters per leaf. Statistical analysis was carried out, PCA (prin‐ cipal 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 measure‐ ments proved to be complementary more than comparable methods. The results of the eval‐ uation 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 similari‐ ties. 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 na‐ tional'showed shorter lateral veins (L1d, L1g) in comparison with the main vein (L). The morphological descriptions and morphometric reconstructions of leaves gave a significant

164 The Mediterranean Genetic Code - Grapevine and Olive

contribution to understanding the grapevine phenotypical biodiversity.

**Figure 8.** Dendrogram of grapevine accessions linked to the designations 'Glera' and 'Rebula' grouped on the basis of

Furthermore, [40] studied 28 morphometric characteristics of leaves in detail and their contri‐ bution 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

the 28 morphometric characteristics of mature leaves [40].

**Figure 9.** Graphical reconstruction of leaves of studied grapevine varieties according to determined phyllometric char‐ acteristics [39].

#### *<IP_ADDRESS>. 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 signifi‐ cant characteristic, which offers a possibility of an accurate description of the main morpho‐ logical 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 de‐ scription 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 descrip‐ tion. 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 descrip‐ tion 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.

**Figure 10.** Dendrogram of grapevine varieties grouped according to the morphological evaluation, OIV codes [43].

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 de‐ scriptors 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).

*<IP_ADDRESS>. Morphological descriptions*

166 The Mediterranean Genetic Code - Grapevine and Olive

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 signifi‐ cant characteristic, which offers a possibility of an accurate description of the main morpho‐ logical 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 de‐ scription 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 descrip‐ tion. 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.

were conducted according to the instructions of the individual descriptive codes.

**Figure 10.** Dendrogram of grapevine varieties grouped according to the morphological evaluation, OIV codes [43].

The aim of the first study was the evaluation of selected OIV codes for grapevine descrip‐ tion 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

The further studies was focused on the variations, differences and possible mistakes linked to the subjective evaluations regarding OIV codes, where different descriptions of the same variety/accession were collected and compared. Therefore we selected accessions where many misunderstanding still provoke - accessions regarding the designations 'Vitovska' and 'Garganja' (Table 5) [18]. The misunderstandings in variety nomination are generally a re‐ sult of false and misleading facts, which can partly be explained by neglect in observations and descriptions of variety characteristics, especially among those with a similar phenotype.

OIV

code **Descriptor VG VGI\* V\* G\* M\*,**

 Shoot: length of tendrils 5 5 Young leaf: colour of upper side of blade (4th leaf) 2,41 3 1 3 2-3, (2-3\*\*)

 Mature leaf: size of blade 7 7\*\* Mature leaf: shape of blade 3 3 4 4 4, (1,4\*\*) Mature leaf: number of lobes 3 2-3 3 2 5, (3\*\*) Mature leaf: colour of the upper side of blade 7 7\*\*

Mature leaf: goffering of blade 5 5 5 5 3, (7\*\*)

 Mature leaf: profile of blade in cross section 4 5 5 5 1 Mature leaf: blistering of upper side of blade 5 3 7 3 3 Mature leaf: shape of teeth 3 2,4 4 5 3, (2\*\*) Mature leaf: length of teeth compared with their width 3 5\*\*

 Mature leaf: shape of base of petiole sinus 1 3 1 3 1, (2\*\*) 081-1 Mature leaf: teeth in the petiole sinus 1 1 1 1 1 081-2 Mature leaf: petiole sinus base limited by vein 1 1 1 1 3

083-2 Mature leaf: teeth in the upper lateral sinus 1 1 1 1 1

Young leaf: density of prostrate hairs between main veins

Young leaf: density of erect hairs between main veins on

Young leaf: density of prostrate hairs on main veins on

Young leaf: density of erect hairs on main veins on lower

Mature leaf: area of anthocyanin coloration of main veins

Mature leaf: area of anthocyanin coloration of main veins

Mature leaf: undulation of blade between main or lateral

Mature leaf: degree of opening / overlapping of petiole

Mature leaf: degree of opening/overlapping of upper

083-1 Mature leaf: shape of the base of upper lateral sinuses 1

Mature leaf: density of prostrate hairs between main veins

Mature leaf: density of prostrate hairs on main veins on

on lower side of blade (4th leaf)

The Mediterranean Genetic Code - Grapevine and Olive

lower side of blade (4th leaf)

lower side of blade (4th leaf)

side of blade (4th leaf)

on upper side of blade

on lower side of blade

veins

sinus

lateral sinuses

on lower side of blade

lower side of blade

**\*\***

7 8 7 5-7

1 2 2 1

5 2 7 7

1 6 5 7

2-3

OIV

170 The Mediterranean Genetic Code - Grapevine and Olive

code **Descriptor VG VGI\* V\* G\* M\*,**

 Berry: formation of seed 3 3 3 3 3, (3\*\*) Berry: length of seeds 5,7 5,7\*\* Berry: weight of seeds 3 3-5\*\* Berry: transversal ridges on dorsal side of seeds 1 1 1 Time of bud burst 7 3 3

303 Time of beginning of berry ripening (vérasion) 5 5 5 304 Time of full physiological maturity of the berry 5 7\*\*

351 Vigour of shoot growth 7 8 5, (5-7\*\*)

353 Length of internodes 3-5 3\*\* 354 Diameter of internodes 3 3\*\*

 Bunch: single bunch weight (g) 3 3 5, (3\*\*) Berry: single berry weight (g) 1-3 3 1, (1-3\*\*) Yield per m2 (kg) 3-53 5, (3\*\*) Sugar content of must 3 3 5 5, (5\*\*) Total acidity of must 3-54 3 5 5, (5\*\*) Must specific pH 3,55 3 7 Mature leaf: length of vein N1 (mm) = L1 \*\*\* 5 7 5\*\* Mature leaf: length of vein N2 (mm) = L2 \*\*\* 5 9 5\*\* Mature leaf: length of vein N3 (mm) = L3 \*\*\* 5-7 7 5-7\*\* Mature leaf: length of vein N4 (mm) = L4 \*\*\* 9 9 9\*\*

5 7 7\*\*

7 7 5\*\*

302 Time of full bloom 5-7

305 Time of beginning of wood maturity 7 306 Autumn coloration of leaves 1

352 Growth of lateral shoot 5

 Resistance to iron chlorosis 1 Resistance to chlorides (salt) 1 Resistance to drought 7 Leaf: degree of resistance to *Plasmopara* 5 Cluster: degree of resistance to *Plasmopara* 5 Leaf: degree of resistance to *Oidium* 1,3 Cluster: degree of resistance to *Oidium* 1,3 Leaf: degree of resistance to *Botrytis* 5 Cluster: degree of resistance to *Botrytis* 5 Percentage of berry set 5

607 Mature leaf: angle between N1 and N2 measured at the

608 Mature leaf: angle between N2 and N3 measured at the

first ramification (°) = α\*\*\*

first ramification (°) = β\*\*\*

**\*\***

**Table 5.** Ampelographic description according to O.I.V. descriptor codes and morphometric characteristics of 'Vitovska grganja' (VG), 'Vitovska grganija' (VGI), 'Vitouska' (V), 'Grganja' (G) and 'Maraština' (M) varieties [18]. \*-[44]; \*\*-[45]; \*\*\*-parameters of phyllometry [18]; 1-copper-reddish between veins; yellow around veins; 2-yellow and reddish; 3-0.8-1 kg m-2; 4-total acidity 5.6 - 8.2 g L-1; 5-pH 3.10 - 3.45.

The study was carried out on grapevine (*Vitis vinifera* L.) varieties cultivated in Slovenia, Croatia and Italia, mostly known as 'Vitovska', 'Vitouska', 'Grganja' and 'Garganija' but also referred to as indigenous or landrace varieties from the mentioned countries [1,7,28]. The studied varieties, with an additional focus on the 'Vitovska grganja' variety, were mostly taken from germplasm collections: 'Vitovska grganja' from Slovenia, 'Vitouska', 'Ribolla gialla' and 'Prosecco' from Italy, 'Garganja' and 'Maraština' from Croatia.

The comparison of the ampelographic descriptions, morphometric and phyllometric charac‐ teristics among the 'Vitovska grganja' (VG), 'Vitovska grganija' (VGI), 'Vitouska' (V), 'Grganja' (G) and 'Maraština' (M) varieties is presented in Table 5. The ampelographic de‐ scriptions 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 re‐ ported [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 nar‐ rower. 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 da‐ ta 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 sug‐ gests that a consistent attention to the ampelographic distinguishing among varieties, espe‐ cially among those with the same designations or synonyms, has to be emphasized in the future.

#### **3. Conclusions**