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

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].


Introduction 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 important role either as a border culture or as an extensive crop.The surface area used by vineyards 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 million 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 Indication (Figure 1) and 31 wine areas with Designation of Origin status including Porto, established 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 terroirs.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 number of cultivars increased until the tree waves of destruction from North American pest and diseases: powdery mildew (Uncinula necator Schweinf.Burrill ) in 1851, phylloxera (Dactylosphaera 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 simply 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 region 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 cultivars 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 century 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 number of cultivars in use is unknown but from the 340 allowed for wine production, 240 are thought to be autochthonous [ 8,9].

The Mediterranean Genetic Code -Grapevine and Olive
Traditionally morphological descriptors were used to characterize cultivars until the advent 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 identification and pedigree studies [12], [13].Microsatellites (SSR) are being used to characterize 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 chloroplastidial 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 single parent inheritance [18].All this range of tools is useful to make decisions on the strategies for conservation.

Wild vine populations: Geographical distribution, morphological and molecular characterization
Wild vine populations of Vitis vinifera L. subspecies sylvestris [(Gmelin) Hegi)] is closely related 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 distributed 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.Human populations since the early settlements in the Iberian Peninsula collected and consumed 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 Portuguese), 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 considered as a continuum [23,24].
In these riparian woods the plants species most frequently found as tutors of Vitis vinifera L. ssp.sylvestris are: Adenocarpus complicatus, Alnus glutinosa, Fraxinus angustifolia, Nerium oleander, Olea europea, Quercus faginea subsp.Broteroi, Quercus suber, Rubus ulmifolius, Salix atrocinerea, 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].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.The characterized wild vine plants featured the particularly morphological characteristics of the subspecies sylvestris: i) open young shoots, which is a characteristic allowing to differentiate 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 suggested by the OIV [31,32].The diversity founded in wild vine genotypes (Table 2) reveals 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] .The values of the Fixation Index (F) range from 0.005 to 0.28, showing the existence of inbreeding in some wild vine populations, since F is expected to be close to zero under random mating [34].

Locus
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 molecular data confirmed that some of the collected plants were clones due to vegetative propagation (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.The Mediterranean Genetic Code -Grapevine and Olive 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).

Cultivated grapevine: Morphological and molecular diversity
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 different 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 collection of accessions all over the country.All CAN accessions were grafted into SO 4 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 grapevine 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 (Table 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 established 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 barrier 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 foreign material as exemplified by important wine cultivars like Touriga Franca and Trincadeira that show the presence of the D chlorotype.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).The Mediterranean Genetic Code -Grapevine and Olive

Overall diversity of the Portuguese grapevine germplasm
Portuguese wild vine populations are in an apparent geographic fringe of the species distribution but the country richness in cultivar diversity [8,9] and the importance in allele contribution 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 include 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 vinifera 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].

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 cultivated subspecies the ideal situations should be maintaining the agro-systems where its diversity 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 habitats.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 authorities 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 subsequently.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 status for the subspecies sylvestris, in Portugal no such protection exists.An formal requirement 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 worldwide viticulture relies in a very restricted number of cultivars an even in a country like Portugal 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 arrival in Europe of Dactylosphaera vitifoliae 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 methodology used to establish these collections was recently reviewed in [55].The existing collections continue to perform several functions.These functions were initially related to the characterization and identification of cultivars using classic ampelography including: i) standardization of the morphological descriptors of Vitis; ii) morphological de-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 pedigrees and finding unsuspected ones.It also allowed tracing the remote history of grapevine domestication including the existence of several secondary domestication centers.The availability 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, morphological, molecular and field performance data will be useful in establishing core collections aiming a better management of the germplasm available.

Figure 2 .
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.

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

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.

Table 3 .
AMOVA 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 domestication of Vitis vinifera occurred.The distribution of chlorotypes in four Southern Portuguese 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

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.

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

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