1.1. Economical, cultural and historical importance of grapevine in Portugal
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 (
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 , linkage mapping , cultivar identification and pedigree studies , . Microsatellites (SSR) are being used to characterize grapevine cultivars and wild vines [10, 14] and to carry out genetic diversity analyses . Usually six
2. Diversity of the Portuguese grape germplasm
2.1. Wild vine populations: Geographical distribution, morphological and molecular characterization
Wild vine populations of
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 V
The characterized wild vine plants featured the particularly morphological characteristics of the subspecies
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 .
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 (
Chloroplastidial microsatellites (cpSSRs) have been used to study the genetic relationships among grapevine cultivars , wild vines  and relations between both subspecies [37, 38 ]. Analysis of chloropastidial microsatellites (Figure 3) revealed the expected situation for the Iberian Peninsula  with the presence of chlorotypes A and B, being chlorotype A the most frequent within the wild vine populations (66%) of Portugal.
Chlorotype A is the most frequent in Western Europe and absent in Near East where the domestication of
2.2. 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 SO4 rootstock and each access is represented by seven plants from the same original mother plant. This collection holds 691 accessions of
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 (
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  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 . The static methods used were recently reviewed in . 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).
2.3. 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  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  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 ). 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
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 cultivated subspecies the ideal situations should be maintaining the agro-systems where its diversity was buildup. However these
Even though some European countries like France and Germany have a legal protection status for the subspecies
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
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