Genetic Origin of Iberian Peninsula Grapevine Cultivars
Currently wild relict populations of Vitis vinifera subspecies silvestris can still be found in a wide geographic range of the Iberian Peninsula following river bank forests and sea shore areas (See following article by Rafael Ocete Rubio). These populations were much larger and occupied a wider geographic range before the end of the 19th century (Ocete et al., 2007). Their drastic reduction is the result of two main factors: the development of human populations and infrastructure; and the arrival in Europe and the Iberian Peninsula of pathogens and pests with a high incidence both in the vineyard and in the natural population.
The Iberian Peninsula as other Mediterranean peninsulas served as refugia to many plant species during the Quaternary glaciations that froze Northern and Central Europe (Goméz and Lunt, 2006) and became a major part of the largest biodiversity centre in Europe, the Mediterranean basin (Myers et al. 2000). Human Palaeolithic populations in the Iberian Peninsula collected grape berries along the river forests and consumed them as fruits (Rivera and Walker, 1989) at the time when the wine culture was first brought to the area by Phoenician sailors. Evidence exists that the Phoenicians not only imported wine but brought cultivars and knowledge of viticulture with them and established the first vineyards in the Iberian colonies (Hidalgo, 2002). Not only the Phoenicians, but also the Greeks and Romans brought their viticultural practices and their cultivars to the Iberian Peninsula as described in other chapters. Moreover, a few centuries after the end of the Western Roman Empire, the Iberian Peninsula was colonized by Muslims from Northern Africa, a cultural interaction that remained in some parts of the Iberian Peninsula for over eight centuries (from the 7th to 15th century). That brought with it special viticultural practices and uses, as well as a number of Eastern cultivars intended primarily for table grape consumption. Consequently, geographical, historical, commercial, political, and religious relationships between the Iberian Peninsula and other geographic areas throughout history have resulted in a large exchange and dissemination of grapevine cultivars, thereby increasing the genetic diversity of the Iberian cultivar genetic pool. On the other hand, some of those factors, together with the introduction of new diseases and pests in the 19th century, and the new rules of economic development, have limited this genetic diversity. Altogether these interacting forces have shaped the mixture of grapevine genotypes that can be found nowadays in the Spanish and Portuguese vineyards, as well as germplasm stock centres. Fortunately, the rapid development of genetic and genomic tools for grapevine analysis in the last two decades allows for a reconstruction of the history of grapevine genotypes that even today bear testimony to the common history and development of grapevine and human populations.
A Genetic and Genomic View of the Origin of Iberian Cultivars
Apart from the nuclear genome, the plant cell has two subcellular organelles that bear their own genome, the chloroplasts and the mitochondria. All three chloroplast, nuclear and mitochondrial genomes of Vitis vinifera have recently been sequenced in reference genotypes (Jansen et al., 2006; Jaillón et al., 2007; Velasco et al., 2007; Goremykin et al., 2009) providing a basic tool for studies in sequence diversity and domestication of the grapevine. Variation in DNA sequences can be classified into two basic types:
i) nucleotide substitutions causing single nucleotide polymorphisms or SNP; and
ii) insertions or deletions known as INDEL. This second group includes all sorts of insertion‑deletions, from single nucleotides to long events. Repetitions of simple nucleotide sequences, known as microsatellites, can also be considered a special type of INDEL. Genetic diversity at the grapevine mitochondrial genome has not been investigated so far. However, information on the amount and distribution of genetic diversity of the chloroplast and nuclear genomes in cultivated and wild Vitis vinifera genotypes can provide information on their origins and genetic relationships. On one hand, nuclear plant genomes evolve at a rate four times faster than the chloroplast genome (Wolfe et al., 1987) making nuclear DNA markers the tools of choice for studies on plant domestication processes (Zeder et al., 2006). On the other hand, chloroplast genomes are uniparentally transmitted in most species (usually maternal in angiosperms and paternal in gymnosperms) and therefore useful to elucidate the relative contribution of seed and pollen flow to the genetic structure of populations (Provan et al. 2001), to test hypotheses of crop‑wild gene flow or to establish the maternal origin of specific genotypes.
Chloroplast genome diversity supports the existence of secondary domestication events in the Iberian Peninsula. The grapevine chloroplast genome is a circular DNA molecule 160,928 bp long with identical gene content and order to other angiosperm chloroplast genomes (Jansen et al. 2006). Chloroplasts are maternally inherited in grapevine (Strefeler et al. 1992; Arroyo García et al. 2002) indicating that any chloroplast genome polymorphism can be transmitted to next generation plants either by seeds or cuttings but never through pollen. Chloroplast genetic diversity in grapevine has been studied through the analysis of polymorphisms at microsatellite loci (Arroyo‑García et al., 2002; Imazio et al., 2006). Chloroplast microsatellites are mononucleotide repeats showing variation in the number of repetitions in different genotypes and consequently in their length. Up to 34 different loci were tested in the grapevine chloroplast genome to find that only five of them showed polymorphism (Arroyo‑García et al. 2006; see also This et al., 2011). The analyses of variation at those polymorphic loci in a large sample of 1201 wild and cultivated grapevine plants identified 2‑3 alleles per microsatellite locus which are combined in eight different chlorotypes or chloroplast genome types. Therefore in the grapevine genotypes analysed, only eight different maternal types could be differentiated and, among them, only four had a frequency higher than 5%. These four chlorotypes were named as A, B, C and D.
Chlorotype distribution is not homogeneous in wild grapevine populations along its native area. Chlorotype A, that is a very divergent chlorotype from chlorotypes B, C and D, is mainly found in Western and Central European wild populations as well as those of the Maghreb ones, but it is absent in Near East and Asian populations. In addition, chlorotypes C and D are highly frequent in Near East and Asian populations but are absent in Western European populations (Arroyo‑García et al., 2006; ). The distribution of chlorotypes in cultivars of grapevine follows similar patterns, with chlorotype A being highly abundant in Western Europe and particularly in wine cultivars from the Iberian Peninsula and chlorotype C being characteristic of Eastern cultivars and highly abundant among table grape cultivars (Arroyo‑García et al., 2006). This pattern of distribution has also been confirmed for the same chlorotypes in cultivated and wild samples from Portugal (Cunha et al., 2010). In Spain and Portugal up to 75% of the wine cultivars carry chlorotype A, which is the most abundant chlorotype found in Iberian natural populations (Arroyo‑García et al., 2006; Cunha et al., 2010). A similar situation is also found in the Maghreb in Northern Africa, where samples collected in wild natural populations generally bear chlorotype A. However, in this region, most of the cultivated accessions are table grape cultivars that frequently bear chlorotype C (Snoussi et al. 2004; Zinelabidine et al., 2010). Although using different chlorotype names, other authors also reported chlorotype VI (equivalent to Chlorotype A) being highly frequent in the Iberian Peninsula (Imazio et al., 2006) and not detected in cultivated or wild samples from Iran where chlorotypes I and III (equivalent to D and C) were the most abundant (Baneh et al., 2007).
Nuclear DNA sequence variation suggests the existence of introgression from wild populations to cultivated accessions in Western Europe. In the nuclear genome, the high evolutionary rate of microsatellite loci has been useful to trace ancestry and measure genetic diversity (Aradhya et al., 2003; Cipriani et al., 2010; Laucou et al., 2011). However, microsatellites are rapidly being substituted by SNPs (Lijavetzky et al., 2007; Myles et al., 2010) that can provide thousands of genotyped markers per experiment. All nuclear microsatellite studies comparing Western wild populations of Vitis vinifera ssp. silvestris with different sets of Vitis vinifera cultivars (from Western, Central Europe or Northern African locations) have shown that wild and cultivated genotypes cluster in different unrelated genetic groups (Grassi et al., 2003; Snoussi et al., 2004; Dzhambazova et al., 2009; Zinelabidine et al., 2010). Those results have generated the idea that in Western Europe wild populations of V. vinifera ssp. silvestris and cultivars belong to different compartments with different genetic origins. Only a recent study combines the analysis of cultivated accessions with samples of Vitis vinifera ssp. silvestris from both Western and Eastern ends of the species distribution area. The results of this study suggest a closer genetic similarity between Eastern cultivars and Eastern silvestris than between Western cultivars and Western silvestris (Myles et al., 2011). These results represent the first genetic evidence supporting an initial Eastern domestication of the grapevine followed by its dissemination in an East to West direction. In addition, these studies also support the existence of introgression of genetic materials from Western silvestris into Western cultivars what would explain the slightly higher genetic similarity shown between Western cultivars and Western silvestris genotypes than between Eastern cultivars and Western silvestris genotypes. Although interesting, and in agreement with the similar chlorotypes observed between wild and cultivated genotypes of Western Europe, these results should be interpreted with caution given the small number of samples analyzed so far.
Pedigree analyses uncover a putative Middle Age cultivar melting pot giving rise to many of today’s cultivars. What happened from the initial cultivation of the first imported materials and the result of those predicted secondary domestication events taking place in the Iberian Peninsula to the cultivars that are used in viticulture today? Unfortunately no records are sufficiently clear to allow the identification of the cultivated grapevine varieties in different times. Some cultivars, such as the Muscatels which are spread all over the Mediterranean region since Greek and Roman times, could have been introduced to the Iberian Peninsula more than one thousand years ago. In fact, these cultivars can be recognized among the grapes described by the 12th century Andalusian agronomist, Ahmad Ibn al‑Awwam al‑Ishbili (Abu Zakaria), in the Kitab al Filaha, or Book of Agriculture (Abu Zacaria 1988), an important medieval text, in which he describes grapevine cultivars of the time (Milla Tapia et al., 2007). Still, the first ampelographic descriptions that allow identification of some of current the Iberian varieties were written by Alonso de Herrera in the 16th century in Spain (de Herrera, 1988) (Milla Tapia et al., 2007).
The study of parentage relationships among grapevine cultivars using microsatellite molecular markers was initiated in 1997 with the discovery of the pedigree of Cabernet Sauvignon (Bowers and Meredith, 1997) shown to be a hybrid of Cabernet Franc and Sauvignon Blanc. This study opened the way to similar analyses in different regions showing that genetically related cultivars within a given geographic area were in fact close relatives (Sefc et al 2000). This extent has been demonstrated for French varieties (Bowers et al., 1989; Boursiquot et al. 2009; This et al., 2006), Italian varieties (Vouillamoz et al., 2007, Crespan et al., 2008; Cipriani et al., 2010), and central Europe varieties (Sefc et al., 1998; Vouillamoz et al., 2003). A similar situation is evident within the Iberian Peninsula from the first pedigree analyses performed so far. Using this method, the Portuguese cultivars Malvasía de Colares, Manteudo and Camarate Tinto seem to be hybrids of the putative Arab female cultivar Gibi and the Portuguese Amaral, the Portuguese cultivars Arinto do Dão and Codega and the Portuguese and Spanish cultivars Alfrocheiro/ Alfrocehiro and Jaen Blanco, respectively (Lacombe et al., 2007). In addition, The Spanish minor cultivar Subirat Parent is a hybrid of Gibi and the Spanish cultivar Tortozón (Planta Nova) (Lacombe, 2007). Gibi has been found to share one allele per locus with another Spanish cultivar, Pedro Ximénez, and it is very likely involved in many additional pedigrees (Vargas et al., 2007). In fact, nuclear microsatellite genotypic analyses of Iberian and also Maghreb cultivars point out the existence of many possible parent‑offspring relationships among cultivars that share at least one allele per locus analyzed (Santana et al., 2010; Zinelabidine et al., 2010). On a larger scale, a 9K SNP array genotyping experiment has shown that up to 75% of the analyzed accessions have a parent offspring relationship with another genotype in the collection, suggesting that a majority of current grapevine cultivars are in fact hybrids derived from other known cultivars (Myles et al., 2011).
Since human‑directed grapevine hybridizations were not performed until the 19th century and many of the hybrid cultivars mentioned have been known for centuries, it has to be assumed that they derived from spontaneous hybridization followed either by the collection of seeds for reproduction of grapes or by the careless management of vineyards allowing hybrid plants to prosper among the vines and to be incorporated into culture in the following growing seasons. Interestingly, varieties derived from selfing of a given plant are not commonly detected, thus revealing the existence of a selection process that eliminated plants with low vigour and weak production traits characteristic of inbreeding depression in the grapevine. From the end of the 19th century until today, additional grapevine cultivars now produced by directed breeding have been incorporated into the Iberian germplasm. Cases in point are the teinturier varieties developed by Henri Bouschet and now cultivated in Portugal and Spain, with synonymous names, moreover (Cabezas et al., 2003).
A well‑documented case of the generation of new cultivars by spontaneous hybridizations within a given timeframe corresponds to the origin of the so‑called creole cultivars within American viticulture, which are also related with the Iberian Peninsula. This case can provide an example of what happened with the original cultivars introduced and domesticated in the Iberian Peninsula. Vitis vinifera cultivars were first introduced in Mexico and Peru by Franciscan and Jesuit friars at the beginning of the 16th century, because of the problems encountered in wine preservation (Hidalgo, 2002). These cultivars were planted in mission vineyards and spread as new missions were established, adopting different names in different regions. Recently, the genotypic analyses of ancient cultivars still cultivated in North and South America revealed that three main cultivars probably widely cultivated in the 16th century in the Iberian Peninsula were originally widespread in traditional American viticulture under different names. These corresponded to Muscat of Alexandria, Listán Prieto and Mollar Cano. The most widespread were the Listán Prieto, a red wine cultivar, known as País, Uva Negra Vino, and Viña Negra in Chile, Criolla Chica in Argentina, Rosa del Perú and Negra Corriente in Peru, Misión in Mexico, and Mission in California. In addition, in the five hundred years that have elapsed since their first introduction, they have produced a first generation of creole cultivars by spontaneous hybridization. In this way, hybridization between Listán Prieto and Muscat of Alexandria has given rise to typical creole cultivars such as Torrontés Riojano, Torrontés Sanjuanino, Torontel, Cereza, Moscatel Amarillo, Criolla Grande, Criolla Mediana, or Huasquina Pisquera (Agüero et al. 2003; This et al., 2006; Milla‑Tapia et al., 2007).
Considering the origin of viticulture in the Iberian Peninsula within the time range of between 2000 and 3000 years before our days, the results of the preliminary studies using different nuclear and chloroplast molecular markers support a mixed origin of Iberian cultivars from imported oriental germplasm mainly bearing chlorotypes C or D, together with the use of local secondarily domesticated genotypes bearing chlorotype A. Since chloroplasts are maternally inherited, domestication is the only possible way to get chloroplast A transmitted into cultivated materials, by cultivation of cuttings or seeds from plants of Western populations of Vitis vinifera ssp. silvestris. These secondary domestication events taking place in the Western region of the Mediterranean basin might have been influenced by the cultivars spread over the area by the Oriental cultures but also derives from the previous knowledge and use of local silvestris populations by the Iberian inhabitants. A few cycles of spontaneous pollination of newly domesticated plants (chlorotype A) with pollen from imported cultivars and selection of hybrids with oriental phenotypes (larger clusters and berries) would have resulted in chlorotype
A cultivars showing phenotypes and nuclear genotypes more closely related to oriental characteristics. The results of the analysis of grapevine genotypes with the 9K SNP arrays support these introgressions of genetic material from Western silvestris in the current Western cultivars (Myles et al., 2011) in agreement with the chlorotype results. Spontaneous hybridizations among cultivated grapevine plants are likely to have been a recurrent situation generating the hybrids that currently exist as cultivars today. Given the time scales provided by the origin of creole cultivars (one generation in 500 years), it is possible to estimate that only between 5‑10 sexual generations would have taken place in the 2000‑ 3000 years of Iberian viticulture to give rise to the current varieties. In any case, it is improbable that originally domesticated or imported plants have been maintained to the present day, since their traits are likely to have been improved by the spontaneous hybrids generated over the course of viticultural history.
Chlorotype A is the most frequently found (>90%) in current natural populations of Vitis vinifera ssp. silvestris on the Iberian Peninsula and the Maghreb countries; its distribution in Europe reaches as far as the Balkans. Chlorotype D is frequently found (almost 50%) in the Middle East, while in Europe it extends as far as the Italian Peninsula. Chlorotype B is detected with low frequency in all regions mentioned above. Finally, chlorotype C is as frequent as chlorotype D in the Middle East but in Europe its distribution in natural populations has not been detected beyond the Balkans. Chlorotype C is very frequent in table grape cultivars and in French wine cultivars derived from the Gouais as the maternal parent.