Douro Vineyards: A Perspective for the Valorization and Conservation of Grapevine Genetic Resources

Abstract

The wine industry is one of the most important industries globally, particularly for Portugal, making a significant contribution to the Portuguese Bioeconomy. The Douro Demarcated Region (DDR) stands out as one of the largest wine-producing areas in the country. Its centuries-old culture has fostered a rich diversity, developed through vegetative and genetic breeding. Within the wine market, the highest prices for grapes are attained only under optimal edaphoclimatic conditions and when phenotypic characteristics, such as berry size and color, flowering and seed number, align favorably. Viticulture is influenced by environmental factors, diseases, and pests, impacting its economic value, profitability, and, ultimately, the employment and economic conditions of those dependent on the crops. Genetic improvement of phenotypic traits offers a faster and more cost-effective means of achieving desirable characteristics, translating into enhanced productivity and wine quality. This work focuses on presenting information about Douro grapes (region, varieties, diseases/pest, and economic value) and explores grapevine genetic diversity, along with approaches to identify genes associated with various desired traits.

1. Introduction

The Douro Demarcated Region (DDR) is one of the largest Portuguese areas, with 43,000 ha of vineyards dedicated to the production of wine [1,2]. Also, according to the Centre for the Research, Study, and Advancement of Mountain Viniculture (CERVIM), the DDR not only is one of the oldest demarcated wine regions, but also is the largest and most heterogeneous mountainous wine region in the world [3]. The DDR region is composed of the sub-regions of Lower Corgo, the Upper Corgo, and the Upper Douro [1]. The Lower Corgo has the largest area with grapevines (29%), followed by the Upper Corgo (22%) and the Upper Douro (9%) [2]. Edaphoclimatic conditions such as temperature, soil composition, and water levels are extremely important for the development of this crop. In fact, the temperature variation is a relevant factor for wine production in the DDR. For example, higher temperatures in late spring increase yield and wine production [4,5]. Higher temperatures also will impact the quality of the produced grapes, which will have a higher sugar content, a higher alcoholic potential, and a lower acidity [6]. Likewise, hydrostatic stress is a crucial factor in this region and has a direct correlation with the quality of grapes [7]. The Upper Corgo and Upper Douro are the most affected areas, given their very dry summer, diminished content of water in the soil, and the high gradient of vapor pressure between the leaves and the surrounding atmosphere [7]. The intense seasonal changes in the DDR’s climate are essential for grape production and quality. In a study of Moutinho-Pereira et al., severe water deficit caused expressive non-stomatal effects affecting photosynthesis [8]. On the other hand, excessive precipitation can affect the color of grapes’ skin and the anthocyanin content [9]. However, maintaining a water balance is a difficult task, as increasing the water in soil can increase yield, but produces fruits with a higher sugar content and lower acidity [10]. The implementation of an irrigation system can be a possible solution to increase yield. However, this practice induces organic acid content and a decrease in fructose, glucose, and soluble solids in grapes [10]. According to the study of Oliveira et al., optimal concentrations of sugar and acids can be obtained by irrigating at 50% of the potential evapotranspiration upon flowering [10]. This technique would achieve the best balance among yield, quality, and water conservation [10]. The DDR soil has a high degree of schist and some granite [3]. Schist, due to its frailty, is easily fragmented, promoting rain and root penetration [11]. Given the black color of soil, this terroir absorbs more sunlight, reaching higher soil temperatures [12]. These schist soils produce rich, austere, peppery wines, with pronounced spiciness [12]. Despite its 4.6–5.5 pH, slightly below the ideal soil pH for viticulture (5.5–7.0), DDR soil is rich in potassium (12%) and poor in phosphorus, magnesium, calcium, and organic matter (0.6–1.6%) [13].

Terroir, a major concept in this field, relates the sensory attributes of wine to the environmental conditions [3]. However, such a relationship is difficult to study given its multiple variables (climate, soil, cultivar, human practices) [3]. A study conducted by Jones et al. demonstrated that the best harvests have similar points, such as earlier events, a short interval between the final stages of dormancy and the first stage of budburst, and a longer berry development period, indicating a direct connection with temperature variation [6]. Thus, temperature and soil conditions also influence the sensory attributes of wine [3]. Wine quality, and in turn its price, is also defined by the ‘grape compositions’ in terms of the metabolic composition of each of its varieties. Thus, grape quality is linked to the presence and quantity of sugar, acids, tannins, anthocyanins, and phenolic and volatile compounds. Lower temperatures in higher altitudes, in the last month of maturation (lower altitudes = higher temperatures) increase the accumulation of anthocyanidin monoglucosides (AMGs) [14]. This pigment is a major quality attribute of DDR wine [14]. Corroborating this fact, Mateus et al. showed that a lower altitude (higher temperatures) led to improvements in the AMGs content, in ‘Touriga Nacional’ [14].

2. Cultivated Grapevines, Varieties, and Economical Value

Cultivated grapevines (Vitis vinifera ssp. sativa) are wild populations of Vitis vinifera ssp. sylvestris that were domesticated from Western Europe to central Asia and North Africa [15,16]. Grapevines (family: Vitaceae; order: Rhammales; subclass: Rosidae of Eudicots) include 1000 different species [16]. Despite the seventeen different genera described, only two have agricultural interest (Euvitis Planch and Musadinia Planch) [16,17]. The high number of varieties cultivated is the result of centuries of breeding between the two major forms of vineyard-cultivated V. vinifera subsp. sativa and wild-form V. vinifera subsp. sylvestris [18,19].

Given the optimal conditions previously described in the DDR, 78% of all grapevines are red and 22% white [1]. It is worth highlighting the most produced red varieties: ‘Touriga Nacional’, ‘Touriga Franca’, ‘Tinta Roriz’, ‘Tinto Cão’, ‘Tinta Barroca’, ‘Tinta Amarela’; and white varieties: ‘Malvasia Fina’, ‘Gouveio’, ‘Viosinho’, ‘Donzelinho’, ‘Códega’ and ‘Malvasia Rei’ [2]. Some traits of these grape varieties are described in

Table 1. Different varieties of grape cultivars, area, traits and yield.

Grape Cultivar	Varieties	Area	Berry Color and Size	Aroma	Yield	Reference
Red	‘Touriga Franca’	22%	Blue–black; medium	Flowers, raspberry, cherries, blackberry, herbaceous	2.6 kg grape/vine	[20,21]
	‘Tinta Roriz’	13%	Black; small	Black pepper, wildflowers, wild cherries, vegetables	2.6 kg grape/vine	[21,22,23]
	‘Touriga Nacional’	8%	Blue–black; medium to small	Sweet fruit aromas, leafy freshness, violets or bergamot. (In Porto wine) Mulberry, blackberry, black pepper, floral	0.9 kg grape/vine	[21,22,23]
	‘Tinto Cão’	-	Black–blue; small	Floral	-	[24]
	‘Tinta Barroca’	-	Medium	Floral	-	[25]
	‘Tinta Amarela’	-	-	Spice, herbaceous, fruity	-	[26]
White	‘Códega’/‘Malvasia Grossa’	4.6%	Golden–white; large and oblong	Orange blossom, linden, acacia, peach, tropical fruit, melon and citrus	2.0 kg grape/vine	[21,22,23]
	‘Malvasia Fina’	3.6%	Minute gold; small	Fruity, honey and floral notes	1.4 kg grape/vine	[21,22,23]
	‘Viosinho’	-	Green–yellow; small	-	-	[27]
	‘Malvasia Rei’	-	Green–yellow; medium	Herbaceous and salty	-	[28]

.

Despite having fertile ground for this culture, given its deep valleys (protected by mountains, cold winters, and hot, dry summers), the DDR was responsible for only 20% of the total wine production in Portugal in 2020 [2]. Portugal is one of the biggest worldwide producers of wine, ranking 16th of all producing countries [29]. According to the Organization of Vine and Wine, the Portuguese people are one of the leaders in wine consumption, consuming 5.3 million hectoliters of wine in 2021 [29]. On average, each Portuguese person drinks 51.9 L of wine per year, almost 10 L more than a French citizen, making Portugal a leader in wine consumption [29]. In 2022, the vinicultural products in the DDR were ~128 million products (table grapes and wine among others) [30]. In the same year, 40 million liters were sold corresponding to a business volume of EUR 244,666,555 [31]. Portugal exported 3276 hectoliters and imported 2841 hectoliters in 2022 [29].

3. Main Diseases and Pests

Similarly to other crops, the economic value of this crop can be affected, since it is susceptible to several diseases and pests [32,33,34,35,36,37,38,39,40,41,42,43]. Diseases (powdery mildew, downey mildew, grey mold, anthracnose, black rot, and leaf blight), and pests, (phylloxera, roundworm, grapevine moth, leafhoppers, stem borer, and mealybugs) have a huge impact on vineyard production (

Table 2. Vineyard diseases and pests, organisms, affected areas, and combat measures.

Biotic Stressor	Organism	Affected Areas	Combat Measure	Reference
Downey mildew	Plasmopara viticola	Leaves, grapefruit	Fungicide	[32]
Powdery mildew	Uncinulanecator (Schw.) Burr.	Leaves, grapefruit	Fungicide	[32]
Grey mold	Botrytis cinerea	Grapefruit	Fungicide	[36]
Anthracnose/’Bird’s eye spot’	Elsinoe ampelina	Berries, leaves, tendrils and petioles	Fungicide	[34,35,36]
Black rot	Guignardia bidwellii	Berries and leaves	Fungicide	[37]
Leaf bligh	Xylophilus ampelinus	Leaf, petiole, stem, root, shoot or flowers	Remotion of the diseased plants	[38]
Stem borer	Lepidoptera: Sesiidae	Branches and root	Pheromone-baited traps, chlorpyrifos is the only insecticide	[39,40,41]
Phylloxera	Daktulosphaira vitifoliae	Roots	No treatment, grafting with phylloxera-resistant American rootstock	[42,43]
Roundworm	Xiphinema spp.	Roots	Soil fumigation, nematicides or mycorrhizal fungi	[52,53]
Grapevine moth	Lobesia botrana	Flowers and berries	Insecticides (natural and synthetic)	[54]
Leafhoppers	Erythroneura spp.	Leaves	Green lacewing remove the infected leaves	[54]
Mealybugs	Pseudococcus maritimus Pseudococcus longispinus Pseudococcus viburni	Shoots and berries	Cryptolaemus montrouzieri, Hyperaspis sp. (natural predator) insecticides	[55,56]

). Although yield loss varies across varieties, geographical, and climatic conditions, powdery and downey mildew can affect up to 100%, and antharacnose 10 to 15% wine quality, and in turn, the price is defined by ‘grape composition’ in terms of metabolite composition. Thus, grape quality is linked to the presence and quantity of sugar, acids, tannins, anthocyanins, and phenolic and volatile compounds. Black rot and leaf blight affect up to 80% of worldwide production. Pest such as roundworm, grapevine moth, stem borer and mealybugs can generate a yield loss of <60%, <35%, <80%, and <40%, respectively. Their high yield losses lead to high economic loss. To ensure that the economic loss is kept low, a close phytosanitary control is made [44,45,46,47,48,49,50,51].

The most produced grapevine varieties in DDR, ‘Tinto Cão’ and ‘Touriga National’ (Table 1), are known for their high resistance to anthracnose and powdery mildew [57,58]. Given the economical and subsequent social importance of this culture, and considering that many traits of grapevines, and in turn, the quality of wine, are linked to specific genes, identifying the genes responsible for phenotypic characteristics is of utmost importance. In this perspective, from now on, we aim to point out some future directions in gene identification and their connection to desirable traits to increase productivity and wine quality.

4. V. vinifera Genetics

Nowadays, due to newly developed trials, the identification of candidate genes linked to phenotypical traits is of major importance to this field. Most of the studies in this field are successfully identifying genes associated with desirable phenotypic traits such as berry size and color, seed number and seedless grapes, flowering and flower number and development, fruit set, and millerandage index. Many studies on the genetic part of flowering are focused on Arabidopsis, which has several homologous genes [59,60].

Grapevine development is a two-year complex process influenced by multiple factors (genotype, environment, and humans) [60]. In this work, we will only focus our attention on the genotype. Briefly, flower development in grapevine involves three main steps: (1) formation of the anlagen; (2) formation of the inflorescence primordia; and (3) differentiation of flowers [60]. Since it is a complex process, more detailed information can be found in previous revisions [61,62,63]. In the first season, the shoot apical meristem (SAM) produces the first lateral meristem, the anlagen (uncommitted primordia) [64]. This club-shaped meristematic formation is the first step of inflorescence formation [60]. Then, the anlagen form two arms, the inner and outer arms. Afterwards, the differentiation of the inflorescent primordia begins, this marks the floral initiation [60]. The VvMADS8 is responsible for floral initiation [61]. The Flowering Locus T (FT) (VvFT gene) is responsible for seasonal floral induction in latent buds and for the development of inflorescences, flowers, and fruits [65,66]. LEAFY (LFY) (VFL grape orthologue) and APETALA1 (VAP1 grape orthologue) are responsible for the floral meristem identity. VFL is highly expressed in the anlage, floral meristems, petals, stamen primordia, leaf primordia, and leaf margins [67,68,69]. The VFL genes are responsible for the correct formation of leaves and leaflets [61]. VAP1 and VFUL-L genes are expressed in the early phases of uncommitted lateral meristem. VFUL-L is also responsible for floral and carpel transition and fruit development [70]. VAP1 expression suggests that this gene plays a crucial role in flowering transition and subsequent development [60].

The following step in the flowering process, floral organ identity, is controlled by a complex regulatory network named the ABCDE model [71,72]. A class genes specify the identity of the sepals. B function genes are expressed in petals and stamens. In this class, several genes have been identified such as VvPI, VvAP3, and VvTM6 [73]. All act at different time points and in different tissues, as described in a previous study [73]. The C class gene AGAMOUS in Arabidopsis is responsible for specification of carpels and stamens [74]. The VviAG1 and 2, VviAGL6a and b (homologs to AGL6 and AGL13 in Arabidopsis) genes were identified in grapevines [62]. C (VviAG3) and D function SEED STICK (STK) genes are required for ovule identity [60,74,75]. SEED STICK (VvMADS5), SHPI and SHP2, and class D genes, are also involved in the regulation of fruit development [75]. Finally, E class genes such as VvMADS2 and VvMADS4 are responsible for early inflorescence development. VvMADS4 is also expressed in berry development, and VvMADS3 is responsible for the development of flowers and vegetative organs [75,76]. In short, classes A + E, A + B + E, B + C+E, C + E, C + D + E correspond to sepals, petals, stamens, carpels, and ovules, respectively [77]. Furthermore, other processes like flowering time are controlled by the CONSTANS-like genes and FLOWERING LOCUS C 1 and 2, VvFLC1 and VvFLC2 [72].

The number of flowers produced by grapevines is also a desirable trait for wine production. This phenotype is controlled by several genes, such as Vitvi08g00630, Vitvi10g01358, Vitvi11g01072, and Vitvi18g01884 [78]. After the flowering process, fruit development is also a key point in wine production. In their growth, MADS-box protein SHORT VEGETATIVE PHASE 2 (SVP2) (Vitvi18g00517), MADS-box AGAMOUS-LIKE 21 (AGL21) (Vitvi18g00553), MADS-box AGAMOUS-LIKE 12 (AGL12) (Vitvi18g02145), and Vitvi18g02631, are involved [78].

Berry color is associated with the anthocyanin pigment, encoded by the VvmybA1 and VvmybA2 genes [79]. The insertion of Gret1, a 10,422 bp retrotransposon, in the VvmybA1 impairs normal transcription, altering the berry color [80]. The white allele is characterized by mutations in the genes mentioned above, leading to a decline in anthocyanin biosynthesis [81]. Despite the extensive efforts to present a more complete gene map, some phenotype traits only have one or few studies. This is the case for berry number in grapevines. The berry number trait is related to several genes such as Vitvi01g01038, Vitvi07g00455, Vitvi16g00894, Vitvi03g01320, Vitvi11g00228, Vitvi11g01393, and Vitvi12g00019 [78]. Another desirable trait is seedless grapes, which is related to the VviAGL11 gene [82,83,84]. This gene is located in chromosome 18:26,889,437. Royo et al. reported that the phenotypic trait of seedless grapes can be traced back to missense mutation in the MADS-BOX Gene VviAGL11 by the substitution of Arg-197Leu in the gene [84].

Concerning the reproductive development, some genetic markers can also protect from certain hazards, such as millerandage. This phenomenon occurs when the number of normal berries is similar to the number of small seedless berries (and/or live green ovaries) in the mature cluster [78]. Millerandage is a potential hazard that depends on high and low pre-flowering temperatures, a low volume of water, and inadequate nutrition [78]. Also, genes such as Vitvi17g00229, Vitvi18g02631, Vitvi02g01270, Vitvi02g01288, Vitvi04g00573, Vitvi05g00281, and Vitvi05g00523 are associated with millerandage [78]. These previously identified genes can be key to foreseen future steps.

Genetic Enhancement

Germplasm banks are collections of genes and their alleles that are available to promote improvements in different plants. These banks include cultivated species, sexually compatible and sexually-incompatible species which harbor important genes that influence fruit quality and can be transferred by genetic engineering [85]. Nowadays, plant breeding does not focus on multifactorial change (environment), but on a few traits, as desired by humans. Therefore, phenotypic and genetic diversity is lower in domesticated plants than in their wild relatives [86,87,88,89]. Given the vegetative propagation of grapevines, this reduction is more prone to occur [90]. Breeding choices can impose a serious threat to the future of this cultivar, since sometimes, we improve features that we desire and not features that will allow the plant to cope with environmental changes (global warming and emerging diseases) [91,92,93]. In an effort to prevent genetic erosion, the scientific community has developed germplasm banks, preserving while conserving the genetic and phenotypic diversity of grapevines [90]. Over time, thousands of these banks were created around the world (Algeria, Argentina, Australia, Belgium, Chile, France, Germany, Iran, Italy, Montenegro, Morrocco, Portugal, Romania, Spain, Tunisia, etc.) [94]. Grapevine germplasm holds up to 10,000 cultivars [94]. In Portugal, until 2020, six locations of wild grapevine populations in situ (Castelo Branco, Montemor-o-Novo, Alcácer do Sal, Portel, Ribeira de Toutalga, and Grândola) and one national ampelographic collection (Lisbon) were referenced [95].

Traditionally, these collections are maintained using the whole plant, exposed to field conditions, or in in vitro cultures [96,97,98]. In Portugal, this conservation is carried out in the field, at the experimental pole for the conservation of vine diversity in Pegões. The number of new entries in this pole and in private collections is about 3–4 thousand a year [97]. However, the much-needed conservation and collections, the field genebanks, carry some risks. Maintenance in field conditions can be more expensive and prone to higher losses from attacks by diseases, pests, and mostly by environmental disasters [99,100,101]. Since these collections have millions of grapevines and not all of them react in the same way to the different methods proposed to prevent pests and diseases, establishing a duplicate in a secondary location can be a security measure [100,101]. Nevertheless, this duplicate strategy adds more expense to the maintenance of genebanks [100,101]. In vitro genebanks can also provide a solution for the possible losses in field genebanks, although they come with some challenges of their own [102,103,104]. These collections can therefore be extremely important to a profitable wine market by conservating the genetically endangered and commercially desirable species for top quality and price wines [15]. Studies of genetic variations and comparison are of extreme value for this culture, assuring the authenticity of a produced variety and also the correct variety of commercialized plants. Proven authenticity of a certain variety is important for their thriving in a certain terroir, commercial value, and sensory qualities. Genomic variations can be achieved by Single Nucleotide Polymorphisms (SNPs), insertions or deletions, copies, and the presence or absence of variations (PAV) [105]. Genotyping with SNPs is a useful tool to solve incorrect names, synonyms or homonyms. Restriction Amplified Polymorphic DNA (RLFP), the first technique to identify genetic diversity without genetic protocols, was developed in the 1980s [96]. Afterwards, PCR-based markers such as Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) were developed. During the 1990s and 2000s, the Single Sequence Repeat (SSR), also called microsatellite markers, created a new way to explore genetic diversity. Despite substantial advances, the techniques mentioned above were still very time consuming, expensive, and had a very low mapping resolution [105]. Simultaneously, several quantitative trait loci (QTLs) were able to identify large genomic areas [106]. Chloroplasts microsatellites (cpSSR) are also a valuable technique, given that chloroplasts are maternally inherited. cpSSR analysis allows one to follow the maternal lineage of any grapevine. Different studies mapped the genetic distribution according to the four major chlorotypes (A, B, C, and D) [106,107,108,109,110,111]. Type B, C, and D chlorotypes were detected in the Middle Eastern populations, while in the Western Mediterranean populations, chlorotype A is more dominant. Three-quarters of the Iberian varieties contain chlorotype A [107,108,111].

Genetic evaluation has been extensively used in identification of Portuguese grapevines. In the study of Cunha et al., 288 samples from the Portuguese National Ampelographic Collection were compared to the samples of the Instituto de Ciencias de la Vid y del Vino-SNP database, through SNP, revealing synonymies and homonymies [5]. For example, ‘Alvarelhão Ceitão’ and ‘Tinta Castellõa’ had identical SNP profiles and are therefore synonymous. However, in Portuguese history, these two cultivars were not known as synonymous [5]. Further information on more synonymies and homonymies can be found in the Vivc database (https://www.vivc.de/index.php?r=cultivarname%2Findex, accessed on 20 September 2023) [61]. Also, in this study, a large number of unique genotypes were also found, indicating that they are possibly autochthonous to Portugal [5]. Alifragkis et al. through SSR loci revealed mislabeling of synonym and homonym grapevines. For example, ‘Alvarelhão’, ‘Caitão’, ‘Boal Espinho’, ‘Cornichel Branco’, ‘Malvasia Branca de S. Jorge’, ‘Molar’, ‘Transâncora’, and ‘Verdial’ were considered as minor cultivars with no certified plant material through SSR loci [112]. Multiple techniques can be applied to identify different varieties, obtaining similar results, as verified in the study [110]. In this work, SSR, SNP, and High-Resolution Melting (HRM) assays were compared, and similar identifications were reported [113]. HRM analysis is a quantitative analysis of the melt curves of product DNA fragments following PCR amplification [110]. This technique allows the identification of small variations in nucleic acid sequences [110]. In the studies of Jailon et al. and Velaco et al., the genomes of an inbred line of ‘Pinot Noir’ (PN40024) and a heterozygous genotype (ENTAV115) used by winemakers were sequenced [114,115]. Both studies used the whole genome shotgun (WGS) method, decoding around 30,000 protein-coding genes, distributed by 38 chromosomes [114,115]. The Next Generation Sequencing (NGS) technique increased the high-throughput read lengths and single-base accuracy, reducing the costs and assembly methods. NGS permits whole-genome resequencing. These different techniques allow us to obtain more detailed genetic information, and despite their various differences, remain equally important.

New and advanced genetic tools allow plant breeding evolution. Laborious and time-consuming conventional breeding programs were overcome by the identification of desirable trait genes. Nowadays, breeding techniques are based on marker-assisted selection (MAS), genomic selection (GS), or NGS. NGS uses SNPs to identify and discover genomes. Also, for breeding purposes, it is essential to identify genetic factors that can be translated in field resistance. Several species present different resistances to the most common diseases and pests. For example, V. riparia, V. rupestris, and V. berlandieri are resistant to phylloxera (Daktulosphaira vitifoliae) [116]. In turn, V. bryoniifolia, V. davidii, V. piasezkii and, V. amurensisare are resistant to powdery and/or downy mildew [116]. V. rotundifolia is also a useful source of resistance to phylloxera, nematodes, powdery and downy mildew [116]. Beside the biotic factors, several species also present resistance to abiotic factors such as low temperatures (V. riparia, V. labrusca and V. amurensisare), and high ones (V. lincecummi, V. bourquinianaand and V. rotundifolia) [116].

The Muscadinia rotundifolia, a wild North American grapevine, is resistant to powdery mildew and downy mildew, given by the presence of seven TIR-NB-LRR genes, which are known to be involved in effector-triggered immunity [117]. Downy mildew resistance, for example, is achieved by two systems: (1) a single hypersensitive gene to the reaction of stomatal tissues, and (2) polygenes that inhibit mycelium growth in plant tissues. Resistant species are homozygous dominant for the single hypersensitive gene. Three major loci, namely Rpv1, Rpv2, and Rpv3, are responsible for resistance to downy mildew [118,119,120]. Rpv1, located in M. rotundifolia, on chromosome 12, is closely related to Run1, a resistant locus for powdery mildew. Relying on RAPD, ISSR, and SSR tools, Lin et al. found that 83% of the genetic variation is linked to Rpv1 [120]. The Rpv2, located in chromosome 18, is also responsible for this genetic resistance. In the study of Bellin et al., another locus, named Rpv3 was found in the same chromosome of the Rpv2. Rpv3 explained up to 56% of the phenotypic variance. However, this result can be misleading given the wide confidence interval and the unstable QTL peak found [121]. The authors proposed that in this locus, more than one functional gene can reside, leading to unreliable values [119,120,121]. Other small loci are also involved in resistance to Downy mildew, such as the ones located in chromosomes 4, 5, 6, 7, 8, 9, 11, 12, 14, and 15 (Rpv 4 to Rpv28) [118,119,120,121,122,123,124,125,126,127,128,129,130].

Resistance to grapevine powdery mildew is controlled by a single gene called Run1, which is responsible for 70% of phenotypic variation [117,131]. This dominant gene is present in wild grapevine M. rotundifolia but absent in V. vinifera [109,123]. Through genetic breeding, this gene, located in chromosome 12, was introduced into V. vinifera. In the study of Riaz et al., another locus was found, called Run 2.1. This accounted for 50% of the phenotypic variation on chromosome 18 [132]. Anthracnose resistance is achieved by two dominant genes for susceptibility (An1 and An2) plus a single dominant resistance gene (An3). All traditional European grapes are known to be susceptible to black rot. However, the hybrid of V. riparia and V. cinerea, (‘Börner’) is highly resistant to this disease [133]. Black rot resistance is either controlled by two dominant genes or is quantitatively controlled [116]. QTL analysis between the ‘Börner’ and a breeding line V3125 detected two major QLT. The first one was linked to group 14 (Rgb1), explaining 21.8% of the phenotypic variation and the second one was linked to group 16 (Rgb2), explaining 8.5% of the variation [133]. These two major QTLs, alongside the minor ones, indicate the polygenic nature of black rot resistance in ‘Börner’ [133].

Phylloxera parasite (D. vitifoliae) was spread to Europe in the middle of the nineteenth century, and consequently, resistance breeding was needed. Rootstocks hybrids (American will Vitis × V. riparia) were resistant to this parasite. However, this new commercialized hybrid has a low resistance to phylloxera. In contrast, the cultivar ‘Börner’ (V. riparia × V. cinerea hybrid) presents a high level of resistance [134,135]. Given the economical drawback that this disease can cause, several genetic maps have been published. The first maps were based on dominant RAPD and AFLP markers [116,134,135,136,137,138,139,140]. Also, co-dominant SSR markers were developed in a joint effort by the Vitis Microsatellite Consortium [141,142]. Recent genetic maps are based on SSR loci, SNP, genomic sequencing, and EST analysis [115,143,144,145,146,147,148,149,150,151,152,153]. Resistance to phylloxera is controlled by multiple genes. Genetic mapping showed a major resistant QTL (RDV1) located in chromosome 13 in ‘Börner’ rootstock [153]. Also, RDV3 was found in chromosome 14 of the Vitis hybrid MN1264 [147]. Finally, genetic maps showed four more loci linked to chromosomes 3, 4, 5, and 7, designated as Rdv7, Rdv4, Rdv5, and Rdv6, respectively [154].

Species such as V. arizonica, V. candicans, V. rufotomentosa, V. smalliana, and V. solonis are resistant to Xiphenema index [155]. This phenotype is controlled by a major QTL named XiR1, near marker VMC5a10 on chromosome 19 [155]. The XiR1 explains ~60% of resistance variance [155]. A second strong QTL, named XiR1′, near marker M4F3F on chromosome 19, responsible for 37% of the variance, was also found in the study of Xu et al. [155]. Given the proximity of XiR1 and XiR1′ on chromosome 19, these two were considered the same QTL [155]. In addition to the XiR1, a minor QTL was also identified. On chromosome 17 near marker VMC9g4, it is responsible for ~6% of the phenotypic variance [155].

5. Conclusions

Portuguese viniculture represents one of the most important genetic diversities in the world and a key economic sector for Portugal, with high revenues of EUR 244,666,555 in 2022. Given the high demand and top quality of wines, it is important to maintain production at the required level and quantity, contributing towards the bioeconomy of rural and mountain regions. Over the centuries in wine industry, the genetic pool of diversity that was originally present has diminished. This refinement has meant that only the top grapes with the most desirable characteristics have been able to prevail. At this moment, many genes have been identified as a source of variability for a certain trait, as explored in this work. If controlled, the genes and, in turn, their effects on the quality and quantity of wine produced, could achieve controlled production, bringing socioeconomic advantages. Increased and consistent production, uniform ripening, and resistance and tolerance to diseases, pests, and sudden changes in climate are, in our opinion, the most important characteristics for reducing variability in production and quality. Exploration of Portuguese vineyards genes is of extreme importance given the significant contribution to gene mapping and breeding programs. Breeding improvement is the next step to produce grapevines with known and specific traits as desired by humans. However, caution must be exercised as this breeding improvement reduces natural genetic diversity. Considering that genes encode possible future traits, more extensive work is needed to map grapevines.