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Grapevine is one of the most important fruit crops in the world. However, the effect of climate change on Vitis vinifera L. could dramatically affect the productivity of vineyards, causing serious economic and ecological damage. Some of the most important factors are drought and water availability. Varietal sensitivity to these abiotic stresses is more pronounced in commercial cultivars like 'Merlot' than local cultivars like 'Callet'. In order to better understand the adaptive differences between these two cultivars, we used transcriptomic, metabolic and hormonal analyses, at three different levels of drought stress (mild, high and extreme), using Richter 110 as rootstock. Differential gene expression (DGE) analysis was performed using RNA-seq data for each condition in both roots and leaves. DGE results showed that the response started in roots, with more differential expression at mild drought stress in these two cultivars. However, more differentially-expressed genes (DEGs) were found out in 'Merlot' than 'Callet', with the most significant response to mild drought in 'Merlot'. However, in high and extreme drought conditions, DGE analysis in leaves showed an increase of DEGs in the two cultivars, being more pronounced in 'Callet' than 'Merlot', suggesting a different the strategy of response between them. Hormonal and metabolic results displayed two different pathways in drought responses. While 'Merlot' allocated more resources to maintain cellular turgor, increasing sugar and alditol concentration in cells, 'Callet' produced more secondary metabolites like mono- or sesqui-terpenes, assisting with sugar transport to the fruit and thus increasing berry quality. Furthermore, a higher photosynthetic activity was found in 'Callet' than 'Merlot', showing higher water use efficiency.

The domestication process in grapevines has facilitated the fixation of desired traits. Nowadays, vegetative propagation through cuttings enables easier preservation of these genotypes compared to sexual reproduction. Nonetheless, even with vegetative propagation, various phenotypes are often present within the same vineyard due to the accumulation of somatic mutations. These mutations are not the sole factors influencing phenotype. Alongside somatic variations, epigenetic variation has been proposed as a pivotal player in regulating phenotypic variability acquired during domestication. The emergence of these epialleles might have significantly influenced grapevine domestication over time. This study aims to investigate the impact of domestication on methylation patterns in cultivated grapevines. Reduced-representation bisulfite sequencing was conducted on 18 cultivated and wild accessions. Results revealed that cultivated grapevines exhibited higher methylation levels than their wild counterparts. Differential Methylation Analysis between wild and cultivated grapevines identified a total of 9955 differentially methylated cytosines, of which 78% were hypermethylated in cultivated grapevines. Functional analysis shows that core methylated genes (consistently methylated in both wild and cultivated accessions) are associated with stress response and terpenoid/isoprenoid metabolic processes. Meanwhile, genes with differential methylation are linked to protein targeting to the peroxisome, ethylene regulation, histone modifications, and defense response. Collectively, our results highlight the significant roles that epialleles may have played throughout the domestication history of grapevines.

In Andalusia (Spain), there are different wine regions that have a great recognized tradition. In these regions, the cultivation of the vine is ancient and there are still vineyards planted with local varieties of Vitis vinifera L. that have not yet been identified. The aim of this research study was to identify 49 accessions of grapevine collected in the districts of four provinces in Andalusia (Spain). All samples were genotyped with 20 microsatellite markers in order to ascertain the identity and analyze the genetic diversity of the collected material. In total, 30 different genotypes were obtained, 22 of them which were identified with named, known varieties by comparison to the Spanish or European microsatellite databases, and eight which are referred to as new genotypes. All loci were polymorphic, and a total of 159 alleles were detected, ranging from 4 to 12 alleles per locus, with an average allele number of 7.95. The overall observed heterozygosity was 0.763 and was slightly higher than expected (0.715), while the gene diversity per locus varied between 0.167 (VVIN73) and 0.967 (VVMD5). A dendrogram representing the genetic similarities among cultivars was depicted using the UPGMA method to investigate their relationships. The eight new genotypes identified in this research work could represent ancient local varieties in danger of extinction. These new cultivars may be used to determine original wines.

We elucidate grapevine evolution and domestication histories with 3525 cultivated and wild accessions worldwide. In the Pleistocene, harsh climate drove the separation of wild grape ecotypes caused by continuous habitat fragmentation. Then, domestication occurred concurrently about 11,000 years ago in Western Asia and the Caucasus to yield table and wine grapevines. The Western Asia domesticates dispersed into Europe with early farmers, introgressed with ancient wild western ecotypes, and subsequently diversified along human migration trails into muscat and unique western wine grape ancestries by the late Neolithic. Analyses of domestication traits also reveal new insights into selection for berry palatability, hermaphroditism, muscat flavor, and berry skin color. These data demonstrate the role of the grapevines in the early inception of agriculture across Eurasia.

Variegation is a rare type of mosaicism not fully studied in plants, especially fruits. We examined red and white sections of grape (Vitis vinifera cv. 'Béquignol') variegated berries and found that accumulation of products from branches of the phenylpropanoid and isoprenoid pathways showed an opposite tendency. Light-responsive flavonol and monoterpene levels increased in anthocyanin-depleted areas in correlation with increasing MYB24 expression. Cistrome analysis suggested that MYB24 binds to the promoters of 22 terpene synthase (TPS) genes, as well as 32 photosynthesis/light-related genes, including carotenoid pathway members, the flavonol regulator HY5 HOMOLOGUE (HYH), and other radiation response genes. Indeed, TPS35, TPS09, the carotenoid isomerase gene CRTISO2, and HYH were activated in the presence of MYB24 and MYC2. We suggest that MYB24 modulates ultraviolet and high-intensity visible light stress responses that include terpene and flavonol synthesis and potentially affects carotenoids. The MYB24 regulatory network is developmentally triggered after the onset of berry ripening, while the absence of anthocyanin sunscreens accelerates its activation, likely in a dose-dependent manner due to increased radiation exposure. Anthocyanins and flavonols in variegated berry skins act as effective sunscreens but for different wavelength ranges. The expression patterns of stress marker genes in red and white sections of 'Béquignol' berries strongly suggest that MYB24 promotes light stress amelioration but only partly succeeds during late ripening.

Climatic change is affecting grapevines growth and development with clear effects on plant production and grape quality. Drought is becoming a serious threat and water availability is becoming a main limitation for vineyard sustainability. Search for grapevine genotypes of enhanced drought resistance is a priority to face the climatic change because it is an environmental friendly solution. The present review shows different genetic variability sources, which could be of major interest to identify more drought-resistant cultivars. A summary of different sources of genetic diversity is shown from wild grapevines to cultivars, clones and rootstocks, showing wide variability in different drought-tolerant traits, such as water use efficiency. The interest of those genetic resources is enhanced by the progress in genomic tools opening a wide field of research and also the expectative for new cultivars of enhanced drought resistance.

Increase in soil salinity, driven by climate change, is a widespread constrain for viticulture across several regions, including the Mediterranean basin. The implementation of salt-tolerant varieties is sought after to reduce the negative impact of salinity in grape production. An accession of wild grapevine (Vitis vinifera L. ssp. sylvestris), named AS1B, found on the coastline of Asturias (Spain), could be of interest toward the achievement of salt-tolerant varieties, as it demonstrated the ability to survive and grow under high levels of salinity. In the present study, AS1B is compared against widely cultivated commercial rootstock Richter 110, regarding their survival capabilities, and transcriptomic profiles analysis allowed us to identify the genes by employing RNA-seq and gene ontology analyses under increasing salinity and validate (via RT-qPCR) seven salinity-stress-induced genes. The results suggest contrasting transcriptomic responses between AS1B and Richter 110. AS1B is more responsive to a milder increase in salinity and builds up specific mechanisms of tolerance over a sustained salt stress, while Richter 110 maintains a constitutive expression until high and prolonged saline inputs, when it mainly shows responses to osmotic stress. The genetic basis of AS1B's strategy to confront salinity could be valuable in cultivar breeding programs, to expand the current range of salt-tolerant rootstocks, aiming to improve the adaptation of viticulture against climate change.

Background and Aims Wild grapevine, considered the ancestor relative of cultivated vines, has a large gene pool that is currently endangered in Europe. These plants can contribute to improving adaptation capacity to stresses due to climate change. The aim of this study was to evaluate the key ampelographic traits to identify true wild individuals supporting the preservation and use of wild populations. Methods and Results Prospections performed since 2002 enabled the inventory of 51 localities with wild grapevines, most of which were located along Spanish riverbank forests. A morphological study of 192 individuals grafted in the grapevine collection of El Encín (Alcalá de Henares, Spain) was carried out ex situ, and results were compared with data from 182 Spanish commercial cultivars grown in the same collection. Wild individuals presented morphological differences with cultivars, but only a few significant differences were found within wild individuals when comparing their geographic origin and plant sex. Ten morphological traits were relevant to discriminate wild and cultivated specimens. Conclusions Ampelography, supported by previous molecular screening, is recommended to identify wild grape plants, although it is not advisable to establish relationships among wild genotypes by geographical location or gender. Significance of the Study Results are expected to contribute to improved discovery, preservation and use of this important phytogenetic resource. © 2016 Australian Society of Viticulture and Oenology Inc.

‘Vinhão’ is an autochthonous Portuguese cultivar with an intense black-bluish skin color, highly appreciated due to this feature. This study aimed to give the first insights into the genetic background that may be responsible for the skin color properties of cv. ‘Vinhão’. For this purpose, the allelic composition of MYBA1 and MYBA2 genes was investigated, along with quantification of the expression levels of structural and regulatory genes involved in the anthocyanin biosynthetic pathway via qRT-PCR. The molecular characterization of MYBA1 and MYBA2 loci revealed that cv. ‘Vinhão’ is homozygous for the functional allele in both genes, corresponding to the most ancestral haplotype, which is consistent with the high colored phenotype that characterizes this cultivar. There were no differences in the DNA sequence of the MYBA1 promoter region between cv. ‘Vinhão’ and the grapevine reference genome Pinot Noir. The expression patterns of genes playing key functional roles in anthocyanin biosynthesis was analyzed in four developmental stages. The dynamics occurring throughout grape berry development revealed the involvement of these genes in the progression of key development events, mainly from veraison to mature berries. These findings provide the first molecular characterization focused on the skin color feature of cv. ‘Vinhão’ to improve our understanding of the genetics behind its intense skin pigmentation.

Even though it is one of the oldest perennial domesticated fruit crops in the world, grapevine (Vitis vinifera L.) cultivation today is the result of both conventional breeding practices (i.e. hybridizations adopted during the last century) and vegetative propagation. Human-assisted asexual propagation has allowed the maintenance of desired traits but has largely impacted the frequency of spontaneous somatic mutations observed in the field. Consequently, many grapevine fruit attributes to date have been artificially selected, including: fruit yield, compactness, size and composition, the latter being greatly diversified in the pursuit of altering berry skin coloration. The present review provides an overview of various aspects related to grapevine diversity, with a special emphasis on grape berry skin color variation and will discuss the current knowledge of how grape skin color variation is affected by the synthesis of phenolic compounds, particularly anthocyanins and their underlying genetic factors. We hope this knowledge will be useful in supporting the importance of the berry color trait diversity in cultivated grapevines, which is used as basis for selection during breeding programs because of its application for vine growers, winemakers and consumers.

[BACKGROUND], During grapevine domestication somatic variation has been used as a source of diversity for clonal selection. [OBJECTIVE], This work provides additional information on the molecular mechanisms responsible for berry skin color reversion on a subset of somatic variants for berry skin color never investigated before. [METHODS], The berry color locus and its surrounding genomic regionwere genetically characterized through a layer-specific approach, which has already been proven to be a successful method to decipher the molecular mechanisms responsible for color reversions on somatic variants. [RESULTS], Deletions of different extent and positions were detected among less-pigmented/unpigmented variants derived from a pigmented wild-type. These deletions affected only the inner cell layer in the less pigmented variants and both cell layers in the unpigmented variants. Regarding the pigmented variants derived from an unpigmented wild-type, only one group was distinguished by the Gret1 retrotransposon partial excision from the VvMybA1 promoter. Moreover, within this latter group, VvMybA2 showed an important role regarding the phenotypic variation, through the recovery of the functional G allele. [CONCLUSIONS], This investigation focused on the berry color locus using somatic variants for berry skin color, promotes a better understanding of the evolutionary events behind their origin and variability, opening an opportunity for their use in the genetic improvement of varieties.

A collection of 317 table-grape accessions preserved in the Vitis Germplasm Bank at the Rancho de la Merced (IFAPA, Jerez de la Frontera, Spain) were genotyped at 20 microsatellite loci. A total of 264 different genotypes were obtained. Among them, 66 are presented for the first time. Twenty-one accession names are considered new synonyms, fourteen homonyms and 48 can be considered as "denomination mistakes" in the Rancho de la Merced collection.

[Background], The mountainous region between the Caucasus and China is considered to be the center of domestication for grapevine. Despite the importance of Central Asia in the history of grape growing, information about the extent and distribution of grape genetic variation in this region is limited in comparison to wild and cultivated grapevines from around the Mediterranean basin. The principal goal of this work was to survey the genetic diversity and relationships among wild and cultivated grape germplasm from the Caucasus, Central Asia, and the Mediterranean basin collectively to understand gene flow, possible domestication events and adaptive introgression. [Results], A total of 1378 wild and cultivated grapevines collected around the Mediterranean basin and from Central Asia were tested with a set of 20 nuclear SSR markers. Genetic data were analyzed (Cluster analysis, Principal Coordinate Analysis and STRUCTURE) to identify groups, and the results were validated by Nei’s genetic distance, pairwise FST analysis and assignment tests. All of these analyses identified three genetic groups: G1, wild accessions from Croatia, France, Italy and Spain; G2, wild accessions from Armenia, Azerbaijan and Georgia; and G3, cultivars from Spain, France, Italy, Georgia, Iran, Pakistan and Turkmenistan, which included a small group of wild accessions from Georgia and Croatia. Wild accessions from Georgia clustered with cultivated grape from the same area (proles pontica), but also with Western Europe (proles occidentalis), supporting Georgia as the ancient center of grapevine domestication. In addition, cluster analysis indicated that Western European wild grapes grouped with cultivated grapes from the same area, suggesting that the cultivated proles occidentalis contributed more to the early development of wine grapes than the wild vines from Eastern Europe. [Conclusions], The analysis of genetic relationships among the tested genotypes provided evidence of genetic relations

Understanding grape berry development and the metabolism of different classes of compounds responsible for traits like berry's color is imperative to control and improve quality aspects of grapes. A colorimetric, biochemical and molecular characterization allowed the comprehensive description of the pigment-related characteristics of nine berry skin color somatic variants, belonging to four different varieties. Although the observed berry skin color variability was not fully explained by MybA locus, the phenolic profiles allowed inferring about specific interferences among the biosynthetic pathways. Data were consistent concerning that grapes showing cyanidin-3-O-glucoside as the major anthocyanin and flavonols with two substituent groups in the lateral B-ring are generally originated by a white ancestor. After retro-mutation, these grapes seem to keep the dysfunction on flavonoid hydroxylases enzymes, which negatively affect the synthesis of both flavonols and anthocyanins with three substituent groups in the lateral B-ring. Overall, the obtained results indicate that the color differences observed between somatic variants are not solely the result of the total amount of compounds synthesized, but rather reflect a different dynamics of the phenolic pathway among the different color variants of the same variety. Chemical compounds Gallic acid (PubChem CID 370); Caftaric acid (PubChem CID 6,440,397); Catechin (PubChem CID 73,160); Epigallocatechin gallate (PubChem CID 65,064); Quercetin-3-O-galactoside (PubChem CID 5,281,643); Quercetin-3-O-glucoside (PubChem CID 25,203,368); Malvidin-3-O-glucoside (PubChem CID 443,652); Peonidin-3-O-p-coumaroylglucoside (PubChem CID: 44,256,849); Malvidin-3-O-p-coumaroylglucoside (PubChem CID: 44,256,988); Resveratrol-3-O-glucoside (PubChem CID: 25,579,167). © 2017 Elsevier Ltd

Wild grapevine is becoming a threatened species in the Iberian Peninsula due to human impacts. The aim of this work was to carry out a holistic study for six years of the largest wild grapevine population found up to date in SW Iberian Peninsula. This population has 115 vines. Ampelographic and soil characteristics have been studied. Evaluation of its environment has also been studied by describing the main parasitic species and natural enemies of pests. The ability of this plant material for its micropropagation and storage in slow-growth conditions has been tested. Microvinification resulted in a wine with good acidity and medium color intensity, two interesting characteristics under a warm climatology. Finally, the identification of private alleles in this wild population, absent in other locations from the Northern and Southern Iberian territories, is a very valuable feature and confirms the importance of establishing conservation programs. The population here studied is genetically unique and potentially useful for commercial rootstocks and cultivars breeding that would improve viticulture and enology.

In this work, a variety of biochemical properties of Enterococcus faecium CECT 8849, which had been isolated from breast milk, were analyzed. Its acidifying capacity and proteolytic activity were low but, in contrast, remarkable peptidase and esterase activities were observed. Ethanol and 3-hydroxy-2-butanone were the most abundant volatile compounds found in experimental model cheese manufactured with E. faecium CECT 8849. This strain inhibited the growth of several Listeria monocytogenes and Listeria innocua strains in vitro. Enterocin A and B structural genes were detected in E. faecium CECT 8849. Model fermented milk and cheeses were manufactured from milk inoculated or not with L. innocua CECT 8848 (2.5–3 log10 colony forming units mL−1) using E. faecium CECT 8849 or Lactococcus lactis ESI 153 as starter cultures. Although E. faecium CECT 8849 controlled Listeria growth in both dairy models, it led to lower reduction in Listeria counts when compared with L. lactis ESI 153. © 2016, Springer-Verlag Berlin Heidelberg.

Wild grapevine is becoming a threatened species in the Iberian Peninsula due to human impacts. The aim of this work was to carry out a holistic study for six years of the largest wild grapevine population found up to date in SW Iberian Peninsula. This population has 115 vines. Ampelographic and soil characteristics have been studied. Evaluation of its environment has also been studied by describing the main parasitic species and natural enemies of pests. The ability of this plant material for its micropropagation and storage in slow-growth conditions has been tested. Microvinification resulted in a wine with good acidity and medium color intensity, two interesting characteristics under a warm climatology. Finally, the identification of private alleles in this wild population, absent in other locations from the Northern and Southern Iberian territories, is a very valuable feature and confirms the importance of establishing conservation programs. The population here studied is genetically unique and potentially useful for commercial rootstocks and cultivars breeding that would improve viticulture and enology. © 2016 INIA.

A germplasm set of twenty-five grapevine accessions, forming eleven groups of possible berry skin color mutants, were genotyped with twelve microsatellite loci, being eleven of them identified as true color mutants. The polyphenolic profiling of the confirmed mutant cultivars revealed a total of twenty-four polyphenols, comprising non-colored compounds (phenolic acids, flavan-3-ols, flavonols and a stilbene) and anthocyanins. Results showed differences in the contribution of malvidin-3-O-glucoside to the characteristic Pinot Noir anthocyanins profile. Regarding the two Pique-Poul colored variants, the lighter variant was richer than the darker one in all classes of compounds, excepting anthocyanins. In Moscatel Galego Roxo the F3′H pathway seems to be more active than F3′5′H, resulting in higher amounts of cyanidin, precursor of the cyanidin derivatives. As far as we are aware, this is the first time that a relationship between the content of polyphenolic compounds is established in groups of grape berry skin color mutant cultivars. © 2015 Elsevier Ltd. All rights reserved.

Grape skin color is among the most important qualitative traits on which selection is based in wine and table grape breeding programmes. Skin color is determined by the quantity and composition of anthocyanins. In prior work on cultivated forms, it was shown that polymorphisms in the grape transcription factor family VvMYBA are responsible for anthocyanin content variation in the berries of cultivated grapevine (Vitis vinifera subsp. vinifera). Wild grapevine (V. vinifera subsp. sylvestris) is the ancestor of the cultivated V. vinifera subsp. sativa and has black-colored berries. The purpose of this study was to determine how the VvmybA1 and VvmybA2 polymorphisms emerged and affected the genetic diversity of wild grapevines in the Mediterranean basin by examining samples from the Iberian Peninsula, Italian Peninsula and Caucasian region. Our observations provide evidence that variation in the two transcriptional regulators generated a novel allele series via length polymorphisms in VvmybA1 and a point mutation in VvmybA2, which is lacking in cultivated grapevine. Further, correlation was detected between allele composition and anthocyanin contents. According to polymorphisms in both VvMYBA genes at the color locus, we were able to identify several haplotypes. The most ancestral haplotype (HapN) was found in wild grapevine in the western Mediterranean region and corresponded to wine grape cultivars, whereas recent haplotypes were detected in eastern regions. These eastern zones showed the most diverse haplotypes, which appeared in table cultivars where intense breeding practices may have replaced the original haplotype diversity. These findings provide information about the evolution of grapes since their domestication and have direct implications for wine quality. © 2014, Springer-Verlag Wien.

Berry texture is a determinant quality trait in the breeding of new table grape varieties. We present the first mapping quantitative trait loci (QTL) results for berry firmness in table grape. Two segregating progenies, Muscat Hamburg × Sugraone (153 offspring) and Ruby Seedless × Moscatuel (78 offspring), were evaluated for this trait during two and four productive seasons, respectively. Firmness was scored at harvest as the force (newtons) required for a 20 % deformation of the berries. Parental and consensus genetic maps were built for each population. QTL analyses revealed a complex genetic control of firmness. Significant QTLs at genome-wide level were detected in seven genomic regions on linkage groups (LGs) 1, 4, 5, 9, 10, 13, and 18, individually explaining up to 19.8 % of the total phenotypic variance. The combined effect of all the QTLs detected in one season explained up to 44.5 % of the total phenotypic variance. Some annotated genes colocating with the LOD-1 support intervals of those QTLs are proposed as putative candidate genes that might be responsible for the QTLs that affect berry firmness. © 2014, Springer-Verlag Berlin Heidelberg.

Aim Assessing levels and patterns of population genetic variation in combination with morphological characterization represents an important step for evaluating rare or endangered species and determining appropriate conservation strategies. This is particularly important for ensuring the preservation of valuable genetic variation in wild relatives of crops, which could provide beneficial alleles for plant breeding and improvement. Methods and results A survey of the relict wild grapevine population was carried out in the province of Burgos, in the Castilian and Leon region (Spain). Genetic diversity, inbreeding extent and possible hybridization with cultivated grapevine were investigated using molecular markers. Results showed that overall, genetic diversity was low but inbreeding was not present. Private alleles were found in the Burgos wild genotypes, suggesting the potential value of these accessions. Comparisons of morphological traits and molecular features among the wild population showed a close genetic relationship among them and no genetic relationship to Castilian grapevine cultivars. Conclusion The genetic differentiation observed between wild and domesticated forms of grapevine points out the interest to characterize and conserve the existing populations as a source of novel alleles for future grapevine breeding programs. At the same time, the observed enological differences between wild and domesticated grapevine populations could be interesting for the wine industry. Finally, given that these populations are at severe risk of extinction, we recommend that this population be prioritized for ex situ and possibly on-farm conservation as well as in situ protection. © 2014 Vigne et Vin Publications Internationales (Bordeaux, France).

Background In Vitis vinifera L.;domestication induced a dramatic change in flower morphology the wild sylvestris subspecies is dioecious while hermaphroditism is largely predominant in the domesticated subsp. V. v. vinifera. The characterisation of polymorphisms in genes underlying the sex-determining chromosomal region may help clarify the history of domestication in grapevine and the evolution of sex chromosomes in plants. In the genus Vitis, sex determination is putatively controlled by one major locus with three alleles, male M, hermaphrodite H and female F, with an allelic dominance M > H > F. Previous genetic studies located the sex locus on chromosome 2. We used DNA polymorphisms of geographically diverse V. vinifera genotypes to confirm the position of this locus, to characterise the genetic diversity and traces of selection in candidate genes, and to explore the origin of hermaphroditism. Results In V. v. sylvestris, a sex-determining region of 154.8 kb, also present in other Vitis species, spans less than 1% of chromosome 2. It displays haplotype diversity, linkage disequilibrium and differentiation that typically correspond to a small XY sex-determining region with XY males and XX females. In male alleles, traces of purifying selection were found for a trehalose phosphatase, an exostosin and a WRKY transcription factor, with strikingly low polymorphism levels between distant geographic regions. Both diversity and network analysis revealed that H alleles are more closely related to M than to F alleles. Conclusions Hermaphrodite alleles appear to derive from male alleles of wild grapevines, with successive recombination events allowing import of diversity from the X into the Y chromosomal region and slowing down the expansion of the region into a full heteromorphic chromosome. Our data are consistent with multiple domestication events and show traces of introgression from other Asian Vitis species into the cultivated grapevine gene pool. © 2014 Picq et al.

Our current research is focused on the anthocyanin composition of female grape accessions, mostly Spanish, preserved at El Encín Germplasm Bank. In 2008, berries of 126 accessions were taken at maturity. After the extraction from grape skins, total anthocyans were determined by spectrophotometry, and the anthocyanin fingerprint of grapes by HPLC, considering the relative amount of 15 anthocyanins. Among those 126 accessions, 23 genotypes (18.25%) did not present acylated anthocyanins. Thus, those 23 genotypes presented a characteristic anthocyanin fingerprint, similar at a certain extent to that presented by some Rhine basin and Italian grape cultivars, e.g.;'Pinot Noir' and 'Gaglioppo'. Nevertheless, the absence of acylated anthocyanins has not been described in any Spanish grape cultivar. The examination of the anthocyanin fingerprint of wild grapes without acylated anthocyanins reveals that the regulation of the anthocyanin biosynthesis may differ in various wild grape accessions.

The wild grapevine, Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi, considered as the ancestor of the cultivated grapevine, is native from Eurasia. In Spain, natural populations of V. vinifera ssp. sylvestris can still be found along river banks. In this work, we have performed a wide search of wild grapevine populations in Spain and characterized the amount and distribution of their genetic diversity using 25 nuclear SSR loci. We have also analysed the possible coexistence in the natural habitat of wild grapevines with naturalized grapevine cultivars and rootstocks. In this way, phenotypic and genetic analyses identified 19% of the collected samples as derived from cultivated genotypes, being either naturalized cultivars or hybrid genotypes derived from spontaneous crosses between wild and cultivated grapevines. The genetic diversity of wild grapevine populations was similar than that observed in the cultivated group. The molecular analysis showed that cultivated germplasm and wild germplasm are genetically divergent with low level of introgression. Using a model-based approach implemented in the software structure, we identified four genetic groups, with two of them fundamentally represented among cultivated genotypes and two among wild accessions. The analyses of genetic relationships between wild and cultivated grapevines could suggest a genetic contribution of wild accessions from Spain to current Western cultivars. © 2011 Blackwell Publishing Ltd.

A wild grapevine population of 76 vines of Vitis vinifera L. subspecies sylvestris (Gmelin) Hegi was found along the Iregua river valley (Northeastern Spain), located in the Ibérica mountain-range. The characterization of this phytogenetic resource was based on an ampelographic description of the male and female individuals and an evaluation of their sanitary state. This shows that the imported North American downy and powdery mildews are the main pathogens, but no symptoms caused by phylloxera on roots were found. Different degrees of infestation caused by Eryophid mites, Colomerus vitis and Calipitrimerus vitis, were also registered on leaves. On the other hand, the red wines obtained show an alcoholic degree situated between 8.5 and 10.8, pH around 3.5 and a high intensity of colour, between 10.2 and, 11.3. The vinegar had only 3.5° of acetic acid. Its phenolic composition is similar to those reported from red and Sherry vinegars produced from cultivars. The genetic analysis based on 18 samples, using 16 nuclear microsatellites, shows a low genetic diversity (He = 0.45). This might be due to inbreeding caused by mating among siblings in this isolated population. The genetic comparison with the allowed cultivars of this Guarantee of Origin showed two distinct gene pools. So it indicated that there is no genetic contribution of these native vines to current instead of actual local varieties. It is necessary to preserve this phytogenetic resource to be used in breeding programs and to restore its destructed habitats by human different impacts.

Background In the Peruvian Coastal Desert, an archipelago of fog oases, locally called lomas, are centers of biodiversity and of past human activity. Fog interception by a tree canopy, dominated by the legume tree tara (Caesalpinia spinosa), enables the occurrence in the Atiquipa lomas (southern Peru) of an environmental island with a diverse flora and high productivity. Although this forest provides essential services to the local population, it has suffered 90% anthropogenic reduction in area. Restoration efforts are now getting under way, including discussion as to the most appropriate reference ecosystem to use. Methodology/Principal Findings Genetic diversity of tara was studied in the Atiquipa population and over a wide geographical and ecological range. Neither exclusive plastid haplotypes to loma formations nor clear geographical structuring of the genetic diversity was found. Photosynthetic performance and growth of seedlings naturally recruited in remnant patches of loma forest were compared with those of seedlings recruited or planted in the adjacent deforested area. Despite the greater water and nitrogen availability under tree canopy, growth of forest seedlings did not differ from that of those recruited into the deforested area, and was lower than that of planted seedlings. Tara seedlings exhibited tight stomatal control of photosynthesis, and a structural photoprotection by leaflet closure. These drought-avoiding mechanisms did not optimize seedling performance under the conditions produced by forest interception of fog moisture. Conclusions/Significance Both weak geographic partitioning of genetic variation and lack of physiological specialization of seedlings to the forest water regime strongly suggest that tara was introduced to lomas by humans. Therefore, the most diverse fragment of lomas is the result of landscape management and resource use by pre-Columbian cultures. We argue that an appropriate reference ecosystem for ecological restoration of

Anatolia (Asia Minor) is considered to be the cradle of viticulture, but wild grape accessions from this region have not been subjected to any genetic analysis. We present the first genetic characterization of wild grapes (Vitis vinifera subsp. sylvestris) from this region. Using 15 nuclear microsatellites, we genotyped 84 wild grape accessions collected from three Anatolian locations. The unweighted pair group method with arithmetic mean analysis revealed four genetic clusters that partially separated Anatolian wild grape populations. In general, accessions from geographically closer locations showed higher genetic similarities than those from more distant locations. In some cases, accessions from one population showed close genetic relationships to accessions from a different population. The genetic diversity between natural populations from both ends of the Mediterranean basin has shown higher genetic diversity in the Anatolian Peninsula than peripheral populations, suggesting that this area could be one centre of diversity of the species. The genetic relationship between wild and cultivated grapevine from Anatolian Peninsula indicated a clear separation between them. However, we cannot discarded a local genetic contribution. Overall, this study reveals useful information for management and potential utilization of Anatolian wild grape germplasm. © 2011 NIAB.

Tempranillo is one of the most widely cultivated grapevine varieties in Spain. After several years of clone selection, some highly recommended old clones have been identified in terms of both their qualitative and production characteristics. This study was designed to discriminate among 28 ancient clones of the cultivar Tempranillo (Vitis vinifera). DNA samples from clones were analysed using two different molecular markers; microsatellites or simple sequence repeats (SSR) and retrotransposons. The results of this study indicate that one variant genotype was expressed as three alleles. Further analysis revealed the presence of a chimera, in which the third allele was present in the leaf but not root or wood tissue, indicating a functionally double-layered apical meristem. The present research also showed that one of the retrotranposon marker was able to discriminate one grapevine clone (VP1) from the remaining clones.

This study investigated the anthocyanin composition of 21 mostly Spanish wild grapevine accessions preserved at El Encín Germoplasm Bank and selected in consideration of observed ampelographic differences and molecular characterization. Sampling was carried out in 2006, 2007, and 2008. After extraction from grape skins, total anthocyanins was determined by spectrophotometry and the anthocyanin fingerprint of grapes, based on 15 anthocyanin variables, was determined by HPLC. Total anthocyanin concentration was similar to that found in winegrape cultivars. The accessions studied showed considerable variability in their anthocyanin fingerprints and it was possible to distinguish several groups, similar to previous reports on the anthocyanin fingerprint of winegrapes. The anthocyanin composition of wild grapevine accessions was similar to that of cultivated grapes. Nevertheless, the presence of wild accessions with anthocyanin fingerprints uncommon or nonexistent in Spanish cultivated varieties suggests that the genetic variability related to anthocyanins in Spanish wild grapevine populations may be higher than that of cultivated varieties commonly considered of Spanish origin. © 2010 by the American Society for Enology and Viticulture. All rights reserved.

Nuclear and chloroplast microsatellite loci were used to genotype Moroccan grapevine accessions (Vitis vinifera L.) from germplasm collections and from plants cultivated in farm fields or found under wild conditions. The genetic diversity of the global Moroccan sample was equivalent to what has been described for cultivated grapevines from other regions around the Mediterranean basin. The predominant chlorotypes among the analyzed samples were A and C, which are detected at higher frequencies in western and eastern regions of the V. vinifera range. Comparison of Moroccan accession genotypes with published genotypes for other grape-vine cultivars shows the existence of multiple synonyms, especially between cultivars grown in Morocco and Spain, as expected given the long history of interaction in the region. Analysis of genetic relationships among Moroccan samples distinguished groups of cultivated and wild samples. The wild samples had chlorotype A, which could represent remnants of natural populations of V. vinifera ssp. sylvestris (Gmelin) Hegi, or chloro-type C, which could represent cultivar derivatives or ancient cultivars generated by spontaneous hybridization among cultivated grapevines. Together the results point out the diverse genetic origin of cultivated and wild samples of grapevine in Morocco and emphasize the need to further collect and characterize its cultivated and wild grapevine germplasm. © 2010 by the American Society for Enology and Viticulture. All rights reserved.

Microsatellite variation at the nuclear and chloroplast genomes was evaluated for wild European and wild American hops, in order to assess the genetic diversity and origin of cultivated hops. Seven nuclear loci and 32 chloroplast loci were used in the analysis of 182 hop accessions including wild European (68), wild American (48), and cultivars (66). A total of 116 alleles were identified using 7 nuclear microsatellites showing different averages of polymorphism and distribution in the wild American and European accessions and cultivars. Two main groups were established as revealed by several statistical analyses; one including European wild accessions and cultivars and a second group consisting of American wild accessions. Three polymorphic chloroplast microsatellite loci were detected, six alleles were scored which defined a total of five haplotypes that were exclusive or presented different distribution between American and European wild accessions. A major influence of the wild European haplotypes was detected among hop cultivars. To the best of our knowledge, this is the first work reporting the use of chloroplast microsatellites in hops. © 2009 Springer Science+Business Media B.V.

The genetic and epigenetic stability of hop accessions cryopreserved for one year or cold stored for three years was evaluated using several molecular markers (RAPD, AFLP, and MSAP). Clear, repetitive patterns were obtained among accessions and between control and treated samples. Although no genetic changes were detected among the control plants grown in the greenhouse and in vitro plants regenerated from slow-cooling cryopreserved shoot tips or cold stored in vitro shoots, MSAP analysis detected methylation changes in 36% of the loci. Nevertheless, only 2.6 to 9.8% of the detected changes could be ascribed to the conservation procedure and most of them seemed to be generated as a result of the in vitro introduction. Due to the number of accessions analysed (51) we can cautiously deduce that the genetic behaviour described in this work after cryopreservation or cold-storage protocols is common to most hop genotypes and these storage procedures are suitable for standard use. However, it is important to keep in mind the epigenetic changes produced, particularly during any in vitro processes. © Agricultural and Food.

Micropropagation is a widely used technique in hops (Humulus lupulus L.). However, to the best of our knowledge, the genetic and epigenetic stability of the microplants has never been tested before. In the present study, two hop accessions were established in vitro and micropropagated for 2 years. The genetic and epigenetic stability of the in vitro plants was analyzed with several molecular techniques random amplified DNA polymorphism (RAPD), retrotransposon microsatellite amplified polymorphism (REMAP), and methylation-sensitive amplification polymorphism (MSAP). No genetic variation among control and treated plants was found, even after 12 cycles of micropropagation. Epigenetic variation was detected, first, when field and in vitro samples were compared. Nearly a 30% of the detected fragments presented the same pattern of alterations in all the vitroplants. Second, lower levels of epigenetic variation were detected among plants from the different subcultures. Part of this detected variation seemed to be accumulated along the 12 sequential subcultures tested. © 2009 Elsevier GmbH. All rights reserved.

Conventional cold storage and cryopreservation methods for hops (Humulus lupulus L.) are available but, to our knowledge, the genetic and epigenetic stability of the recovered plants have not been tested. This study analyzed 51 accessions of hop using the molecular techniques, Random Amplified DNA Polymorphism (RAPD) and Amplified Fragment Length Polymorphism (AFLP), revealing no genetic variation among greenhouse-grown controls and cold stored or cryopreserved plants. Epigenetic stability was evaluated using Methylation Sensitive Amplified Polymorphism (MSAP). Over 36% of the loci were polymorphic when the cold and cryo-treated plants were compared to greenhouse plants. The main changes were demethylation events and they were common to the cryopreserved and cold stored plants indicating the possible effect of the in vitro establishment process, an essential step in both protocols. Protocol-specific methylation patterns were also detected indicating that both methods produced epigenetic changes in plants following cold storage and cryopreservation. © 2008 Elsevier Inc. All rights reserved.

The domestication of the Eurasian grape (Vitis vinifera ssp. sativa) from its wild ancestor (Vitis vinifera ssp. sylvestris) has long been claimed to have occurred in Transcaucasia where its greatest genetic diversity is found and where very early archaeological evidence, including grape pips and artefacts of a 'wine culture', have been excavated. Whether from Transcaucasia or the nearby Taurus or Zagros Mountains, it is hypothesized that this wine culture spread southwards and eventually westwards around the Mediterranean basin, together with the transplantation of cultivated grape cuttings. However, the existence of morphological differentiation between cultivars from eastern and western ends of the modern distribution of the Eurasian grape suggests the existence of different genetic contribution from local sylvestris populations or multilocal selection and domestication of sylvestris genotypes. To tackle this issue, we analysed chlorotype variation and distribution in 1201 samples of sylvestris and sativa genotypes from the whole area of the species' distribution and studied their genetic relationships. The results suggest the existence of at least two important origins for the cultivated germplasm, one in the Near East and another in the western Mediterranean region, the latter of which gave rise to many of the current Western European cultivars. Indeed, over 70% of the Iberian Peninsula cultivars display chlorotypes that are only compatible with their having derived from western sylvestris populations. © 2006 The Authors.

Organogenic calli induced from internodal segments were subcultured three times. Regenerated plants obtained from each subculture were analysed by molecular methods. No major genetic rearrangements were detected in the callus-derived plants since none of the amplified fragment-length polymorphism (AFLP) loci were found to be polymorphic. However, epigenetic changes due to a demethylation process were detected by methylation-sensitive amplified polymorphism (MSAP) technique. The results allowed inference of the possible relationship among the plants derived from different calli subcultures and the in vitro control. The plants recovered from the first and second callus subcultures clustered with the in vitro control pools in the phenogram while the regenerants from the third callus subculture showed the highest genetic distance with the controls. This is the first study reporting data about the genetic stability of callus-derived Humulus lupulus L. plants.

Two sequence tagged sites (STS) detection techniques were used in the identification of 25 hop cultivars. In cultivar identification, capillary electrophoresis is more efficient than autoradiography. First, it was more sensitive in detection of fragments that allowed observation of the instability in the locus 7a82 due to the small size differences of its alleles. Additionally, the fluorescent detection of fragments was a nonsubjective technique suitable of automation that increased the flexibility of work because no radioactive labeling or long vertical polyacrylamide electrophoresis was needed. Only two cultivars, Hersbrucker and Hallertauer, were indistinguishable, but further molecular analysis revealed that both were included in a separate cluster within the European hop cultivars. The statistical analysis of fluorescence and radioactive detection data including allelic frequencies, heterozygosity, and probability of identity are displayed in this study, which corroborates the following advantages of the capillary electrophoresis technique high sensitivity, amenable to automation, and its nonsubjectivity. Therefore, fluorescent-labeled primer microsatellite detection by capillary electrophoresis is a powerful tool in cultivar identification for hop breeders, merchants, and brewers. © 2005 American Society of Brewing Chemists, Inc.

Thirty five new grape microsatellite markers were developed under an international consortium involving AGROGENE. These loci were amplified in 41 Spanish cultivars of V. vinifera. Eleven of the markers were polymorphic and informative in V. vinifera. Twelve were monomorphic and of the remaining markers one was polymorphic but less useful because individuals amplified more than two bands and the rest had amplification problems. The number of alleles detected for the 11 informative markers ranged from 4 to 12, with heterozygosity values ranging from 0.6 to 0.8. Primer sequences are reported for these markers.

We have used nuclear and chloroplast molecular markers to genotype cultivated and wild accessions of Vitis vinifera L. from Tunisia and assess their genetic relationships. Fifty-five distinct genotypes were identified among 80 cultivated accessions, including 18 genotypic groups containing between 2 and 5 accessions per group. They could represent a total of 60 distinct cultivars owing to berry colour variation found within identical genotype groups. Most of the 55 genotypes represent unique table grape genotypes except for one of them that was found identical to the genotype of table grape cultivar Rosseti. Hybridization among cultivars as well as self pollinations seems to have played an important role in their origin since several groups of closely related cultivars were observed. Furthermore, a parentage analysis showed a high probability for a parent hybrid relationship within two groups of three cultivars. No strong genetic similarities were found between cultivated and wild samples indicating that the cultivated accessions do not derive from local Vitis vinifera L. populations but could have been introduced from other regions in historic times. © 2004 NRC Canada.

El hongo Fusarium oxysporum Sch.:Fr. presenta una elevada variabilidad biológica y genética que se manifiesta por la existencia de numerosas formas especializadas y razas. Con el fin de evaluar la relación genética entre diferentes formas especializadas se aplicaron dos tipos de marcadores genéticos. En primer lugar se secuenció la región ITS (internal transcribed spacer) del ADN ribosomal de 17 aislados pertenecientes a siete de dichas formas: F.o. melonis (4 aislados), F.o. dianthi (2), F.o. niveum (4), F.o. lycopersici (2), F.o. radicis-lycopersici (3), F.o. lagenaria (1) y F.o. luffae (1). El análisis de estas secuencias reveló que aislados de diferentes formas presentan una secuencia idéntica, mientras que aislados de la misma forma aparecen en grupos diferentes del correspondiente dendrograma. Esta observación se confirmó mediante marcadores AFLP de las mismas formas especializadas y 10 más pertenecientes a F.o. ciceris, F.o. cucumerinum, F. proliferatum y F.o. asparagi, así como dos aislados no patogénicos. El dendrograma obtenido no reveló ninguna estructuración genética de las 10 formas especializadas. Estos datos, que están en la línea de los obtenidos por otros autores, parecen indicar que algunas formas especializadas de F. oxysporum no constituyen linajes monofiléticos, ya que evolucionan de forma divergente. Parece más bien que genotipos muy diferentes podrían compartir factores similares de especifidad y especialización patogénica

Representative grapevine accessions (Vitis vinifera) cultivated in Spain under the names Garnacha and Garnacha Tintorera, as well as their synonyms, were analyzed to determine genetic diversity and relationships. Both varieties are characterized by high levels of intravarietal morphological variation. Results confirmed the monophyletic origin of the Garnacha variety, which is represented by a main genotype with several phenotypic variants, likely corresponding to somatic mutations. In contrast, Garnacha Tintorera was characterized as a genetically heterogeneous group, which included three different teinturier genotypes. Possible parentage relationships among the teinturier varieties were identified and further confirmed using microsatellites, showing that all are derived from crosses performed in the nineteenth century to improve color intensity of well-known red wine varieties.

The use of consensus chloroplast microsatellites primers for dicotyledonous chloroplast genomes revealed the existence of intra and interspecific length variation within the genus Vitis. Three chloroplast microsatellite loci were found to be polymorphic in samples of Vitis vinifera, Vitis berlandieri, Vitis riparia, and Vitis rupestris out of a total of 10 consensus primer pairs tested. These polymorphisms were always due to a variable number of mononucleotide residues within A and (or) T stretches in the amplified regions. Chloroplast microsatellite polymorphisms were used to demonstrate the maternal inheritance of chloroplast in V. vinifera and to characterise the chloroplast haplotypes present in wine grape cultivars of this species grown in Spain and Greece. The different distribution of haplotype frequencies in the two ends of the Mediterranean growth area suggests the existence of independent domestication events for grapevine. © 2002 NRC.

Plants of red fescue (Festuca rubra), a commercially important turf grass, are infected by the fungal endophyte Epichloë festucae in semiarid natural grasslands, known as dehesas, in western Spain. We used amplified fragment length polymorphism (AFLP) markers to analyse the genetic polymorphism existing in two natural populations of Epichloë festucae. Linkage disequilibrium and the presence of clonal lineages indicated that nonrecombinant asexual reproduction predominates in both populations. However, most genetic variation detected was found to occur within populations, with only a moderate amount of genetic differentiation between populations (FST 0.197). Overall, the study suggests that dehesa grasslands are useful reservoirs of Epichloë festucae endophytes, and provides information on population structure which is relevant to design sampling strategies.

Using reverse transcriptase-amplified fragment length polymorphism (RT-AFLP) analysis of differential mRNA expression and semiquantitative reverse transcriptase-polymerase chain reaction, we compared mRNA expression in bovine blastocysts from 4 sources, known to differ in quality in terms of their ability to withstand cryopreservation 1) in vitro culture in synthetic oviduct fluid of in vitro-matured (IVM)/in vitro fertilized (IVF) zygotes; 2) in vitro culture in TCM-199 supplemented with granulosa cells (coculture) of IVM/IVF zygotes; 3) in vivo culture in the ewe oviduct of IVM/IVF zygotes; or 4) superovulation, artificial insemination, and nonsurgical embryo recovery. Total mRNA was isolated from pools of blastocysts and reverse transcription was performed. Triplicate reactions from each sample were displayed, and only consistent banding variations were recorded. Using AFLP-differential display assay, we found that cDNA banding patterns are highly conserved between the 4 groups of blastocysts studied; however, there was a difference of 7% in bands either missing or expressed across the groups. Fifty bands were reamplified, and a sequence comparison search revealed similarity of 14 isolated fragments to ribosomal and mitochondrial genes, 16 matched to described cDNA, and 20 corresponded to unknown sequences that may represent novel genes. The study of 7 differentially expressed mRNAs known to be involved in developmental process in the embryo suggests roles for apoptosis, oxidative stress, gap junctions, and differentiation in the determination of embryo quality. The aberrant transcription patterns detected in in vitro-produced bovine embryos compared with those produced in vivo may explain their reduced quality in terms of viability after cryopreservation.

Two species have traditionally been considered within the genus Laurus L. nobilis L.;L. azorica (Seub.) Franco. The first is characterized by the presence of glabrous twig leaves and is located in the Mediterranean region. It can be found as cultivated or naturalized, and has been reported in Spain, France, Italy and Greece. L. azorica is characterized by the presence of densely tomentose to hirsute twig leaves and has been described in the Azores, Madeira and the Canary Islands. We have found that some natural populations of Laurus in Northern Spain, which are considered to belong to L. nobilis, have hirsute young buds, with wide variation in hair number and density, in contradiction with taxonomical descriptions reported for this species. In order to evaluate the genetic similarity between these Laurus populations and the two reported species, we have analyzed 14 populations of L. nobilis and L. azorica from different geographical areas, including the Iberian peninsula, the Canary and Madeira Islands, France and Italy, using amplified fragment length polymorphisms (AFLPs). UPGMA clustering and principal component analysis (PCA) of the AFLP data revealed a low genetic similarity between the Iberian populations, including populations from Northern Spain, and the rest of the populations analyzed from France and Italy. Moreover, laurel accessions from the Iberian peninsula showed higher genetic similarity to those from the Canary Islands and Madeira, originally identified as L. azorica, than to samples from populations along the Mediterranean area, morphologically classified as L. nobilis.

Diversity was analyzed in wild and cultivated Lactuca germplasm using molecular markers derived from resistance genes of the NBS-LRR type. Three molecular markers, one microsatellite marker and two SCAR markers that amplified LRR-encoding regions, were developed from sequences of resistance gene homologs at the main resistance gene cluster in lettuce. Variation for these markers were assessed in germplasm including accessions of cultivated lettuce, Lactuca sativa L.;three wild Lactuca spp.;L. serriola L.;L. saligna and L. virosa L. Diversity was also studied within and between natural populations of L. serriola from Israel and California; the former is close to the center of diversity for Lactuca spp. while the latter is an area of more recent colonization. Large numbers of haplotypes were detected indicating the presence of numerous resistance genes in wild species. The diversity in haplotypes provided evidence for gene duplication and unequal crossing-over during the evolution of this cluster of resistance genes. However, there was no evidence for duplications and deletions within the LRR-encoding regions studied. The three markers were highly correlated with resistance phenotypes in L. sativa. They were able to discriminate between accessions that had previously been shown to be resistant to all known isolates of Bremia lactucae. Therefore, these markers will be highly informative for the establishment of core collections and marker-aided selection. A hierarchical analysis of the population structure of L. serriola showed that countries, as well as locations, were significantly differentiated. These differences may reflect local founder effects and/or divergent selection.

The resistance mechanism to potato virus Y (PVY) in pepper plants homozygous for the recessive resistance allele ya (pr21) was investigated. The results of the fluorescence immunochemical analysis of protoplasts prepared from virus-inoculated plants or virus-inoculated protoplasts indicate that this resistance is due to impaired cellto-cell movement of the virus, rather than to an interference with virus replication.