Genetic Characterization by SSR Markers of a Comprehensive Wine Grape Collection Conserved at Rancho de la Merced (Andalusia, Spain).

The IFAPA research center "Rancho de la Merced" (Jerez, Spain) hosts one of the oldest and most diverse grapevine germplasm repositories in Europe, and is aimed at providing feasible solutions to deal with any agronomic trait by exploring its genetic variability and by means of association and Deoxyribonucleic Acid (DNA) editing studies. In this work, we focused on a wine and dual-use grapevine subcollection that consists of 930 accessions. Genetic analysis allowed to identify 521 unique genotypes. After comparing them with several databases, matches were found for 476 genetic profiles while the remaining 45 have not been previously described. Combination with clustering analysis suggested a total pool of 481 Vitis vinifera accessions that included some table cultivars. Several synonymies, homonymies and mislabeling have also been detected. Structure analysis allowed identifying six clusters according to eco-geographic cultivation areas and one additional group including non-vinifera accessions. Diversity analysis pointed out that Spanish Mediterranean varieties are genetically closer to oriental genotypes than to European varieties typical of oceanic and continental climates. The origin of Spanish varieties is discussed in depth considering our data and previous studies. Analysis of molecular variance partition confirmed a well-structured germplasm, although differentiation among groups had a much lower effect on genetic variability than differences within groups, which are strongly related to a very high heterozygosity. A core collection that covers all allele richness is proposed. It is constituted of about 13% of total accessions, and each cluster inferred by structure analysis is represented.

Somatic variants for seed and fruit set in grapevine.

BACKGROUND: Grapevine reproductive development has direct implications on yield. It also impacts on berry and wine quality by affecting traits like seedlessness, berry and bunch size, cluster compactness and berry skin to pulp ratio. Seasonal fluctuations in yield, fruit composition and wine attributes, which are largely driven by climatic factors, are major challenges for worldwide table grape and wine industry. Accordingly, a better understanding of reproductive processes such as gamete development, fertilization, seed and fruit set is of paramount relevance for managing yield and quality. With the aim of providing new insights into this field, we searched for clones with contrasting seed content in two germplasm collections. RESULTS: We identified eight variant pairs that seemingly differ only in seed-related characteristics while showing identical genotype when tested with the GrapeReSeq_Illumina_20K_SNP_chip and several microsatellites. We performed multi-year observations on seed and fruit set deriving from different pollination treatments, with special emphasis on the pair composed by Sangiovese and its seedless variant locally named Corinto Nero. The pollen of Corinto Nero failed to germinate in vitro and gave poor berry set when used to pollinate other varieties. Most berries from both open- and cross-pollinated Corinto Nero inflorescences did not contain seeds. The genetic analysis of seedlings derived from occasional Corinto Nero normal seeds revealed that the few Corinto Nero functional gametes are mostly unreduced. Moreover, three genotypes, including Sangiovese and Corinto Nero, were unexpectedly found to develop fruits without pollen contribution and occasionally showed normal-like seeds. Five missense single nucleotide polymorphisms were identified between Corinto Nero and Sangiovese from transcriptomic data. CONCLUSIONS: Our observations allowed us to attribute a seedlessness type to some variants for which it was not documented in the literature. Interestingly, the VvAGL11 mutation responsible for Sultanina stenospermocarpy was also discovered in a seedless mutant of Gouais Blanc. We suggest that Corinto Nero parthenocarpy is driven by pollen and/or embryo sac defects, and both events likely arise from meiotic anomalies. The single nucleotide polymorphisms identified between Sangiovese and Corinto Nero are suitable for testing as traceability markers for propagated material and as functional candidates for the seedless phenotype.

Genetic Resources of Olea europaea L. in the Garda Trentino Olive Groves Revealed by Ancient Trees Genotyping and Parentage Analysis of Drupe Embryos.

The area of the Garda Lake within the Trentino province (north of Italy) is the northernmost part of Europe where the Mediterranean species Olea europaea L. is traditionally cultivated. 'Casaliva' is claimed as the main variety traditionally grown in the Garda Trentino area (GT) from which a world renowned niche extra virgin olive oil is produced. Since a dominant presence of 'Casaliva' would link the fruit set success and yield to a self-pollination compatibility system, a deep genetic survey of the olive tree population in the GT has been performed with the aim of establishing the actual varietal composition and of understanding from which pollen donor the 'Casaliva' olives originate. Forty-four different genetic profiles were observed among the 205 leaf samples collected from 106 ancient trees through the analysis of 20 nuclear microsatellite markers. The varietal composition in modern orchards was also explored and the vast majority of the additional 151 trees analyzed showed the same genotype as the ancient accessions of 'Casaliva'. The results support the long historical link of 'Casaliva' with the GT and, besides a high varietal homogeneity, they also revealed the presence of olive genetic resources essential to fruit production. In fact, the parentage analysis of 550 embryos from drupes of 'Casaliva' evidenced that a cross-fertilization system is favored and a list of candidate cultivars most suitable as local pollinizers of 'Casaliva' was identified.

Induction of Terpene Biosynthesis in Berries of Microvine Transformed with VvDXS1 Alleles.

Terpenoids, especially monoterpenes, are major aroma-impact compounds in grape and wine. Previous studies highlighted a key regulatory role for grapevine 1-deoxy-D-xylulose 5-phosphate synthase 1 (VvDXS1), the first enzyme of the methylerythritol phosphate pathway for isoprenoid precursor biosynthesis. Here, the parallel analysis of VvDXS1 genotype and terpene concentration in a germplasm collection demonstrated that VvDXS1 sequence has a very high predictive value for the accumulation of monoterpenes and also has an influence on sesquiterpene levels. A metabolic engineering approach was applied by expressing distinct VvDXS1 alleles in the grapevine model system "microvine" and assessing the effects on downstream pathways at transcriptional and metabolic level in different organs and fruit developmental stages. The underlying goal was to investigate two potential perturbation mechanisms, the former based on a significant over-expression of the wild-type (neutral) VvDXS1 allele and the latter on the ex-novo expression of an enzyme with increased catalytic efficiency from the mutated (muscat) VvDXS1 allele. The integration of the two VvDXS1 alleles in distinct microvine lines was found to alter the expression of several terpenoid biosynthetic genes, as assayed through an ad hoc developed TaqMan array based on cDNA libraries of four aromatic cultivars. In particular, enhanced transcription of monoterpene, sesquiterpene and carotenoid pathway genes was observed. The accumulation of monoterpenes in ripe berries was higher in the transformed microvines compared to control plants. This effect is predominantly attributed to the improved activity of the VvDXS1 enzyme coded by the muscat allele, whereas the up-regulation of VvDXS1 plays a secondary role in the increase of monoterpenes.

Experimental Review of DNA-Based Methods for Wine Traceability and Development of a Single-Nucleotide Polymorphism (SNP) Genotyping Assay for Quantitative Varietal Authentication.

The genetic varietal authentication of wine was investigated according to DNA isolation procedures reported for enological matrices and also by testing 11 commercial extraction kits and various protocol modifications. Samples were collected at different stages of the winemaking process of renowned Italian wines Brunello di Montalcino, Lambruschi Modenesi, and Trento DOC. Results demonstrated not only that grape DNA loss is produced by the fermentation process but also that clarification and stabilization operations contribute to the reduction of double-stranded DNA content on wine. Despite the presence of inhibitors, downstream PCR genotyping yielded reliable nuclear and chloroplast SSR markers for must samples, whereas no amplification or inconsistent results were obtained at later stages of the vinification. In addition, a TaqMan genotyping assay based on cultivar-specific single-nucleotide polymorphisms (SNPs) was designed, which allowed assessment of grapevine DNA mixtures. Once the wine matrix limitations are overcome, this sensitive tool may be implemented for the relative quantification of cultivars used for blend wines or frauds.

Quantitative trait loci affecting pathogen resistance and ripening of grapevines.

Grapevines (Vitis vinifera L.) form the basis of viticulture, and are susceptible to diseases such as downy mildew (Plasmopara viticola) and powdery mildew (Erysiphe necator). Therefore, successful viticulture programs require the use of pesticides. Breeding for resistance is the only eco-friendly solution. Marker-assisted selection is currently widely used for grapevine breeding. Consequently, traits of interest must be tagged with molecular markers linked to quantitative trait loci (QTL). We herein present our findings regarding genetic mapping and QTL analysis of resistance to downy and powdery mildew diseases in the progenies of the GF.GA-47-42 ('Bacchus' × 'Seyval') × 'Villard blanc' cross. Simple sequence repeats and single nucleotide polymorphisms of 151 individuals were analyzed. A map consisting of 543 loci was screened for QTL analyses based on phenotypic variations observed in plants grown in the field or under controlled conditions. A major QTL for downy mildew resistance was detected on chromosome 18. For powdery mildew resistance, a QTL was identified on chromosome 15. This QTL was replaced by a novel QTL on chromosome 18 in 2003 (abnormally high temperatures) and 2004. Subsequently, both QTLs functioned together. Additionally, variations in the timing of the onset of veraison, which is a crucial step during grape ripening, were studied to identify genomic regions affecting this trait. A major QTL was detected on linkage group 16, which was supplemented by a minor QTL on linkage group 18. This study provides useful information regarding novel QTL-linked markers relevant for the breeding of disease-resistant grapevines adapted to current climatic conditions.

Molecular identification and genetic relationships of Palestinian grapevine cultivars.

Palestine has a wide range of agro-ecological concerns and hosts a large variety of plants. Grapes are part of the cultural heritage and provide an indispensable food ingredient. Local cultivars have been traditionally identified on the basis of morphological traits, geographical origin, or names of the vineyard owner; therefore, the occurrence of homonymy, synonymy, and misnaming significantly prevents their valorization. DNA profiling by 22 common SSR markers was used to characterize 43 putative cultivars grown mainly for local table grape consumption at the southern highland regions of West-Bank, to further evaluate genetic diversity and relationships of the population. Consistent matching of SSR markers with grapevines cultivated in neighboring countries or maintained in European germplasm collections was found for 8 of the 21 different non-redundant genotypes discovered, suggesting possible synonyms as well as the occurrence of breeding selections formerly developed in the USA. Genetic relationships inferred from SSR markers clearly assigned Palestinian cultivars to the Proles orientalis subpr. Antasiatica ancestral population, and they even remarked the connection between local resources and cultivars generated from international table grape breeding. This study supports the value of collection and conservation of vines endemic to a region of immense historical importance for viticulture.

Linkage mapping and molecular diversity at the flower sex locus in wild and cultivated grapevine reveal a prominent SSR haplotype in hermaphrodite plants.

Cultivars used for wine and table grape have self-fertile hermaphrodite flowers whereas wild European vines and American and Asian species are dioecious, having either male or female flowers. Consistent with previous studies, the flower sex trait was mapped as a single major locus on chromosome 2 based on a pure Vitis vinifera population segregating for hermaphrodite and female progeny, and a hybrid population producing all three flower sex types. The sex locus was placed between the same SSR and SNP markers on both genetic maps, although abnormal segregation hampered to fine map the genomic region. From a total of 55 possible haplotypes inferred for three SSR markers around the sex locus, in a population of 132 V. sylvestris accessions and 171 V. vinifera cultivars, one of them accounted for 66 % of the hermaphrodite individuals and may be the result of domestication. Specific size variants of the VVIB23 microsatellite sequence within the 3'-UTR of a putative YABBY1 gene were found to be statistically significantly associated with the sex alleles M, H and f; these markers can provide assistance in defining the status of wild grapevine germplasm.