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.

Ethylene Production Affects Blueberry Fruit Texture and Storability.

Ethylene, produced endogenously by plants and their organs, can induce a wide array of physiological responses even at very low concentrations. Nevertheless, the role of ethylene in regulating blueberry (Vaccinium spp.) ripening and storability is still unclear although an increase in ethylene production has been observed in several studies during blueberry ripening. To overcome this issue, we evaluated the endogenous ethylene production of a Vaccinium germplasm selection at different fruit ripening stages and after cold storage, considering also textural modifications. Ethylene and texture were further assessed also on a bi-parental full-sib population of 124 accessions obtained by the crossing between "Draper" and "Biloxi", two cultivars characterized by a different chilling requirement and storability performances. Our results were compared with an extensive literature research, carried out to collect all accessible information on published works related to Vaccinium ethylene production and sensitivity. Results of this study illustrate a likely role of ethylene in regulating blueberry shelf life. However, a generalisation valid for all Vaccinium species is not attainable because of the high variability in ethylene production between genotypes, which is strictly genotype-specific. These differences in ethylene production are related with blueberry fruit storage performances based on textural alterations. Specifically, blueberry accessions characterized by the highest ethylene production had a more severe texture decay during storage. Our results support the possibility of tailoring ad hoc preharvest and postharvest strategies to extend blueberry shelf life and quality according with the endogenous ethylene production level of each cultivar.

Candidate genes and SNPs associated with stomatal conductance under drought stress in Vitis.

BACKGROUND: Understanding the complexity of the vine plant's response to water deficit represents a major challenge for sustainable winegrowing. Regulation of water use requires a coordinated action between scions and rootstocks on which cultivars are generally grafted to cope with phylloxera infestations. In this regard, a genome-wide association study (GWAS) approach was applied on an 'ad hoc' association mapping panel including different Vitis species, in order to dissect the genetic basis of transpiration-related traits and to identify genomic regions of grape rootstocks associated with drought tolerance mechanisms. The panel was genotyped with the GrapeReSeq Illumina 20 K SNP array and SSR markers, and infrared thermography was applied to estimate stomatal conductance values during progressive water deficit. RESULTS: In the association panel the level of genetic diversity was substantially lower for SNPs loci (0.32) than for SSR (0.87). GWAS detected 24 significant marker-trait associations along the various stages of drought-stress experiment and 13 candidate genes with a feasible role in drought response were identified. Gene expression analysis proved that three of these genes (VIT_13s0019g03040, VIT_17s0000g08960, VIT_18s0001g15390) were actually induced by drought stress. Genetic variation of VIT_17s0000g08960 coding for a raffinose synthase was further investigated by resequencing the gene of 85 individuals since a SNP located in the region (chr17_10,497,222_C_T) was significantly associated with stomatal conductance. CONCLUSIONS: Our results represent a step forward towards the dissection of genetic basis that modulate the response to water deprivation in grape rootstocks. The knowledge derived from this study may be useful to exploit genotypic and phenotypic diversity in practical applications and to assist further investigations.

Development of a Novel Phenotypic Roadmap to Improve Blueberry Quality and Storability.

Improved fruit quality and prolonged storage capability are key breeding traits for blueberry (Vaccinium spp.) fruit. Until now, breeding selection was mostly oriented on the amelioration of agronomic traits, such as flowering time, chilling requirement, or plant structure. Up until now, however, the storage effect on fruit quality has not been extensively studied, mostly because objective and handy phenotyping tools to evaluate quality traits were not available. In this study we are proposing a novel phenotyping protocol to support breeding selection and quality control within the entire blueberry production chain. Volatile organic compounds (VOCs) and texture traits, were measured by Proton Transfer Reaction- Time of Flight- Mass Spectrometry (PTR-ToF-MS) and a texture analyzer respectively, taking into consideration the influence of prolonged storage. The exploitation of the genetic variability existing within the investigated blueberry germplasm collection (including both southern and northern highbush, hybrids, and rabbiteyes) allowed the identification of the best performing cultivars, based on texture and VOCs variability, to be used as superior parental lines for future breeding programs. The comprehensive characterization of blueberry aroma allowed the identification of a wide array of spectrometric features, mostly related to aldehydes, alcohols, terpenoids, and esters, that can be used as putative biomarkers to rapidly evaluate the blueberry aroma variations related to genetic differences and storability. In addition, this study revealed a lack of straightforward relationship between harvest and postharvest quality features, that might be genotype-dependent.

Drawing Links from Transcriptome to Metabolites: The Evolution of Aroma in the Ripening Berry of Moscato Bianco (Vitis vinifera L.).

Monoterpenes confer typical floral notes to "Muscat" grapevine varieties and, to a lesser extent, to other aromatic non-Muscat varieties. Previous studies have led to the identification and functional characterization of some enzymes and genes in this pathway. However, the underlying genetic map is still far from being complete. For example, the specific steps of monoterpene metabolism and its regulation are largely unknown. With the aim of identifying new candidates for the missing links, we applied an integrative functional genomics approach based on the targeted metabolic and genome-wide transcript profiling of Moscato Bianco ripening berries. In particular, gas chromatography-mass spectrometry analysis of free and bound terpenoid compounds was combined with microarray analysis in the skins of berries collected at five developmental stages from pre-veraison to over-ripening. Differentially expressed metabolites and probes were identified in the pairwise comparison between time points by using the early stage as a reference. Metabolic and transcriptomic data were integrated through pairwise correlation and clustering approaches to discover genes linked with particular metabolites or groups of metabolites. These candidate transcripts were further checked for co-localization with quantitative trait loci (QTLs) affecting aromatic compounds. Our findings provide insights into the biological networks of grapevine secondary metabolism, both at the catalytic and regulatory levels. Examples include a nudix hydrolase as component of a terpene synthase-independent pathway for monoterpene biosynthesis, genes potentially involved in monoterpene metabolism (cytochrome P450 hydroxylases, epoxide hydrolases, glucosyltransferases), transport (vesicle-associated proteins, ABCG transporters, glutathione S-transferases, amino acid permeases), and transcriptional control (transcription factors of the ERF, MYB and NAC families, intermediates in light- and circadian cycle-mediated regulation with supporting evidence from the literature and additional regulatory genes with a previously unreported association to monoterpene accumulation).

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.

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.

Genetic diversity and population structure assessed by SSR and SNP markers in a large germplasm collection of grape.

BACKGROUND: The economic importance of grapevine has driven significant efforts in genomics to accelerate the exploitation of Vitis resources for development of new cultivars. However, although a large number of clonally propagated accessions are maintained in grape germplasm collections worldwide, their use for crop improvement is limited by the scarcity of information on genetic diversity, population structure and proper phenotypic assessment. The identification of representative and manageable subset of accessions would facilitate access to the diversity available in large collections. A genome-wide germplasm characterization using molecular markers can offer reliable tools for adjusting the quality and representativeness of such core samples. RESULTS: We investigated patterns of molecular diversity at 22 common microsatellite loci and 384 single nucleotide polymorphisms (SNPs) in 2273 accessions of domesticated grapevine V. vinifera ssp. sativa, its wild relative V. vinifera ssp. sylvestris, interspecific hybrid cultivars and rootstocks. Despite the large number of putative duplicates and extensive clonal relationships among the accessions, we observed high level of genetic variation. In the total germplasm collection the average genetic diversity, as quantified by the expected heterozygosity, was higher for SSR loci (0.81) than for SNPs (0.34). The analysis of the genetic structure in the grape germplasm collection revealed several levels of stratification. The primary division was between accessions of V. vinifera and non-vinifera, followed by the distinction between wild and domesticated grapevine. Intra-specific subgroups were detected within cultivated grapevine representing different eco-geographic groups. The comparison of a phenological core collection and genetic core collections showed that the latter retained more genetic diversity, while maintaining a similar phenotypic variability. CONCLUSIONS: The comprehensive molecular characterization of our grape germplasm collection contributes to the knowledge about levels and distribution of genetic diversity in the existing resources of Vitis and provides insights into genetic subdivision within the European germplasm. Genotypic and phenotypic information compared in this study may efficiently guide further exploration of this diversity for facilitating its practical use.

Functional effect of grapevine 1-deoxy-D-xylulose 5-phosphate synthase substitution K284N on Muscat flavour formation.

Grape berries of Muscat cultivars (Vitis vinifera L.) contain high levels of monoterpenols and exhibit a distinct aroma related to this composition of volatiles. A structural gene of the plastidial methyl-erythritol-phosphate (MEP) pathway, 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS), was recently suggested as a candidate gene for this trait, having been co-localized with a major quantitative trait locus for linalool, nerol, and geraniol concentrations in berries. In addition, a structured association study discovered a putative causal single nucleotide polymorphism (SNP) responsible for the substitution of a lysine with an asparagine at position 284 of the VvDXS protein, and this SNP was significantly associated with Muscat-flavoured varieties. The significance of this nucleotide difference was investigated by comparing the monoterpene profiles with the expression of VvDXS alleles throughout berry development in Moscato Bianco, a cultivar heterozygous for the SNP mutation. Although correlation was detected between the VvDXS transcript profile and the accumulation of free monoterpenol odorants, the modulation of VvDXS expression during berry development appears to be independent of nucleotide variation in the coding sequence. In order to assess how the non-synonymous mutation may enhance Muscat flavour, an in vitro characterization of enzyme isoforms was performed followed by in vivo overexpression of each VvDXS allele in tobacco. The results showed that the amino acid non-neutral substitution influences the enzyme kinetics by increasing the catalytic efficiency and also dramatically affects monoterpene levels in transgenic lines. These findings confirm a functional effect of the VvDXS gene polymorphism and may pave the way for metabolic engineering of terpenoid contents in grapevine.

A candidate gene association study on muscat flavor in grapevine (Vitis vinifera L.).

BACKGROUND: The sweet, floral flavor typical of Muscat varieties (Muscats), due to high levels of monoterpenoids (geraniol, linalool and nerol), is highly distinct and has been greatly appreciated both in table grapes and in wine since ancient times. Muscat flavor determination in grape (Vitis vinifera L.) has up to now been studied by evaluating monoterpenoid levels through QTL analysis. These studies have revealed co-localization of 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS) with the major QTL positioned on chromosome 5. RESULTS: We resequenced VvDXS in an ad hoc association population of 148 grape varieties, which included muscat-flavored, aromatic and neutral accessions as well as muscat-like aromatic mutants and non-aromatic offsprings of Muscats. Gene nucleotide diversity and intragenic linkage disequilibrium (LD) were evaluated. Structured association analysis revealed three SNPs in moderate LD to be significantly associated with muscat-flavored varieties. We identified a putative causal SNP responsible for a predicted non-neutral substitution and we discuss its possible implications for flavor metabolism. Network analysis revealed a major star-shaped cluster of reconstructed haplotypes unique to muscat-flavored varieties. Moreover, muscat-like aromatic mutants displayed unique non-synonymous mutations near the mutated site of Muscat genotypes. CONCLUSIONS: This study is a crucial step forward in understanding the genetic regulation of muscat flavor in grapevine and it also sheds light on the domestication history of Muscats. VvDXS appears to be a possible human-selected locus in grapevine domestication and post-domestication. The putative causal SNP identified in Muscat varieties as well as the unique mutations identifying the muscat-like aromatic mutants under study may be immediately applied in marker-assisted breeding programs aimed at enhancing fragrance and aroma complexity respectively in table grape and wine cultivars.

The 1-deoxy-D: -xylulose 5-phosphate synthase gene co-localizes with a major QTL affecting monoterpene content in grapevine.

Muscat flavor is a relevant trait both in winemaking and in fresh grape consumption. From a chemical point of view, it is strongly related to the accumulation of monoterpenes in berries. However, knowledge of the genetic mechanisms underlying its regulation is still limited. The objective of this study was to dissect the genetic determinism of aroma in grapevine by applying the analysis of quantitative trait loci (QTL) and the candidate gene (CG) approach. Two F(1) segregating progenies were evaluated through high-resolution gas chromatography-mass spectrometry (HRGC-MS) for the amounts of individual monoterpenes over 3 and 2 years. In the Italia x Big Perlon cross 34 CGs, chosen according to gene ontology (GO) terms, were placed on a complete map and tested for linkage with QTLs for linalool, nerol and geraniol levels. Two CGs mapped within a QTL for linalool content on LG 10. A third one co-localized with a major QTL for the level of the three monoterpenes on LG 5; this gene encodes 1-deoxy-D: -xylulose 5-phosphate synthase (DXS), which is the first enzyme in the plastidial pathway of terpene biosynthesis. Depending on these findings, we report the first in silico analysis of grapevine DXS genes based on the whole genome sequence. Further research on the functional significance of these associations might help to understand the genetic control of Muscat flavor.