A high-density integrated map for grapevine based on three mapping populations genotyped by the Vitis18K SNP chip.

KEY MESSAGE: We present a high-density integrated map for grapevine, allowing refinement and improved understanding of the grapevine genome, while demonstrating the applicability of the Vitis18K SNP chip for linkage mapping. The improvement of grapevine through biotechnology requires identification of the molecular bases of target traits by studying marker-trait associations. The Vitis18K SNP chip provides a useful genotyping tool for genome-wide marker analysis. Most linkage maps are based on single mapping populations, but an integrated map can increase marker density and show order conservation. Here we present an integrated map based on three mapping populations. The parents consist of the well-known wine cultivars 'Cabernet Sauvignon', 'Corvina' and 'Rhine Riesling', the lesser-known wine variety 'Deckrot', and a table grape selection, G1-7720. Three high-density population maps with an average inter-locus gap ranging from 0.74 to 0.99 cM were developed. These maps show high correlations (0.9965-0.9971) with the reference assembly, containing only 93 markers with large order discrepancies compared to expected physical positions, of which a third is consistent across multiple populations. Moreover, the genetic data aid the further refinement of the grapevine genome assembly, by anchoring 104 yet unanchored scaffolds. From these population maps, an integrated map was constructed which includes 6697 molecular markers and reduces the inter-locus gap distance to 0.60 cM, resulting in the densest integrated map for grapevine thus far. A small number of discrepancies, mainly of short distance, involve 88 markers that remain conflictual across maps. The integrated map shows similar collinearity to the reference assembly (0.9974) as the single maps. This high-density map increases our understanding of the grapevine genome and provides a useful tool for its further characterization and the dissection of complex traits.

Functional characterisation of three members of the Vitis vinifera L. carotenoid cleavage dioxygenase gene family.

BACKGROUND: In plants, carotenoids serve as the precursors to C13-norisoprenoids, a group of apocarotenoid compounds with diverse biological functions. Enzymatic cleavage of carotenoids catalysed by members of the carotenoid cleavage dioxygenase (CCD) family has been shown to produce a number of industrially important volatile flavour and aroma apocarotenoids including ß-ionone, geranylacetone, pseudoionone, α-ionone and 3-hydroxy-ß-ionone in a range of plant species. Apocarotenoids contribute to the floral and fruity attributes of many wine cultivars and are thereby, at least partly, responsible for the "varietal character". Despite their importance in grapes and wine; carotenoid cleavage activity has only been described for VvCCD1 and the mechanism(s) and regulation of carotenoid catabolism remains largely unknown. RESULTS: Three grapevine-derived CCD-encoding genes have been isolated and shown to be functional with unique substrate cleavage capacities. Our results demonstrate that the VvCCD4a and VvCCD4b catalyse the cleavage of both linear and cyclic carotenoid substrates. The expression of VvCCD1, VvCCD4a and VvCCD4b was detected in leaf, flower and throughout berry development. VvCCD1 expression was constitutive, whereas VvCCD4a expression was predominant in leaves and VvCCD4b in berries. A transgenic population with a 12-fold range of VvCCD1 expression exhibited a lack of correlation between VvCCD1 expression and carotenoid substrates and/or apocarotenoid products in leaves, providing proof that the in planta function(s) of VvCCD1 in photosynthetically active tissue is distinct from the in vitro activities demonstrated. The isolation and functional characterisation of VvCCD4a and VvCCD4b identify two additional CCDs that are functional in grapevine. CONCLUSIONS: Taken together, our results indicate that the three CCDs are under various levels of control that include gene expression (spatial and temporal), substrate specificity and compartmentalisation that act individually and/or co-ordinately to maintain carotenoid and volatile apocarotenoid levels in plants. Altering the expression of VvCCD1 in a transgenic grapevine population illustrated the divergence between the in vitro enzyme activity and the in planta activity of this enzyme, thereby contributing to the efforts to understand how enzymatic degradation of carotenoids involved in photosynthesis occurs. The identification and functional characterisation of VvCCD4a and VvCCD4b suggest that these enzymes are primarily responsible for catalysing the cleavage of plastidial carotenoids.

The genes and enzymes of the carotenoid metabolic pathway in Vitis vinifera L.

BACKGROUND: Carotenoids are a heterogeneous group of plant isoprenoids primarily involved in photosynthesis. In plants the cleavage of carotenoids leads to the formation of the phytohormones abscisic acid and strigolactone, and C13-norisoprenoids involved in the characteristic flavour and aroma compounds in flowers and fruits and are of specific importance in the varietal character of grapes and wine. This work extends the previous reports of carotenoid gene expression and photosynthetic pigment analysis by providing an up-to-date pathway analysis and an important framework for the analysis of carotenoid metabolic pathways in grapevine. RESULTS: Comparative genomics was used to identify 42 genes putatively involved in carotenoid biosynthesis/catabolism in grapevine. The genes are distributed on 16 of the 19 chromosomes and have been localised to the physical map of the heterozygous ENTAV115 grapevine sequence. Nine of the genes occur as single copies whereas the rest of the carotenoid metabolic genes have more than one paralogue. The cDNA copies of eleven corresponding genes from Vitis vinifera L. cv. Pinotage were characterised, and four where shown to be functional. Microarrays provided expression profiles of 39 accessions in the metabolic pathway during three berry developmental stages in Sauvignon blanc, whereas an optimised HPLC analysis provided the concentrations of individual carotenoids. This provides evidence of the functioning of the lutein epoxide cycle and the respective genes in grapevine. Similarly, orthologues of genes leading to the formation of strigolactone involved in shoot branching inhibition were identified: CCD7, CCD8 and MAX1. Moreover, the isoforms typically have different expression patterns, confirming the complex regulation of the pathway. Of particular interest is the expression pattern of the three VvNCEDs: Our results support previous findings that VvNCED3 is likely the isoform linked to ABA content in berries. CONCLUSIONS: The carotenoid metabolic pathway is well characterised, and the genes and enzymes have been studied in a number of plants. The study of the 42 carotenoid pathway genes of grapevine showed that they share a high degree of similarity with other eudicots. Expression and pigment profiling of developing berries provided insights into the most complete grapevine carotenoid pathway representation. This study represents an important reference study for further characterisation of carotenoid biosynthesis and catabolism in grapevine.