Grapevine European catalogue: towards a comprehensive list

Starting from the 1950’s, different European countries have established official lists of grapevine varieties authorized to be cultivated. The objectives of such lists were to limit ambiguous denomination of varieties and the related trade conflicts, as well as the implementation of viticulture recovery politics through the support of certain varieties and the limitation of others. In the same direction, in 1968 the European Union (EU) decided to create a “common catalogue of varieties”, defined as the sum of current national catalogues (Council Directive of 9 April 1968 “on the marketing of material for the vegetative propagation of the vine”, 68/193/EEC, modified). These catalogues (also called “registers”) include all the wine varieties “classified” by each Member State within the framework of the Common Organisation of the Market in wine (Council Directive No 479/2008), as well as all the non-classified varieties (table grape, rootstocks, etc.). For this work, we took the opportunity of the European research program “GrapeGen06”, focused on the study and the preservation of grapevine genetic resources, to contribute to the establishment and improvement of such an European catalogue. The comprehensive survey aims at two objectives: to support the implementation of European regulations on propagation of grapevine plants within the EU, and to assess priority actions, by technical and research institutes, for the conservation of grapevine genetic resources.Peer reviewe

Bases moléculaires de la variation clonale chez la vigne (Vitis vinifera L.) (approche pangénomique)

L'exploitation de la variation clonale est une des voies d'amélioration utilisée chez un grand nombre de plantes d'intérêts agronomiques telles que la pomme de terre, le café et la vigne. En effet, après plusieurs cycles de reproduction végétative, des caractéristiques agronomiques stables apparaissent donnant naissance à une diversité phénotypique remarquable, appelée diversité clonale . Chez la vigne, cette diversité clonale est d'une importance majeure pour les viticulteurs puisqu'elle permet une amélioration variétale sans changer d'identité de cépage en conformité avec la réglementation fixée par Appellations d'Origine Protégée. L'hypothèse la plus parcimonieuse expliquant cette diversité phénotypique clonale est l'accumulation de mutations somatiques au cours des cycles de reproduction végétative. L'objectif de cette thèse a été de dresser un panorama le plus exhaustif possible des différents polymorphismes moléculaires entre les génomes de plusieurs clones. Dans un premier temps trois clones de Pinot ont été séquencés par la technique 454 GS-FLX puis dans un second temps 11 clones de quatre cépages ont été séquencés la technique Illumina HiSeq 2000. Afin d'analyser la grande quantité de données obtenues, nous avons construit un pipeline d'analyse (Bacchus pipeline) permettant d'identifier tous les types de polymorphismes moléculaires entre les différents génomes.Nos résultats permettent, pour la première fois un inventaire exhaustif des polymorphismes moléculaires dans un contexte multiplication végétatif. L'ensemble des mutations polymorphes entre deux clones a pu être identifié, SNPs, indels (2,5 SNPs et 11,5 indels par Mb en moyenne) ainsi que des variations d'ordre structural (larges insertions ou délétions) représentant la classe la plus fréquente (129 évènements par Mb entre deux clones en moyenne). Afin d'évaluer le polymorphisme d'insertion généré par ces éléments nous en avons étudié quatre par une approche S-SAP sur plusieurs niveaux de diversité (inter-espèces, inter-cépages, inter-clones et entre plusieurs tissus d'un même individu). L'analyse phylogénétique au niveau des espèces est conforme à celle réalisée avec d'autres types de marqueurs moléculaires (SSR, SNP). Cependant, une forte instabilité de ces insertions a été confirmée entre les clones et entre les tissus d'un même d'individu. L'identification des clones par une méthodologie moléculaire serait d'une grande importance pour la filère. Pour cet objectif, nos résultats indiquent que les mutations de types SNP et petits indels qui sont certes moins fréquentes que les variations structurales mais qui sont plus stables semblent plus pertinentes pour la mise en place d'une méthodologie d'identification des clonesClonal variation is considered as an effective contribution to breeding programs of vegetatively propagated species with major agronomical interest such as banana, potato, coffee and grape. Indeed, after several propagation cycles, stable and heritable phenotypic variations appear giving rise to a phenotypic variation termed clonal diversity . This clonal diversity is very important for wine-growers because it allows preserving cultivars identity in the strict respect of Appellation (A.O.P) wines specifications The most parsimonious hypothesis explaining clonal phenotypic diversity is the accumulation of somatic mutations. The objective of my thesis was to provide a broad description of molecular polymorphisms in the context of vegetative propagation. Three clones were first sequenced by 454 GS-FLX technology and eleven clones were then sequenced with Illumina Hiseq2000 technique. To analyse the high quantity of data obtained, we built a pipeline (Bacchus pipeline) allowing the identification of all existing molecular polymorphisms between different genomes.All polymorphism types were observed: indels and SNPs which have a low polymorphism frequency (2.5 SNPs and 11.5 indels per Mb between two clones in average) and structural variations (large insertions or deletions) which have a high polymorphism frequency (129 per Mb between two clones in average) but are unstable. To evaluate stability and polymorphism level of these transposable elements, we have studied 4 elements using S-SAP method at different diversity levels (inter-species, inter-cultivars, inter-clones and between organs/tissues of a single individual). Our interspecific phylogenetic analysis is similar to other phylogenies performed with SSR or SNPs markers. However, we confirm the high instability of these elements between clones and between tissues in single individuals.Clone identification through molecular methods would be of high significance for the wine industry. SNP or small indels mutations are less frequent but more stable than structural variation and could be used for accurate clone identification.MONTPELLIER-SupAgro La Gaillarde (341722306) / SudocSudocFranceF

Beyond seek and destroy: How to generate allelic series using genome editing tools

Genome editing tools have greatly facilitated the functional analysis of genes of interest by targeted mutagenesis. Many usable genome editing tools, including different site-specific nucleases and editor databases that allow single-nucleotide polymorphisms (SNPs) to be introduced at a given site, are now available. These tools can be used to generate high allelic diversity at a given locus to facilitate gene function studies, including examining the role of a specific protein domain or a single amino acid. We compared the effects, efficiencies and mutation types generated by our LbCPF1, SpCAS9 and base editor (BECAS9) constructs for the OsCAO1 gene. SpCAS9 and LbCPF1 have similar efficiencies in generating mutations but differ in the types of mutations induced, with the majority of changes being single-nucleotide insertions and short deletions for SpCAS9 and LbCPF1, respectively. The proportions of heterozygotes also differed, representing a majority in our LbCPF1, while with SpCAS9, we obtained a large number of biallelic mutants. Finally, we demonstrated that it is possible to specifically introduce stop codons using the BECAS9 with an acceptable efficiency of approximately 20%. Based on these results, a rational choice among these three alternatives may be made depending on the type of mutation that one wishes to introduce, the three systems being complementary. SpCAS9 remains the best choice to generate KO mutations in primary transformants, while if the desired gene mutation interferes with regeneration or viability, the use of our LbCPF1 construction will be preferred, because it produces mainly heterozygotes. LbCPF1 has been described in other studies as being as effective as SpCAS9 in generating homozygous and biallelic mutations. It will remain to be clarified in the future, whether the different LbCFP1 constructions have different efficiencies and determine the origin of these differences. Finally, if one wishes to specifically introduce stop codons, BECAS9 is a viable and efficient alternative, although it has a lower efficiency than SpCAS9 and LbCPF1 for creating KO mutations

A SNP transferability survey within the genus Vitis

<p>Abstract</p> <p>Background</p> <p>Efforts to sequence the genomes of different organisms continue to increase. The DNA sequence is usually decoded for one individual and its application is for the whole species. The recent sequencing of the highly heterozygous <it>Vitis vinifera </it>L. cultivar Pinot Noir (clone ENTAV 115) genome gave rise to several thousand polymorphisms and offers a good model to study the transferability of its degree of polymorphism to other individuals of the same species and within the genus.</p> <p>Results</p> <p>This study was performed by genotyping 137 SNPs through the SNPlex™ Genotyping System (Applied Biosystems Inc.) and by comparing the SNPlex sequencing results across 35 (of the 137) regions from 69 grape accessions. A heterozygous state transferability of 31.5% across the unrelated cultivars of <it>V. vinifera</it>, of 18.8% across the wild forms of <it>V. vinifera</it>, of 2.3% among non-<it>vinifera Vitis </it>species, and of 0% with <it>Muscadinia rotundifolia </it>was found. In addition, mean allele frequencies were used to evaluate SNP informativeness and develop useful subsets of markers.</p> <p>Conclusion</p> <p>Using SNPlex application and corroboration from the sequencing analysis, the informativeness of SNP markers from the heterozygous grape cultivar Pinot Noir was validated in <it>V. vinifera </it>(including cultivars and wild forms), but had a limited application for non-<it>vinifera Vitis </it>species where a resequencing strategy may be preferred, knowing that homology at priming sites is sufficient. This work will allow future applications such as mapping and diversity studies, accession identification and genomic-research assisted breeding within <it>V. vinifera</it>.</p

High quality phased assembly of grape genome offer new opportunities in chimera detection

In perennial plants and especially those propagated through cuttings, several genotypes can coexist in a single individual, thus leading to chimeras. When the variant induces a noticeable phenotype modification, it can lead to a new cultivar. Viticulture already took economic advantage of this natural phenomenon: for instance, the berry skin of ‘Pinot gris’ derived from ‘Pinot noir’ by the selection of a chimera. Chimeras could also impact other crucial traits without being visually identified. Periclinal chimera where the variant has entirely colonized a cell layer is the most stable and can be propagated through cuttings. In grapevine, two functional cell layers are present in leaves, L1 and L2. However, lateral roots are formed from the L2 cell layer only. Thus, comparing DNA sequences of roots and leaves could allow chimera detection. In this study we used new generation Hifi long reads sequencing and recent bioinformatics tools applied to ‘Merlot’ to detect periclinal chimeras. Sequencing of ‘Magdeleine Noire des Charentes’ and ‘Cabernet franc’, the parents of ‘Merlot’, allowed haplotype resolved assembly. Pseudomolecules were built with few contigs, in some occasions only one per chromosome. This high resolution allowed haplotype comparison. Annotation from PN40024 was transferred to all pseudomolecules. Through variant detection, periclinal chimeras were found on both haplotypes. These results open new perspectives on chimera detection, which is an important resource to improve cultivars through clonal selection or breed new ones. Detailed results will be presented and discussed

Construction of nested genetic core collections to optimize the exploitation of natural diversity in Vitis vinifera L. subsp. sativa

Background: The first high quality draft of the grape genome sequence has just been published. This is a critical step in accessing all the genes of this species and increases the chances of exploiting the natural genetic diversity through association genetics. However, our basic knowledge of the extent of allelic variation within the species is still not sufficient. Towards this goal, we constructed nested genetic core collections (G-cores) to capture the simple sequence repeat (SSR) diversity of the grape cultivated compartment (Vitis vinifera L. subsp. sativa) from the world's largest germplasm collection (Domaine de Vassal, INRA Hérault, France), containing 2262 unique genotypes. Results: Sub-samples of 12, 24, 48 and 92 varieties of V. vinifera L. were selected based on their genotypes for 20 SSR markers using the M-strategy. They represent respectively 58%, 73%, 83% and 100% of total SSR diversity. The capture of allelic diversity was analyzed by sequencing three genes scattered throughout the genome on 233 individuals: 41 single nucleotide polymorphisms (SNPs) were identified using the G-92 core (one SNP for every 49 nucleotides) while only 25 were observed using a larger sample of 141 individuals selected on the basis of 50 morphological traits, thus demonstrating the reliability of the approach. Conclusion: The G-12 and G-24 core-collections displayed respectively 78% and 88% of the SNPs respectively, and are therefore of great interest for SNP discovery studies. Furthermore, the nested genetic core collections satisfactorily reflected the geographic and the genetic diversity of grape, which are also of great interest for the study of gene evolution in this species

A small XY chromosomal region explains sex determination in wild dioecious V. vinifera and the reversal to hermaphroditism in domesticated grapevines

Publis014-agap-029Background 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. La vigne domestiquée, Vitis vinifera ssp. vinifera, cultivée pour la production de fruit et de vin à travers le monde, dérive de la vigne sauvage, Vitis vinifera ssp. sylvestris, sous-espèce endémique de l'Eurasie. Au cours de la domestication, le système de reproduction a évolué de la diécie à l'hermaphrodisme. Nous montrons que la région du sexe est sous le contrôle d'une région chromosomique qui couvre au maximum 154kpbp, moins de 1% du chromosome 2. La caractérisation de ce locus en terme de diversité haplotypique, de signature de sélection et de déséquilibre de liaison a permis de révéler un système de détermination sexuelle de type XY. La petite taille de cette région chromosomique semble indiquer un stade très précoce dans l'évolution de chromosomes sexuels, malgré que la diécie soit le trait ancestral chez toutes les espèces de Vitis, ayant divergé du sous-genre Muscadinia il y a plusieurs millions d'années. L'analyse des distances génétiques entre haplotypes dans le locus du sexe a révélé que l'hermaphrodisme observé chez la vigne domestiquée résulte de la mutation de l'allèle mâle présent chez la vigne sauvage. Le réseau d'haplotypes a montré qu'en plus de la contribution de V. sylvestris, une autre espèce de Vitis asiatique a pu contribuer à la constitution du génome actuel de la vigne cultivée moderne. Ces travaux résultent d'une collaboration entre l'équipe DAAV d'AGAP et l'UMR CBAE (Montpellier)

Dissecting genetic architecture of grape proanthocyanidin composition through quantitative trait locus mapping

<p>Abstract</p> <p>Background</p> <p>Proanthocyanidins (PAs), or condensed tannins, are flavonoid polymers, widespread throughout the plant kingdom, which provide protection against herbivores while conferring organoleptic and nutritive values to plant-derived foods, such as wine. However, the genetic basis of qualitative and quantitative PA composition variation is still poorly understood. To elucidate the genetic architecture of the complex grape PA composition, we first carried out quantitative trait locus (QTL) analysis on a 191-individual pseudo-F1 progeny. Three categories of PA variables were assessed: total content, percentages of constitutive subunits and composite ratio variables. For nine functional candidate genes, among which eight co-located with QTLs, we performed association analyses using a diversity panel of 141 grapevine cultivars in order to identify causal SNPs.</p> <p>Results</p> <p>Multiple QTL analysis revealed a total of 103 and 43 QTLs, respectively for seed and skin PA variables. Loci were mainly of additive effect while some loci were primarily of dominant effect. Results also showed a large involvement of pairwise epistatic interactions in shaping PA composition. QTLs for PA variables in skin and seeds differed in number, position, involvement of epistatic interaction and allelic effect, thus revealing different genetic determinisms for grape PA composition in seeds and skin. Association results were consistent with QTL analyses in most cases: four out of nine tested candidate genes (<it>VvLAR1</it>, <it>VvMYBPA2</it>, <it>VvCHI1</it>, <it>VvMYBPA1</it>) showed at least one significant association with PA variables, especially <it>VvLAR1 </it>revealed as of great interest for further functional investigation. Some SNP-phenotype associations were observed only in the diversity panel.</p> <p>Conclusions</p> <p>This study presents the first QTL analysis on grape berry PA composition with a comparison between skin and seeds, together with an association study. Our results suggest a complex genetic control for PA traits and different genetic architectures for grape PA composition between berry skin and seeds. This work also uncovers novel genomic regions for further investigation in order to increase our knowledge of the genetic basis of PA composition.</p

New stable QTLs for berry weight do not colocalize with QTLs for seed traits in cultivated grapevine (Vitis vinifera L.)

International audienceBACKGROUND: In grapevine, as in other fruit crops, fruit size and seed content are key components of yield and quality; however, very few Quantitative Trait Loci (QTLs) for berry weight and seed content (number, weight, and dry matter percentage) have been discovered so far. To identify new stable QTLs for marker-assisted selection and candidate gene identification, we performed simultaneous QTL detection in four mapping populations (seeded or seedless) with various genetic backgrounds. RESULTS: For berry weight, we identified five new QTLs, on linkage groups (LGs) 1, 8, 11, 17 and 18, in addition to the known major QTL on LG 18. The QTL with the largest effect explained up to 31% of total variance and was found in two genetically distant populations on LG 17, where it colocalized with a published putative domestication locus. For seed traits, besides the major QTLs on LG 18 previously reported, we found four new QTLs explaining up to 51% of total variance, on LGs 4, 5, 12 and 14. The previously published QTL for seed number on LG 2 was found related in fact to sex. We found colocalizations between seed and berry weight QTLs only for the major QTL on LG 18 in a seedless background, and on LGs 1 and 13 in a seeded background. Candidate genes belonging to the cell number regulator CNR or cytochrome P450 families were found under the berry weight QTLs on LGs 1, 8, and 17. The involvement of these gene families in fruit weight was first described in tomato using a QTL-cloning approach. Several other interesting candidate genes related to cell wall modifications, water import, auxin and ethylene signalling, transcription control, or organ identity were also found under berry weight QTLs. CONCLUSION: We discovered a total of nine new QTLs for berry weight or seed traits in grapevine, thereby increasing more than twofold the number of reliable QTLs for these traits available for marker assisted selection or candidate gene studies. The lack of colocalization between berry and seed QTLs suggests that these traits may be partly dissociated