Caractérisation de la variation phénotypique de la taille de la baie chez la vigne Vitis vinifera L. et approches de génétique d'association et de recherche de traces de sélection pour ce caractère

La taille du fruit est un caractère agronomique important pour le rendement des espèces cultivées.Or, excepté chez la tomate, les mécanismes impliqués dans la variation de la taille des fruits charnus sont encore mal connus. L'objectif de ma thèse était d'identifier les principaux facteurs anatomiques impliqués dans la variation de la taille des baies, de réaliser des tests d'association génétique sur des gènes candidats et de rechercher des possibles traces de domestication liées à la variation de la taille des baies. Le nombre de cellules déjà déterminé dans l ovaire avant l anthèse et la capacité des cellules du mésocarpe à grossir après l anthèse se sont révélés les principaux déterminants de la variation de la baie entre les variétés. Trois gènes potentiellement impliqués dans ce caractère ont été identifiés par des approches de génétique d'association. La MADS boxVvAGL11 est associée au nombre de pépins de la baie. VvHMGR1, une HMGR impliquée dans la synthèse des isoprénoïdes précurseurs de régulateurs de croissance, est associée à la longueur des baies. Enfin, VvJMJ un facteur de transcription de type jumonji est à la fois associé à la variation du poids et à la longueur des baies. La caractérisation du locus Fleshless berry (Flb) a également été poursuivie avec (i) l analyse du polymorphisme de séquence de 69 fragments de gènes entre les vignes sauvages et cultivées, ce qui a permis l identification d un gène présentant une trace de sélection et une diversité nucléotidique plus élevée dans le compartiment sauvage que dans lecultivé. (ii) La mise en évidence d une homozygotie complète d une région de 1 Mb contenant le locus Flb chez certains cultivars comme l Ugni Blanc, rendant impossible le clonage positionnel du gène dans la population F1. L ensemble de ce travail a permis de mettre en évidence la complexité des mécanismes à l origine de la variation de la taille de la baie chez la vigne. Nous avons montré la faisabilité des études de génétique d association chez la vigne en utilisant des gènes candidats pour le poids de baies, et identifié les candidats les plus intéressants pour la sélection assistée par marqueurs et les études fonctionnelles ultérieures.Fruit size is an important trait in fruit crops. However, excepted in tomato, little is known about the genetic and molecular control of fleshy fruit size variation. The aim of my thesis was to identify themain anatomical factors underlying berry size variation, to carry out an association genetics based approach with candidate genes and to search for possible signatures of domestication linked toberry size variation. Cell divisions before anthesis and cell expansion after anthesis were found tobe the major determinants of flesh weight variation between varieties in grapevine. Three geneswere identified by association genetics approaches. The VvAGL11 MADS box was found associated only with the seed number per berry. VvHMGR1, a putative HMGR involved in the synthesis of isoprenoid, a precursor of plant growth regulators, was found associated with berry length. Finally,VvJMJ encoding a putative transcription factor jumonji was associated both with berry weight andberry length variation. The characterization of the Fleshless berry locus (Flb) was also pursued with(i) the analysis of the sequence polymorphism of 69 regions between wild and cultivated grapevines,that leaded to the identification of one gene showing a trace of selection and a higher nucleotide diversity in the wild compartment than in the cultivated compartment. (ii) The discovery of acomplete homozygosity of a 1 Mb region containing the Flb locus in some cultivars such as theUgni Blanc, making impossible the fine mapping of the Flb locus in a F1 population. This study has highlighted the complexity of the mechanisms underlying the berry size variation in grapevines. We demonstrated the feasibility to screen in grapevine by association genetics candidate genes for berryweight and to select the most interesting ones for marker-assisted selection and for furtherfunctional studies.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine

<p>Abstract</p> <p>Background</p> <p>Stenospermocarpy is a mechanism through which certain genotypes of <it>Vitis vinifera </it>L. such as Sultanina produce berries with seeds reduced in size. Stenospermocarpy has not yet been characterized at the molecular level.</p> <p>Results</p> <p>Genetic and physical maps were integrated with the public genomic sequence of <it>Vitis vinifera </it>L. to improve QTL analysis for seedlessness and berry size in experimental progeny derived from a cross of two seedless genotypes. Major QTLs co-positioning for both traits on chromosome 18 defined a 92-kb confidence interval. Functional information from model species including <it>Vitis </it>suggested that <it>VvAGL11</it>, included in this confidence interval, might be the main positional candidate gene responsible for seed and berry development.</p> <p>Characterization of <it>VvAGL11 </it>at the sequence level in the experimental progeny identified several SNPs and INDELs in both regulatory and coding regions. In association analyses performed over three seasons, these SNPs and INDELs explained up to 78% and 44% of the phenotypic variation in seed and berry weight, respectively. Moreover, genetic experiments indicated that the regulatory region has a larger effect on the phenotype than the coding region. Transcriptional analysis lent additional support to the putative role of <it>VvAGL11's </it>regulatory region, as its expression is abolished in seedless genotypes at key stages of seed development. These results transform <it>VvAGL11 </it>into a functional candidate gene for further analyses based on genetic transformation.</p> <p>For breeding purposes, intragenic markers were tested individually for marker assisted selection, and the best markers were those closest to the transcription start site.</p> <p>Conclusion</p> <p>We propose that <it>VvAGL11 </it>is the major functional candidate gene for seedlessness, and we provide experimental evidence suggesting that the seedless phenotype might be caused by variations in its promoter region. Current knowledge of the function of its orthologous genes, its expression profile in <it>Vitis </it>varieties and the strong association between its sequence variation and the degree of seedlessness together indicate that the D-lineage MADS-box gene <it>VvAGL11 </it>corresponds to the <it>Seed Development Inhibitor locus </it>described earlier as a major locus for seedlessness. These results provide new hypotheses for further investigations of the molecular mechanisms involved in seed and berry development.</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

Patterns of sequence polymorphism in the fleshless berry locus in cultivated and wild Vitis vinifera accessions

<p>Abstract</p> <p>Background</p> <p>Unlike in tomato, little is known about the genetic and molecular control of fleshy fruit development of perennial fruit trees like grapevine (<it>Vitis vinifera </it>L.). Here we present the study of the sequence polymorphism in a 1 Mb grapevine genome region at the top of chromosome 18 carrying the <it>fleshless berry </it>mutation (<it>flb</it>) in order, first to identify SNP markers closely linked to the gene and second to search for possible signatures of domestication.</p> <p>Results</p> <p>In total, 62 regions (17 SSR, 3 SNP, 1 CAPS and 41 re-sequenced gene fragments) were scanned for polymorphism along a 3.4 Mb interval (85,127-3,506,060 bp) at the top of the chromosome 18, in both <it>V. vinifera cv</it>. Chardonnay and a genotype carrying the <it>flb </it>mutation, <it>V. vinifera cv</it>. Ugni Blanc mutant. A nearly complete homozygosity in Ugni Blanc (wild and mutant forms) and an expected high level of heterozygosity in Chardonnay were revealed. Experiments using qPCR and BAC FISH confirmed the observed homozygosity. Under the assumption that <it>flb </it>could be one of the genes involved into the domestication syndrome of grapevine, we sequenced 69 gene fragments, spread over the <it>flb </it>region, representing 48,874 bp in a highly diverse set of cultivated and wild <it>V. vinifera </it>genotypes, to identify possible signatures of domestication in the cultivated <it>V. vinifera </it>compartment. We identified eight gene fragments presenting a significant deviation from neutrality of the Tajima's D parameter in the cultivated pool. One of these also showed higher nucleotide diversity in the wild compartments than in the cultivated compartments. In addition, SNPs significantly associated to berry weight variation were identified in the <it>flb </it>region.</p> <p>Conclusions</p> <p>We observed the occurrence of a large homozygous region in a non-repetitive region of the grapevine otherwise highly-heterozygous genome and propose a hypothesis for its formation. We demonstrated the feasibility to apply BAC FISH on the very small grapevine chromosomes and provided a specific probe for the identification of chromosome 18 on a cytogenetic map. We evidenced genes showing putative signatures of selection and SNPs significantly associated with berry weight variation in the <it>flb </it>region. In addition, we provided to the community 554 SNPs at the top of chromosome 18 for the development of a genotyping chip for future fine mapping of the <it>flb </it>gene in a F2 population when available.</p

Identification of stable QTLs for vegetative and reproductive traits in the microvine (Vitis vinifera L.) using the 18 K Infinium chip

UMR AGAP - équipe DAAV - Diversité, adaptation et amélioration de la vigne[b]Background[/b] [br/]The increasing temperature associated with climate change impacts grapevine phenology and development with critical effects on grape yield and composition. Plant breeding has the potential to deliver new cultivars with stable yield and quality under warmer climate conditions, but this requires the identification of stable genetic determinants. This study tested the potentialities of the microvine to boost genetics in grapevine. A mapping population of 129 microvines derived from Picovine x Ugni Blanc flb, was genotyped with the Illumina® 18 K SNP (Single Nucleotide Polymorphism) chip. Forty-three vegetative and reproductive traits were phenotyped outdoors over four cropping cycles, and a subset of 22 traits over two cropping cycles in growth rooms with two contrasted temperatures, in order to map stable QTLs (Quantitative Trait Loci). [br/][b]Results[/b] [br/]Ten stable QTLs for berry development and quality or leaf area were identified on the parental maps. A new major QTL explaining up to 44 % of total variance of berry weight was identified on chromosome 7 in Ugni Blanc flb, and co-localized with QTLs for seed number (up to 76 % total variance), major berry acids at green lag phase (up to 35 %), and other yield components (up to 25 %). In addition, a minor QTL for leaf area was found on chromosome 4 of the same parent. In contrast, only minor QTLs for berry acidity and leaf area could be found as moderately stable in Picovine. None of the transporters recently identified as mutated in low acidity apples or Cucurbits were included in the several hundreds of candidate genes underlying the above berry QTLs, which could be reduced to a few dozen candidate genes when a priori pertinent biological functions and organ specific expression were considered. [br/][b]Conclusions[/b] [br/]This study combining the use of microvine and a high throughput genotyping technology was innovative for grapevine genetics. It allowed the identification of 10 stable QTLs, including the first berry acidity QTLs reported so far in a Vitis vinifera intra-specific cross. Robustness of a set of QTLs was assessed with respect to temperature variatio

Characterization of the berry size in grapevine Vitis vinifera L., association genetics approaches and search of traces of selection for this trait

La taille du fruit est un caractère agronomique important pour le rendement des espèces cultivées.Or, excepté chez la tomate, les mécanismes impliqués dans la variation de la taille des fruits charnus sont encore mal connus. L'objectif de ma thèse était d'identifier les principaux facteurs anatomiques impliqués dans la variation de la taille des baies, de réaliser des tests d'association génétique sur des gènes candidats et de rechercher des possibles traces de domestication liées à la variation de la taille des baies. Le nombre de cellules déjà déterminé dans l’ovaire avant l’anthèse et la capacité des cellules du mésocarpe à grossir après l’anthèse se sont révélés les principaux déterminants de la variation de la baie entre les variétés. Trois gènes potentiellement impliqués dans ce caractère ont été identifiés par des approches de génétique d'association. La MADS boxVvAGL11 est associée au nombre de pépins de la baie. VvHMGR1, une HMGR impliquée dans la synthèse des isoprénoïdes précurseurs de régulateurs de croissance, est associée à la longueur des baies. Enfin, VvJMJ un facteur de transcription de type jumonji est à la fois associé à la variation du poids et à la longueur des baies. La caractérisation du locus Fleshless berry (Flb) a également été poursuivie avec (i) l’analyse du polymorphisme de séquence de 69 fragments de gènes entre les vignes sauvages et cultivées, ce qui a permis l’identification d’un gène présentant une trace de sélection et une diversité nucléotidique plus élevée dans le compartiment sauvage que dans lecultivé. (ii) La mise en évidence d’une homozygotie complète d’une région de 1 Mb contenant le locus Flb chez certains cultivars comme l’Ugni Blanc, rendant impossible le clonage positionnel du gène dans la population F1. L’ensemble de ce travail a permis de mettre en évidence la complexité des mécanismes à l’origine de la variation de la taille de la baie chez la vigne. Nous avons montré la faisabilité des études de génétique d’association chez la vigne en utilisant des gènes candidats pour le poids de baies, et identifié les candidats les plus intéressants pour la sélection assistée par marqueurs et les études fonctionnelles ultérieures.Fruit size is an important trait in fruit crops. However, excepted in tomato, little is known about the genetic and molecular control of fleshy fruit size variation. The aim of my thesis was to identify themain anatomical factors underlying berry size variation, to carry out an association genetics based approach with candidate genes and to search for possible signatures of domestication linked toberry size variation. Cell divisions before anthesis and cell expansion after anthesis were found tobe the major determinants of flesh weight variation between varieties in grapevine. Three geneswere identified by association genetics approaches. The VvAGL11 MADS box was found associated only with the seed number per berry. VvHMGR1, a putative HMGR involved in the synthesis of isoprenoid, a precursor of plant growth regulators, was found associated with berry length. Finally,VvJMJ encoding a putative transcription factor jumonji was associated both with berry weight andberry length variation. The characterization of the Fleshless berry locus (Flb) was also pursued with(i) the analysis of the sequence polymorphism of 69 regions between wild and cultivated grapevines,that leaded to the identification of one gene showing a trace of selection and a higher nucleotide diversity in the wild compartment than in the cultivated compartment. (ii) The discovery of acomplete homozygosity of a 1 Mb region containing the Flb locus in some cultivars such as theUgni Blanc, making impossible the fine mapping of the Flb locus in a F1 population. This study has highlighted the complexity of the mechanisms underlying the berry size variation in grapevines. We demonstrated the feasibility to screen in grapevine by association genetics candidate genes for berryweight and to select the most interesting ones for marker-assisted selection and for furtherfunctional studies

Caractérisation de la variation phénotypique de la taille de la baie chez la vigne Vitis vinifera L. et approches de génétique d'association et de recherche de traces de sélection pour ce caractère

Fruit size is an important trait in fruit crops. However, excepted in tomato, little is known about the genetic and molecular control of fleshy fruit size variation. The aim of my thesis was to identify themain anatomical factors underlying berry size variation, to carry out an association genetics based approach with candidate genes and to search for possible signatures of domestication linked toberry size variation. Cell divisions before anthesis and cell expansion after anthesis were found tobe the major determinants of flesh weight variation between varieties in grapevine. Three geneswere identified by association genetics approaches. The VvAGL11 MADS box was found associated only with the seed number per berry. VvHMGR1, a putative HMGR involved in the synthesis of isoprenoid, a precursor of plant growth regulators, was found associated with berry length. Finally,VvJMJ encoding a putative transcription factor jumonji was associated both with berry weight andberry length variation. The characterization of the Fleshless berry locus (Flb) was also pursued with(i) the analysis of the sequence polymorphism of 69 regions between wild and cultivated grapevines,that leaded to the identification of one gene showing a trace of selection and a higher nucleotide diversity in the wild compartment than in the cultivated compartment. (ii) The discovery of acomplete homozygosity of a 1 Mb region containing the Flb locus in some cultivars such as theUgni Blanc, making impossible the fine mapping of the Flb locus in a F1 population. This study has highlighted the complexity of the mechanisms underlying the berry size variation in grapevines. We demonstrated the feasibility to screen in grapevine by association genetics candidate genes for berryweight and to select the most interesting ones for marker-assisted selection and for furtherfunctional studies.La taille du fruit est un caractère agronomique important pour le rendement des espèces cultivées.Or, excepté chez la tomate, les mécanismes impliqués dans la variation de la taille des fruits charnus sont encore mal connus. L'objectif de ma thèse était d'identifier les principaux facteurs anatomiques impliqués dans la variation de la taille des baies, de réaliser des tests d'association génétique sur des gènes candidats et de rechercher des possibles traces de domestication liées à la variation de la taille des baies. Le nombre de cellules déjà déterminé dans l’ovaire avant l’anthèse et la capacité des cellules du mésocarpe à grossir après l’anthèse se sont révélés les principaux déterminants de la variation de la baie entre les variétés. Trois gènes potentiellement impliqués dans ce caractère ont été identifiés par des approches de génétique d'association. La MADS boxVvAGL11 est associée au nombre de pépins de la baie. VvHMGR1, une HMGR impliquée dans la synthèse des isoprénoïdes précurseurs de régulateurs de croissance, est associée à la longueur des baies. Enfin, VvJMJ un facteur de transcription de type jumonji est à la fois associé à la variation du poids et à la longueur des baies. La caractérisation du locus Fleshless berry (Flb) a également été poursuivie avec (i) l’analyse du polymorphisme de séquence de 69 fragments de gènes entre les vignes sauvages et cultivées, ce qui a permis l’identification d’un gène présentant une trace de sélection et une diversité nucléotidique plus élevée dans le compartiment sauvage que dans lecultivé. (ii) La mise en évidence d’une homozygotie complète d’une région de 1 Mb contenant le locus Flb chez certains cultivars comme l’Ugni Blanc, rendant impossible le clonage positionnel du gène dans la population F1. L’ensemble de ce travail a permis de mettre en évidence la complexité des mécanismes à l’origine de la variation de la taille de la baie chez la vigne. Nous avons montré la faisabilité des études de génétique d’association chez la vigne en utilisant des gènes candidats pour le poids de baies, et identifié les candidats les plus intéressants pour la sélection assistée par marqueurs et les études fonctionnelles ultérieures

The microvine, a model to study the effect of temperature on grapevine latent bud development and fruitfulness

International audienceAim: The success of inflorescence primordia initiation and differentiation within latent buds (i.e. bud fruitfulness) is a critical issue for grapevine yield sustainability under climate change. The aim of the present study was to track the timing and rate of inflorescence development in latent buds along the cane and to quantify their responses to elevated day/night (D/N) temperatures. Methods and Results: The experiments were conducted under controlled conditions, using the microvine model, which is suitable for establishment in small areas. Two imagery methods for analyzing bud anatomy were assessed: light microscopy and x-ray microtomography. Light microscopy was laborious, but it was the most accurate method for investigating organogenesis in the primordial shoot of the latent bud. In plants grown in a greenhouse (D/N, 25°C/15°C), the number of phytomer primordia in latent buds increased linearly from the apical to the basal buds on the cane. A maximum of six phytomers and two inflorescence primordia were observed beneath the 20th bud position that is, slightly fewer than usually reported with macrovines. The first and second inflorescences started to differentiate at the 14th and 18th bud position, respectively. Temperature increases in the growth chamber (D/N, 20–30°C/15–25°C) only slightly changed the final number of preformed phytomers and the probability of inflorescence primordia differentiation per bud. However, elevated temperature sharply accelerated and thereby shortened development of the latent bud primordial shoot, resulting in differentiation of the first inflorescence primordia straight from the fifth bud position. Based on the spatiotemporal conversion of bud position into thermal time, the first inflorescence started to differentiate 332 growing degree days (°Cd) (or 41 days) after bud emergence at D/N 20°C/15°C, and only 98°Cd (or 5 days) after bud emergence at D/N 35°C/25°C. Finally, the number of preformed phytomers was shown to correlate with primary bud length and cane diameter, independent of temperature. These easily measured variables may be used as indicators of bud developmental stage and potential bud fruitfulness in further studies using the microvine. Conclusions: The microvine appears to be suitable for parameterizing a developmental model of grapevine latent buds under controlled environmental conditions and when evaluating the response to elevated D/N temperatures. Significance and impact of the study: The precise description of the timing and rate of differentiation of phytomers and inflorescences opens new perspectives for understanding the molecular processes underlying the response of bud fruitfulness to environmental constraints

Microvine: A New Model to Study Grapevine Growth and Developmental Patterns and their Responses to Elevated Temperature

Growing standardized plant material in controlled environment can facilitate the disentangling of the many impacts of climate change on grapevine production and quality. Microvine is a natural gibberellic acid insensitive mutant showing dwarfism, early and continuous flowering along the vegetative axes. It was initially proposed as a model for genetics. In this study, we questioned its suitability to facilitate and hasten the characterization of grapevine vegetative and reproductive growth and development patterns as wells as their responses to temperature elevation. A series of experiments were performed in the greenhouse and in growth chambers under either ‘standard’ (25/15°C days/night) or contrasted (from 22/12°C up to 30/25°C) thermal treatments for several weeks. Under ‘standard’ thermal condition, measured temporal patterns of leaf and berry growth were similar among several phytomers along the main axis allowing us to estimate temporal growth patterns from spatial distribution of organ size. These patterns were stable between independent experiments under similar thermal and irradiance conditions. When plants were exposed to contrasted thermal treatments, leaf emergence rate was found linearly related to average daily temperature allowing us to derive a thermal time based model of development. Under cool thermal conditions (22/12°C), the temporal evolution of biochemical parameters was similar to that classically found for grapevine. However, exposing plants to a + 8°C thermal treatment for 450 °Cd revealed strong alterations of the thermal time based developmental program with either acceleration (leaf and internode growth) or delay (flowering, sugar accumulation in berries), as well as major uncoupling between growth and storage in internodes. These results reveal the potential of Microvine to study grapevine responses to the many facets of climate change.Bases développementales, moléculaires et génétiques de l'adaptation de la vigne à la contrainte thermique

The <em>fleshless</em> mutation: a valuable source to investigate the regulation of pericarp morphogenesis

The FLESHLESS somatic variation was identified in the grapevine as a chimeric phenotype that develops small fruits with a very little amount of flesh (Fernandez et al., 2006). The lack of flesh development was shown to result from the impairment of both cell division and expansion. Interestingly, the phenotype was found inheritable as a dominant trait, even some phenotypic reversions to the wild-type grapevine berry being observed in the original somatic variant and derived populations. Considering the interest of this phenotypic trait, several experimental approaches were performed to: i) decipher the causal genetic mechanism of the misregulation of the flesh morphogenesis due to flb mutation and ii) identify main genes associated with the fleshless phenotype as potential critical players of early fruit development in the grapevine. Fine mapping using DRCF (Dwarf, Rapid Cycling and Flowering) progenies (Chaib et al., 2010) and gene sequencing identified the insertion of an active MITEs (Miniature Inverted-repeat Transposable Elements) in the promoter of the PISTILLATA-like gene as the original event of the fleshless phenotypic variations. The TE insertion causes specific ectopic activation of a VvPI allele during early fruit development that maintains the expression of floral genes within the fruit. The analysis of transcriptomic patterns induced by the flb mutation into the fruit revealed a set of genes which expression is specific to the transition from flower to young berry development. Some of these genes were functionally characterised, in particular through DRCF genetic transformation. Main outputs of this work will be presented and discussed