End-to-end multimodal 3D imaging and machine learning workflow for non-destructive phenotyping of grapevine trunk internal structure.

Quantifying healthy and degraded inner tissues in plants is of great interest in agronomy, for example, to assess plant health and quality and monitor physiological traits or diseases. However, detecting functional and degraded plant tissues in-vivo without harming the plant is extremely challenging. New solutions are needed in ligneous and perennial species, for which the sustainability of plantations is crucial. To tackle this challenge, we developed a novel approach based on multimodal 3D imaging and artificial intelligence-based image processing that allowed a non-destructive diagnosis of inner tissues in living plants. The method was successfully applied to the grapevine (Vitis vinifera L.). Vineyard's sustainability is threatened by trunk diseases, while the sanitary status of vines cannot be ascertained without injuring the plants. By combining MRI and X-ray CT 3D imaging with an automatic voxel classification, we could discriminate intact, degraded, and white rot tissues with a mean global accuracy of over 91%. Each imaging modality contribution to tissue detection was evaluated, and we identified quantitative structural and physiological markers characterizing wood degradation steps. The combined study of inner tissue distribution versus external foliar symptom history demonstrated that white rot and intact tissue contents are key-measurements in evaluating vines' sanitary status. We finally proposed a model for an accurate trunk disease diagnosis in grapevine. This work opens new routes for precision agriculture and in-situ monitoring of tissue quality and plant health across plant species.

Elimination of Eight Viruses and Two Viroids from Preclonal Candidates of Six Grapevine Varieties (Vitis vinifera L.) through In Vivo Thermotherapy and In Vitro Meristem Tip Micrografting.

Viruses and virus-like organisms are a major problem in viticulture worldwide. They cannot be controlled by standard plant protection measures, and once infected, plants remain infected throughout their life; therefore, the propagation of healthy vegetative material is crucial. In vivo thermotherapy at 36-38 °C for at least six weeks, followed by meristem tip micrografting (0.1-0.2 mm) onto in vitro-growing seedling rootstocks of Vialla (Vitis labrusca × Vitis riparia), was successfully used to eliminate eight viruses (grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine Pinot gris virus (GPGV), grapevine fanleaf virus (GFLV), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine fleck virus (GFkV), grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus-1 (GSyV-1), and raspberry bushy dwarf virus (RBDV)), as well as two viroids (hop stunt viroid (HSVd) and grapevine yellow speckle viroid 1 (GYSVd-1)) from preclonal candidates of six grapevine varieties (Vitis vinifera L.). A half-strength MS medium including vitamins supplemented with 30 g/L of sucrose and solidified with 8 g/L of agar, without plant growth regulators, was used for the growth and root development of micrografts and the subsequently micropropagated plants; no callus formation, hyperhydricity, or necrosis of shoot tips was observed. Although the overall regeneration was low (higher in white than in red varieties), a 100% elimination was achieved for all eight viruses, whereas the elimination level for viroids was lower, reaching only 39.2% of HSVd-free and 42.6% GYSVd-1-free vines. To the best of our knowledge, this is the first report of GPGV, GRVFV, GSyV-1, HSVd, and GYSVd-1 elimination through combining in vivo thermotherapy and in vitro meristem tip micrografting, and the first report of RBDV elimination from grapevines. The virus-free vines were successfully acclimatized in rockwool plugs and then transferred to soil.

Genetic diversity, linkage disequilibrium and power of a large grapevine (Vitis vinifera L) diversity panel newly designed for association studies.

BACKGROUND: As for many crops, new high-quality grapevine varieties requiring less pesticide and adapted to climate change are needed. In perennial species, breeding is a long process which can be speeded up by gaining knowledge about quantitative trait loci linked to agronomic traits variation. However, due to the long juvenile period of these species, establishing numerous highly recombinant populations for high resolution mapping is both costly and time-consuming. Genome wide association studies in germplasm panels is an alternative method of choice, since it allows identifying the main quantitative trait loci with high resolution by exploiting past recombination events between cultivars. Such studies require adequate panel design to represent most of the available genetic and phenotypic diversity. Assessing linkage disequilibrium extent and panel power is also needed to determine the marker density required for association studies. RESULTS: Starting from the largest grapevine collection worldwide maintained in Vassal (France), we designed a diversity panel of 279 cultivars with limited relatedness, reflecting the low structuration in three genetic pools resulting from different uses (table vs wine) and geographical origin (East vs West), and including the major founders of modern cultivars. With 20 simple sequence repeat markers and five quantitative traits, we showed that our panel adequately captured most of the genetic and phenotypic diversity existing within the entire Vassal collection. To assess linkage disequilibrium extent and panel power, we genotyped single nucleotide polymorphisms: 372 over four genomic regions and 129 distributed over the whole genome. Linkage disequilibrium, measured by correlation corrected for kinship, reached 0.2 for a physical distance between 9 and 458 Kb depending on genetic pool and genomic region, with varying size of linkage disequilibrium blocks. This panel achieved reasonable power to detect associations between traits with high broad-sense heritability (> 0.7) and causal loci with intermediate allelic frequency and strong effect (explaining > 10 % of total variance). CONCLUSIONS: Our association panel constitutes a new, highly valuable resource for genetic association studies in grapevine, and deserves dissemination to diverse field and greenhouse trials to gain more insight into the genetic control of many agronomic traits and their interaction with the environment.

Genome-wide prediction methods in highly diverse and heterozygous species: proof-of-concept through simulation in grapevine.

Nowadays, genome-wide association studies (GWAS) and genomic selection (GS) methods which use genome-wide marker data for phenotype prediction are of much potential interest in plant breeding. However, to our knowledge, no studies have been performed yet on the predictive ability of these methods for structured traits when using training populations with high levels of genetic diversity. Such an example of a highly heterozygous, perennial species is grapevine. The present study compares the accuracy of models based on GWAS or GS alone, or in combination, for predicting simple or complex traits, linked or not with population structure. In order to explore the relevance of these methods in this context, we performed simulations using approx 90,000 SNPs on a population of 3,000 individuals structured into three groups and corresponding to published diversity grapevine data. To estimate the parameters of the prediction models, we defined four training populations of 1,000 individuals, corresponding to these three groups and a core collection. Finally, to estimate the accuracy of the models, we also simulated four breeding populations of 200 individuals. Although prediction accuracy was low when breeding populations were too distant from the training populations, high accuracy levels were obtained using the sole core-collection as training population. The highest prediction accuracy was obtained (up to 0.9) using the combined GWAS-GS model. We thus recommend using the combined prediction model and a core-collection as training population for grapevine breeding or for other important economic crops with the same characteristics.

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

BACKGROUND: 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.

Effect of shooting medium and source of material on grapevine (Vitis vinifera L.) shoot tip recovery after cryopreservation.

BACKGROUND: Selecting experimental material at the optimal physiological stage is of paramount importance for successful cryopreservation. OBJECTIVE: The study was to investigate the effect of the physiological state of grapevine buds on their regrowth after liquid nitrogen exposure. METHODS: In a first set of experiments, we tested the regrowth of cryopreserved buds sampled from microcuttings cultured on shooting medium containing benzylaminopurine or zeatin riboside for various durations. In a second set of experiments, we studied the regrowth after liquid nitrogen exposure of buds sampled from different positions on the stem of in vitro plantlets. RESULTS: Regrowth of cryopreserved buds sampled from microcuttings was higher (30%), compared to buds sampled directly from in vitro plantlets (23%), for all culture durations of microcuttings on shooting medium tested (2-6 weeks). Addition of cytokinin in the shooting medium improved regrowth of cryopreserved buds compared to buds sampled from microcuttings cultured on medium devoid of growth regulators; however similar results were obtained with the two cytokinins tested. Buds sampled on nodes 3-4 and 6-7 (from the top of the stem) displayed higher regrowth compared to shoot tips. No significant differences were noted in regrowth after cryopreservation between buds sampled from microcuttings produced from the terminal node, or nodes 3-4 and 6-7. CONCLUSION: The physiological state of the plant material is important for cryopreservation success. Actively growing buds sampled from microcuttings displayed higher regrowth compared to buds sampled directly on in vitro plantlets.

Molecular characterization and expression analysis of the Rab GTPase family in Vitis vinifera reveal the specific expression of a VvRabA protein.

As a first step to investigate whether Rab GTPases are involved in grape berry development, the Vitis vinifera EST and gene databases were searched for members of the VvRab family. The grapevine genome was found to contain 26 VvRabs that could be distributed into all of the eight groups described in the literature for model plants. Genetic mapping was successfully performed; VvRabs were mostly located on independent chromosomes, apart from eight that were located on the as yet unassigned portions of the genome clustered in the ChrUn Random chromosome. Conserved and divergent regions between VvRab protein sequences were identified. Transcript expression of 11 VvRabs was analysed by real-time quantitative RT-PCR. Except for VvRabA5b, transcript expression was detected, in general, in all the organs investigated, but with different patterns. In grape berries, VvRab transcripts were expressed at all stages of fruit development, with different profiles, except in the case of members of the A family which displayed generally similar patterns. The response to growth regulators in cell cultures was generally specific to each VvRab, with a differential pattern of expression for ethylene, auxin, and abscisic acid according to the VvRab. Interestingly, and unexpectedly considering transcript expression, western blotting using a monoclonal antibody raised against AtRabA5c (ARA4) showed a specific expression in the exocarp of ripe grape berries, in all seven red and white berry varieties tested. By contrast, no expression was detected in any of the other organs or tissues investigated. This paper contains the first description of Rab GTPases in V. vinifera. The involvement of a specific VvRab in grape berry late development and the potential role of this Rab GTPase are discussed in relation to literature data.

Characterization of Vitis vinifera L. somatic variants exhibiting abnormal flower development patterns.

Mutants have proven to be a key resource for functional genomic studies in model annual plant species. In perennial plant species where mutants are difficult to generate and to screen, spontaneous somatic variants represent a unique resource to understand the genetic control of complex developmental patterns. The morphological and histological characterization of six Vitis vinifera L. somatic variants that display four different abnormal phenotypes of flower development are described here. A phenotype of reiterated reproductive meristems (RRM), with both flower and petal reiteration, was observed in a somatic variant of the cultivar Carignan. An abnormal development of reproductive organs was displayed by the unfused carpels (UFC) somatic variant of cv. Bouchalès, while a somatic variant of cv. Mourvèdre named carpel-less (CLS) developed abnormal ovules in the absence of carpels. Finally, three independent somatic variants in cvs Gamay, Morrastel, and Pinot displayed a phenotype of multiple perianth whorls (MPW). Gene expression studies showed that the expression profiles of VvMADS-box 1, 2, and 3 (putative orthologues of Arabidopsis flowering genes AG, SEP, and AGL13), were altered during grapevine flower development in the somatic variants, whereas the corresponding original cultivars displayed similar VvMADS-box gene expression profiles. Phenotypic and molecular characterization of these variants allowed the development of hypotheses on genetic functions that might be altered in most of the variants in light of the current ABCDE flower model.

The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla.

The analysis of the first plant genomes provided unexpected evidence for genome duplication events in species that had previously been considered as true diploids on the basis of their genetics. These polyploidization events may have had important consequences in plant evolution, in particular for species radiation and adaptation and for the modulation of functional capacities. Here we report a high-quality draft of the genome sequence of grapevine (Vitis vinifera) obtained from a highly homozygous genotype. The draft sequence of the grapevine genome is the fourth one produced so far for flowering plants, the second for a woody species and the first for a fruit crop (cultivated for both fruit and beverage). Grapevine was selected because of its important place in the cultural heritage of humanity beginning during the Neolithic period. Several large expansions of gene families with roles in aromatic features are observed. The grapevine genome has not undergone recent genome duplication, thus enabling the discovery of ancestral traits and features of the genetic organization of flowering plants. This analysis reveals the contribution of three ancestral genomes to the grapevine haploid content. This ancestral arrangement is common to many dicotyledonous plants but is absent from the genome of rice, which is a monocotyledon. Furthermore, we explain the chronology of previously described whole-genome duplication events in the evolution of flowering plants.

Molecular characterization and expression analysis of the Rop GTPase family in Vitis vinifera.

Rop/Rac GTPases are plant-specific signalling proteins with multiple roles, some of which have implications in plant development and in hormone signal transduction. Using expressed sequence tag (EST) and gene database analyses, members of the Rop family were characterized for the first time in a perennial species (Vitis vinifera). The grapevine genome was found to contain seven expressed VvRops. The phylogenetic analyses indicated that VvRops could be distributed into four groups, as described in the literature for model plants. Genetic mapping was successfully performed for five VvRops, which were localized on independent linkage groups. Conserved and divergent regions were identified on the protein sequences. The results of VvRop expression obtained by real-time quantitative reverse transcription-PCR analyses indicated that all the organs investigated displayed VvRop expression, however with different patterns. Whereas no total organ specificity for VvRop expression could be evidenced, VvRop9 displayed high expression in developing berries only. During berry development, the transcript profile was generally similar for all the VvRops, i.e. displaying a peak early in the herbaceous phase followed by a decline towards veraison and thereafter. Western blotting gave a similar expression profile for VvRop proteins. Response to growth regulators was generally specific to each VvRop. The potential involvement of specific VvRops in grapevine development is discussed.

Proteome changes in leaves from grapevine (Vitis vinifera L.) transformed for alcohol dehydrogenase activity.

A proteomic approach has been used to study changes in leaf protein content from plants transformed for alcohol dehydrogenase (ADH) activity. Individual quantitative analysis of 190-436 spots separated by two-dimensional electrophoresis was performed, and spots displaying significant quantitative changes between control (C), sense (S), and antisense (R) transformants were selected using Student's t test. Of the 14 spots selected and further analyzed after trypsic digestion, 9 could be identified by MS analysis and 5 by LC-MS/MS. Identified proteins had mainly a chloroplastic origin: four rubisco large subunits, one rubisco binding protein, two glutamine synthetases, one elongation factor Tu, one ATP synthase beta subunit, and one plastidic aldolase. Proteins with other localization were also identified, such as a UDP-glucose pyrophosphorylase, a mitochondrial aminomethyltransferase, a linalool synthase, which comigrated with the protein identified as elongation factor Tu, an enolase comigrating with a glyceraldehyde 3-phosphate dehydrogenase, and a mixture of eight proteins among which were a dehydroascorbate reductase, a chalcone isomerase, and a rubisco activase. The results emphasize the changes in carbon metabolism-associated proteins linked to the alteration in ADH activity of grapevine transformant leaves.

Effects of genetic manipulation of alcohol dehydrogenase levels on the response to stress and the synthesis of secondary metabolites in grapevine leaves.

The functional role of Adh in regulating susceptibility to abiotic stress and the synthesis of secondary metabolites was investigated in transgenic grapevine plants over- and underexpressing alcohol dehydrogenase (Adh). Plants were transformed with gene constructs containing a sense or antisense orientated grapevine VvAdh2 cDNA under the constitutive cauliflower mosaic virus 35S promoter. Plants transformed with either antisense orientation or the Adh-less construct displayed a low but detectable constitutive ADH activity, whereas plants transformed with the sense-expressed transgene showed a significantly higher (100-fold) ADH activity than the control. Compared with the control, the sense transgene induced an overexpression of VvAdh2 transcripts, whereas a reduced VvAdh2 expression was detected in antisense transformants. Grapevine plants overexpressing Adh displayed a lower sucrose content, a higher degree of polymerization of proanthocyanidins, and a generally increased content of volatile compounds, mainly in carotenoid- and shikimate-derived volatiles. In general, no significant differences between sense/antisense transformants were observed with regard to carotenoid and chlorophyll contents, suggesting a strong metabolic regulation of the synthesis of these compounds.

Involvement of ethylene signalling in a non-climacteric fruit: new elements regarding the regulation of ADH expression in grapevine.

Although grape berries have been classified as non-climacteric fruits, ongoing studies on grape ethylene signalling challenge the role of ethylene in their ripening. One of the significant molecular changes in berries is the up-regulation of ADH (alcohol dehydrogenase, EC 1.1.1.1) enzyme activity at the inception of fruit ripening and of VvADH2 transcript levels. This paper shows that the ethylene signal transduction pathway could be involved in the control of VvADH2 expression in grapevine berries and in cell suspensions. The induction of VvADH2 transcription, either in berries at the inception of ripening or in cell suspensions, was found to be partly inhibited by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene receptors. Treatment of cell suspensions with 2-chloroethylphosphonic acid (2-CEPA), an ethylene-releasing compound, also resulted in a significant increase in ADH activity and VvADH2 transcription under anaerobiosis, showing that concomitant ethylene and anaerobic treatments in cell suspensions could result in changes in VvADH2 expression. All these results associated with the presence in the VvADH2 promoter of regulatory elements for ethylene and anaerobic response, suggest that the ethylene transduction pathway and anaerobic stress could be, in part, involved in the regulation of VvADH2 expression in ripening berries and cell suspensions. These data open new aspects of the expression control of a ripening-related gene in a non-climacteric fruit.