Philippe Chatelet

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

BackgroundIn 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.ResultsIn 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.ConclusionsHermaphrodite 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.

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.

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.

Cryopreservation of grapevine ( Vitis vinifera L.) in vitro shoot tips

In this work, we compared the efficiency of encapsulation-dehydration and droplet-vitrification techniques for cryopreserving grapevine (Vitis vinifera L.) cv. Portan shoot tips. Recovery of cryopreserved samples was achieved with both techniques; however, droplet-vitrification, which was used for the first time with grapevine shoot tips, produced higher regrowth. With encapsulationdehydration, encapsulated shoot tips were precultured in liquid medium with progressively increasing sucrose concentrations over a 2-day period (12 h in medium with 0.25, 0.5, 0.75 and 1.0 M sucrose), then dehydrated to 22.28% moisture content (fresh weight). After liquid nitrogen exposure 37.1% regrowth was achieved using 1 mm-long shoot tips and only 16.0% with 2 mm-long shoot tips. With droplet-vitrification, 50% regrowth was obtained following treatment of shoot tips with a loading solution containing 2 M glycerol + 0.4 M sucrose for 20 min, dehydration with half-strength PVS2 vitrification solution (30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% dimethylsulfoxide and 0.4 M sucrose in basal medium) at room temperature, then with full strength PVS2 solution at 0°C for 50 min before direct immersion in liquid nitrogen. No regrowth was achieved after cryopreservation when shoot tips were dehydrated with PVS3 vitrification solution (50% (w/v) glycerol and 50% (w/v) sucrose in basal medium).

In vitro introduction of healthy and virus-infected genotypes of native Croatian grapevine cultivars

We evaluated the response of eight economically important Croatian grapevine cultivars and studied the impact of their sanitary status on in vitro introduction, by comparing the response of healthy and virus-infected genotypes of one cultivar. Nodal explant survival on three media, M1 (half-strength MS), M2 (full-strength MS) or M3 (full-strength MS with 4.4 µM L−1 benzylaminopurine) was measured after 2 weeks and regrowth after 8 weeks. After 8 weeks, average shoot length and node number were significantly higher on M2 compared to M1 and M3. M3 induced significantly shorter average internode length, compared to M1 and M2. Survival of one healthy and of five cultivar Plavac mali genotypes infected with GFLV, GLRaV-1, GLRaV-3, GLRaV-3+GVA and GLRaV-1+GLRaV-3 was 97.5 and 82.8–87.5%, respectively. Regrowth of the healthy genotype reached 95.5%, but dropped to 5.5–31.4% in infected ones. The healthy genotype showed significantly higher shoot length (6.3 cm) and node number (7.3) compared to infected genotypes, with shoot length between 1.2–2.6 cm and node number between 1.2–3.0. By contrast, internode length was not significantly different between the healthy and the infected genotypes. The present work represents the first successful in vitro introduction for three of the eight native Croatian cultivars studied.

Bottleneck and gene flow effects impact the genetic structure of seed-propagated apricot populations in Moroccan oasis agroecosystems

In order to highlight the genetic status and origin of Moroccan apricot populations, trees were collected from ten oasis agroecosystems and analysed with AFLP markers. A total of 87 accessions and 12 cultivars grown in Moroccan orchards, including ‘Canino’ and ‘Del Patriarca’ cultivars, were surveyed and compared with in situ Tunisian and ex situ Montfavet (France) collections. Our results highlighted a narrow genetic diversity in the Maghreb region (Tunisia and Morocco) associated with a strong differentiation from the other groups, which supports a bottleneck effect. A similar model was illustrated at a finer geographical scale, i.e. the Draa Valley in Morocco. Genetic structure appeared as two major clusters subdivided into six sub-clusters in which Moroccan germplasm constituted specific groups in comparison with other Mediterranean apricots. Moroccan germplasm was classified into three sub-clusters, two of which were formed by genotypes related to ‘Del Patriarca’ and ‘Canino’, respectively. The present study highlights the wide Moroccan apricots diversity in traditional agroecosystems, and also suggests a substantial gene flow occurring from recently introduced cultivars (‘Canino’ and ‘Del Patriarca’) to local apricot populations, thus leading to local germplasm diversification through seedling propagation. If we consider its geographical position, the historical diffusion of the species and farming practices, Morocco could be viewed as an additional centre of secondary diversification for apricot. Understanding the origin and specificity of local apricot populations is crucial for managing local collections in regard to adaptive traits for arid and Saharan conditions as well as for introducing local genetic resources into current breeding programmes.

Integration of cryopreservation in French plant genetic resource collections: the CRYOVEG project

This book represents contributions, oral as well as posters, of the final meeting of COST Action 871, CRYOPLANET (Cryopreservation of crop species in Europe) held in Angers. Local organizers of the meeting were Dr. Agnes Grapin (AGROCAMPUS OUEST – Angers) and Dr. Florent Engelmann (Institut de Recherche pour le Developpement). COST Action 871 started in December 2006 with a Kick-off meeting at the COST office in Brussels and officially ended in December 2010. Twenty-one COST Action Countries (see figure 1) and 3 non-COST institutes (New Zealand Institute for Crop & Food Research (New Zealand), Vavilov Institute of Plant Industry (Russian Federation), Faculte des Sciences de Sfax (Tunisia) participated actively in this initiative. The Action was created because plant cryobiologists realized that plant cryopreservation was hardly applied in Europe. This was mainly due to the fact that efficient and robust cryopreservation protocols applicable to many plant species and diverse germplasm types were not available, plant researchers were unacquainted to recent developments in cryogenic storage methods and there was a lack of coordinated research in Europe on plant cryopreservation. The main objective of this action was therefore “to improve and apply technologically advanced techniques for plant genetic resources conservation of crops that are grown/ and or conserved in Europe with main emphasis on long-term conservation through cryopreservation”. In order to achieve this, 2 working groups (WGs) were established