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The implementation of in vitro tissue culture propagation methods, such as somatic embryogenesis (SE), rely on the amenability of plant cells to undergoing induced reprogrammation (de-differentiation followed by re-differentiation) after treatment by growth regulators. It has been well documented in various species that these processes involve epigenetic regulation pathways that are similar to those associated with responses to abiotic stresses, which makes in vitro-derived material a great system for studying them within (theoretically) genetically homogeneous populations. Originally, our work on SE-derived oil palms stems from the practical necessity of understanding and preventing the emergence of a pervasive somaclonal variation (the mantled floral phenotype) with negative impact on oil production. In doing so, we also aim to leverage the knowledge acquired from this case study to address broader scientific questions related to stress memory and acclimation in perennial species. With this dual objective in mind, we are currently assessing the expression of small non-coding RNAs (sncRNAs) in embryogenic suspensions and their possible roles in the regulation of genome expression and structure. We are using high-throughput transcriptome, small RNAome and degradome datasets to identify both source and target sequences of sncRNA-mediated regulations, and annotate the biological pathways that are affected by these regulations. As our analyses are still under way, we will present a global outline of our project and report on both our progress and a few lessons learned.

Le palmier rônier ou Borassus aethiopum Mart. est une plante aux multiples usages, qui présente des caractéristiques socio-économiques importantes pour les populations locales. Les nombreux avantages dont offre la plante, font qu'elle subit de grandes pressions anthropiques. Dans le but de définir des stratégies de gestion et de conservation à moyen et long terme, une analyse de la diversité a été effectuée grâce aux marqueurs microsatellites spécifiques de l'espèce. Ces derniers ont été développés à partir d'un séquençage partiel du génome de B. aethiopum, puis par la méthode QDD, onze (11) amorces spécifiques ont pu être définies et testées sur 180 échantillons représentant neuf (9) populations au Bénin. L'utilisation de ces marqueurs a permis de distinguer deux groupes génétiques dont l'un est constitué des populations Sud et Centre Bénin et le second groupe représentant l'ensemble des populations du Nord y compris la population de Savè qui est aussi située au Centre Bénin. De plus, dans le souci d'élucider la frontière génétique entre les différentes espèces de Borassus sp., les analyses phylogénétiques conduites sur l'ensemble des échantillons issus des cinq espèces (B. aethiopum, B. akeassii, B. madagascariensis, B. heineaus, B. flabellifer) ont permis de déterminer que l'espèce soeur de B. aethiopum serait B. madagascariensis avec une spéciation intervenue il y a trois (03) millions d'années (Ma). Ces résultats sont d'une grande importance et amélioreront l'état des connaissances aussi bien sur B. aethiopum que les autres espèces. Tout ceci contribuera à une meilleure gestion de l'espèce dans son aire de distribution.

The African fan palm Borassus aethiopum Mart. is widely distributed in savanna ecosystems throughout of the continent. It is an important non-timber forest product with multiple usages of significant socio-economic importance for local populations. As such, it is under strong anthropogenic pressures that have the potential to affect negatively the genetic diversity of the species. However, in spite of its role for fighting both desertification and poverty, the African fan palm remains under-studied, which hampers its sustainable development as a resource. In the present study, we analyze the genetic diversity and population structure of B. aethiopum sampled in different locations across Benin, West Africa. In order to achieve this, we have generated a partial, low-cover genomic sequencing of this species, using a high-throughput sequencing technology, and used it to identify microsatellite loci in the fan palm genome. Among these potential target loci, 11 were found to yield polymorphic amplifications between our test samples and were subsequently used in our analyses. Our results highlight the overall low genetic diversity of Borassus aethiopum populations in Benin, with number of effective alleles ranging from one point forty-four to two point zero sixty-nine per locus, and expected heterozygosity (He) ranging from zero point two hundred and sisty-three to zero point four hundred and fifty-one depending on the population. Moreover, our Bayesian analysis shows that Beninese fan palm populations are mostly structured according to geographic distances, with two population cluster including samples from the Southern and Central geo-climatic regions vs. samples from the Northern region, respectively. The present work constitutes a first step towards larger-scale genetic studies dedicated to B. aethiopum and, beyond that, provides new molecular data to the palm community.

Among the models that are available to plant scientists for studying the impact of epigenetic regulations on agronomically and economically relevant crop phenotypes, the mantled somaclonal variation of the oil palm is one of the oldest, since its association with genome-wide hypomethylation has been established >20 years ago. Since then, significant progress has been made towards elucidating the molecular mechanisms of floral organ feminization in adult plants generated through somatic embryogenesis. Yet, to this day, the origin of this variant phenotype during early stages of the in vitro process remains to be identified, and thus the reliable assessment of clonal phenotype stability still elude us. In order to better understand the epigenetic misregulations involved in the emergence of the mantled phenotype, we are currently investigating the role of small non-coding RNAs (sncRNAs) in embryogenic suspensions of oil palm using high-throughput sequencing techniques. During a first phase of the project, we profiled the different size classes of sncRNAs produced in different embryogenic lines. We then used in silico prediction software to identify likely target transcripts of sequenced microRNAs (miRNAs), some of which were subsequently validated by 5'RACE. A comprehensive degradome analysis is also under way, and will provide us with a comprehensive overview of small RNA-mediated post-transcriptional regulation mechanisms under our experimental conditions. We will discuss our results and their implications with respect to the study of epigenetic regulations in non-model crop genomes.

Additional file 2. List of sampled Borassus aethiopum individuals. M, F: male or female palm, respectively. All geographic coordinates are provided as North from the Equator (latitude) and East from the Greenwich meridian (longitude), respectively.

Additional file 4. Results of the Bayesian cluster analysis with variable values of K. Graphical summary generated from STRUCTURE results by CLUMPAK’s main pipeline with values of K ranging from 1 to 10.

Au-delà des controverses qui l'entourent, le palmier à huile (Elaeis guineensis Jacq.) est une plante dont l'étude s'avère fascinante, car elle nous confronte à la fois à une espèce pérenne tropicale à la biologie complexe et aux réseaux d'influences complexes associés à une culture qui constitue une ressource économique majeure pour de nombreux pays du Sud. L'emploi du clonage in vitro de cette plante, dans le cadre de son amélioration variétale, a conduit à l'apparition d'un phénotype nommé mantled, affectant l'appareil reproducteur ainsi que la production d'huile. Nous avons démontré il y a 20 ans que cette anomalie est étroitement associée à un dérèglement des mécanismes épigénétiques qui sont mis en jeu au cours de la micropropagation. Depuis, les travaux dans cette direction ont permis d'avancer considérablement sur l'identification de la source du phénotype mantled chez la plante adulte. Toutefois, des questions demeurent en suspens quant à la nature et à la chronologie des évènements survenant au cours du processus de culture in vitro du palmier à huile, en amont de l'expression de ce phénotype. Au travers de nos récents résultats, nous illustrons les nouvelles pistes qui se dessinent vers une meilleure compréhension de l'impact des stress in vitro sur les régulations épigénétiques.

The African fan palm or toddy palm (Borassus aethiopum Mart.) plays a significant role in rural village economy because of the many uses of different parts of the plant for food, construction, traditional medicines and crafts. However, some of these uses which specifically target either male individuals (construction beams) or reproductive stages (fruits and hypocotyls) put pressure on the genetic diversity of palm populations and threaten the species as a whole. As a preliminary step to defining conservation strategies for Borassus aethiopum populations in Benin, an assessment of their genetic diversity was conducted through the use of microsatellite markers. In the absence of molecular resources for this species, we first set out to evaluate the transferability of microsatellite primers that had previously been designed in other palm species, including the cousin species from Asia Borassus flabellifer. We show that the portability of such markers, quantified as their ability to successfully amplify B. aethiopum DNA, is very low. Moreover, among the successfully transferred primers we found that the ability to detect polymorphisms, both within or among nine fan palm populations sampled across the Beninese territory, was even lower. In light of these first results, we discuss experimental options that will allow us to overcome this technical difficulty, and the future development of our project throughout the West African subregion.

Epigenetic mechanims are involved in the regulation of gene and genome structure and expression at multiple levels. They respond to both internal signals, notably in the course of differentiation and development, and to external ones, as exemplified through their ability to be affected by environmental cues. Because of their properties, epigenetic processes may well provide insights into how short-term (acclimation, priming) and long-term (adaptation) adjustments to ongoing global changes could impact crop yield in a not-so-distant future, and thereby help us devise new breeding strategies in order to better anticipate them. I will illustrate this talk with practical cases taken from ongoing research on tropical and mediterranean crops, with emphasis on our group's research focusing on oil and date palms. (Texte intégral)

Summary of the contents of the E. oleifera and E. guineensis TE databases. (PDF 9Â kb)

Sequence coverage of LTR retrotransposon lineages in the E. oleifera (Eo) and in E. guineensis (Eg) genomes. (PDF 35Â kb)

The mantled somaclonal variation of oil palm (Elaeis guineensis Jacq.) is morphologically similar to mutants defective in MADS-box genes of the B-class. Indeed in such mutants male floral organs are converted into female ones and this can be observed in oil palm flowers of both sexes. The detrimental consequences of somaclonal variation on fruit set then oil yields and its unpredictable incidence in clonal progenies have prompted the search for early detection markers. More generally, a better understanding of the molecular mechanisms underlying the onset and the maintenance of this variant phenotype is needed. The mantledsomaclonal variation is observed only in clonal populations generated through somatic embryogenesis at a large commercial stage. Thus, because of the nature of the variation, research strategy must involve two parallel approaches aimed at studying simultaneously the in vitro stages of clonal palms production (the flask) and the in plantadevelopment of the reproductive organs which are affected by the phenotype (the flower). On both materials a novel combination of epigenetics approaches, high-throughput sequencing technologies and bioinformatics analyses now provide new insights into a complex developmental phenomenon. Our research work also paves the way for innovation and development oriented towards end-users in the palm oil industry. (Texte intégral)

Sequence coverage of LTR retrotransposon families in the E. oleifera (Eo) and in E. guineensis (Eg) genomes. (PDF 356Â kb)

The mantled somaclonal variation of oil palm (Elaeis guineensis Jacq.) is morphologically similar to B-class MADS-box genes mutants: floral organs undergo a male-to-female homeotic conversion in flowers of both sexes. The negative economic consequences of this somaclonal variation on oil yields, as well as its variable incidence in clonal progenies, have made it necessary to understand the molecular mechanisms responsible for the variant phenotype. We have recently demonstrated that the splicing of EgDEF1, the APETALA3ortholog that is believed to be involved in the mantled floral phenotype, is significantly altered in variant flowers with respect to normal ones, resulting in the over-accumulation of a shorter, and potentially defective, transcript isoform (Jaligot et al., 2014). We are now attempting to determine through In Situ Hybridization whether these alternative transcripts could indeed compete within the same tissues. Also, because EgDEF1is inserted in a region of the oil palm genome that is rich in Transposable Elements (TEs), we are investigating the possibility that TEs might play a role in the onset of the variation in the course of the somatic embryogenesis process. (Texte intégral)

Transcriptional activity of 63 LTR retrotransposons. A: Percentage of normalized read counts per superfamily. B: Percentage of normalized read counts per tissue. (PDF 178Â kb)

Protein coding domain composition of full-length LTR retrotransposons of oil palm E. guineensis . A: Number of protein coding domain identified per retrotransposon (figures correspond to the number of elements displaying either 1, 2, 3, 4 or 5 domains). B: Frequency of the different protein coding domain in the Copia and Gypsy superfamilies. AP = protease, INT = integrase, RT = reverse transcriptase, RH = ribonuclease, GAG = capsid. (PDF 174 kb)

Phylogenetic analysis of full-length LTR retrotransposon sequences predicted from the E. guineensis genome. See Methods for details. (PDF 332Â kb)

Relationship between the density (sequences per Mb) across chromosomes of predicted coding sequences and that of Copia (A) and Gypsy (B) full-length elements. (PDF 153Â kb)

The mantled variation of oil palm (Elaeis guineensis) involves a significant deficit in genome-wide methylation as well as the mis-expression of a variety of genes affecting both the in vitro-propagated material from which the palms originate and the adult regenerants (for a review see [1]). This reversible floral phenotype is morphologically similar to B-class MADS-box mutants: indeed, the abnormal flower displays an homeotic conversion of male floral organs into female ones [2]. In order to investigate the role of epigenetic processes in the transcriptional regulation of EgDEF1, the ortholog of APETALA3/DEFICIENS in the oil palm genome, we have studied the DNA methylation pattern of this gene concomitantly with its transcript production in the developing inflorescence. The exploration of the genomic region surrounding the gene led to the isolation of several retroelements which were also examined. Although our results do not point to a direct implication of DNA methylation in the regulation of the EgDEF1 gene, they raise exciting new questions about the possible mechanisms underlying the variant phenotype. (Texte intégral)