Transferability, development of simple sequence repeat (SSR) markers and application to the analysis of genetic diversity and population structure of the African fan palm (Borassus aethiopum Mart.) in Benin.

BACKGROUND: In Sub-Saharan Africa, Borassus aethiopum Mart. (African fan palm) is an important non-timber forest product-providing palm that faces multiple anthropogenic threats to its genetic diversity. However, this species is so far under-studied, which prevents its sustainable development as a resource. The present work is a first attempt at characterizing the genetic diversity and population structure of B. aethiopum across nine collection sites spanning the three climatic regions of Benin, West Africa, through the use of microsatellite markers. RESULTS: During a first phase we relied on the reported transferability of primers developed in other palm species. We find that, in disagreement with previously published results, only 22.5% of the markers tested enable amplification of B. aethiopum DNA and polymorphism detection is very low. In a second phase, we generated a B. aethiopum-specific genomic dataset through high-throughput sequencing and used it for the de novo detection of microsatellite loci. Among the primer pairs targeting these, 11 detected polymorphisms and were further used for analyzing genetic diversity. Across the nine sites, expected heterozygosity (He) ranges from 0.263 to 0.451 with an overall average of 0.354, showing a low genetic diversity. Analysis of molecular variance (AMOVA) shows that within-site variation accounts for 53% of the genetic variation. Accordingly, the low number of migrants and positive values of the fixation index (F) in sites from both the Central (Sudano-Guinean) and the Southern (Guinean) climatic regions suggest limited gene flow between sites. The global correlation between genetic and geographic distances is weak; however, our clustering analyses indicate that B. aethiopum palms from Savè (Center) are genetically more similar to those from the North than to samples from other Central sites. CONCLUSIONS: In the light of our results, we discuss the use of inter-species transfer vs. de novo development of microsatellite markers in genetic diversity analyses targeting under-studied species, and suggest future applications for our molecular resources. We propose that, while prominent short-range pollen and seed dispersal in Benin explain most of our results, gene flux between the Central and Northern regions, as a result of animal and/or human migrations, might underlie the Savè discrepancy.

MooSciTIC: Training of trainers in West African research and higher education.

The MooSciTIC project is a capacity-building initiative targeting West African research scientists and higher education teachers. The project aimed to improve the self-reliance of researchers and upgrade research practices by providing on-site summer schools on trans-disciplinary topics such as scientific writing, communication, and integrity. Here, we explain how this program was designed and implemented and share the positive responses from our trainees, hoping to inspire similar initiatives.

Gene coexpression network analysis of oil biosynthesis in an interspecific backcross of oil palm.

Global demand for vegetable oils is increasing at a dramatic rate, while our understanding of the regulation of oil biosynthesis in plants remains limited. To gain insights into the mechanisms that govern oil synthesis and fatty acid (FA) composition in the oil palm fruit, we used a multilevel approach combining gene coexpression analysis, quantification of allele-specific expression and joint multivariate analysis of transcriptomic and lipid data, in an interspecific backcross population between the African oil palm, Elaeis guineensis, and the American oil palm, Elaeis oleifera, which display contrasting oil contents and FA compositions. The gene coexpression network produced revealed tight transcriptional coordination of fatty acid synthesis (FAS) in the plastid with sugar sensing, plastidial glycolysis, transient starch storage and carbon recapture pathways. It also revealed a concerted regulation, along with FAS, of both the transfer of nascent FA to the endoplasmic reticulum, where triacylglycerol assembly occurs, and of the production of glycerol-3-phosphate, which provides the backbone of triacylglycerols. Plastid biogenesis and auxin transport were the two other biological processes most tightly connected to FAS in the network. In addition to WRINKLED1, a transcription factor (TF) known to activate FAS genes, two novel TFs, termed NF-YB-1 and ZFP-1, were found at the core of the FAS module. The saturated FA content of palm oil appeared to vary above all in relation to the level of transcripts of the gene coding for ß-ketoacyl-acyl carrier protein synthase II. Our findings should facilitate the development of breeding and engineering strategies in this and other oil crops.

Genome-wide analysis of LTR-retrotransposons in oil palm.

BACKGROUND: The oil palm (Elaeis guineensis Jacq.) is a major cultivated crop and the world's largest source of edible vegetable oil. The genus Elaeis comprises two species E. guineensis, the commercial African oil palm and E. oleifera, which is used in oil palm genetic breeding. The recent publication of both the African oil palm genome assembly and the first draft sequence of its Latin American relative now allows us to tackle the challenge of understanding the genome composition, structure and evolution of these palm genomes through the annotation of their repeated sequences. METHODS: In this study, we identified, annotated and compared Transposable Elements (TE) from the African and Latin American oil palms. In a first step, Transposable Element databases were built through de novo detection in both genome sequences then the TE content of both genomes was estimated. Then putative full-length retrotransposons with Long Terminal Repeats (LTRs) were further identified in the E. guineensis genome for characterization of their structural diversity, copy number and chromosomal distribution. Finally, their relative expression in several tissues was determined through in silico analysis of publicly available transcriptome data. RESULTS: Our results reveal a congruence in the transpositional history of LTR retrotransposons between E. oleifera and E. guineensis, especially the Sto-4 family. Also, we have identified and described 583 full-length LTR-retrotransposons in the Elaeis guineensis genome. Our work shows that these elements are most likely no longer mobile and that no recent insertion event has occurred. Moreover, the analysis of chromosomal distribution suggests a preferential insertion of Copia elements in gene-rich regions, whereas Gypsy elements appear to be evenly distributed throughout the genome. CONCLUSIONS: Considering the high proportion of LTR retrotransposon in the oil palm genome, our work will contribute to a greater understanding of their impact on genome organization and evolution. Moreover, the knowledge gained from this study constitutes a valuable resource for both the improvement of genome annotation and the investigation of the evolutionary history of palms.

Ploidy and domestication are associated with genome size variation in Palms.

PREMISE OF THE STUDY: The genome size of a species (C-value) is associated with growth, development and adaptation to environmental changes. Angiosperm C-values range 1200-fold and frequently vary within species, although little is known about the impacts of domestication on genome size. Genome size variation among related species of palms is of evolutionary significance because changes characterize clades and may be associated with polyploidy, transposon amplifications, deletions, or rearrangements. Further knowledge of genome size will provide crucial information needed for planning of whole genome sequencing and accurate annotations. We studied the genome size of Cocos nucifera and its variation among cultivars, and compared it to values for related palms from the Attaleinae subtribe. METHODS: Flow cytometric analysis of isolated nuclei from young palm leaves was used to estimate genome sizes of 23 coconut cultivars (Talls, Dwarfs, and hybrids) worldwide and 17 Cocoseae species. Ancestral genome size was reconstructed on a maximum likelihood phylogeny of Attaleinae from seven WRKY loci. KEY RESULTS: The coconut genome is large-averaging 5.966 pg-and shows intraspecific variation associated with domestication. Variation among Tall coconuts was significantly greater than among Dwarfs. Attaleinae genomes showed moderate size variation across genera, except polyploids Jubaeopsis caffra, Voanioala gerardii, Beccariophoenix alfredii, and Allagoptera caudescens, which had larger genomes. CONCLUSIONS: Our results contribute to the understanding of the relationship between domestication and genome size in long-lived tree crops and provide a basis for whole-genome sequencing of the coconut and other domesticated plants. Polyploidy evolved independently in two clades within Attaleinae.

DNA methylation and expression of the EgDEF1 gene and neighboring retrotransposons in mantled somaclonal variants of oil palm.

The mantled floral phenotype of oil palm (Elaeis guineensis) affects somatic embryogenesis-derived individuals and is morphologically similar to mutants defective in the B-class MADS-box genes. This somaclonal variation has been previously demonstrated to be associated to a significant deficit in genome-wide DNA methylation. In order to elucidate the possible role of DNA methylation in the transcriptional regulation of EgDEF1, the APETALA3 ortholog of oil palm, we studied this epigenetic mark within the gene in parallel with transcript accumulation in both normal and mantled developing inflorescences. We also examined the methylation and expression of two neighboring retrotransposons that might interfere with EgDEF1 regulation. We show that the EgDEF1 gene is essentially unmethylated and that its methylation pattern does not change with the floral phenotype whereas expression is dramatically different, ruling out a direct implication of DNA methylation in the regulation of this gene. Also, we find that both the gypsy element inserted within an intron of the EgDEF1 gene and the copia element located upstream from the promoter are heavily methylated and show little or no expression. Interestingly, we identify a shorter, alternative transcript produced by EgDEF1 and characterize its accumulation with respect to its full-length counterpart. We demonstrate that, depending on the floral phenotype, the respective proportions of these two transcripts change differently during inflorescence development. We discuss the possible phenotypical consequences of this alternative splicing and the new questions it raises in the search for the molecular mechanisms underlying the mantled phenotype in the oil palm.

Variations in genomic DNA methylation during the long-term in vitro proliferation of oil palm embryogenic suspension cultures.

KEY MESSAGE : The long-term proliferation of embryogenic cell suspensions of oil palm is associated with changes in both genomic methylation rates and embryogenic capacities. In the aim of exploring the relationship between epigenetic stability and the long-term in vitro proliferation of plant tissues, we have studied changes in genomic DNA methylation levels in embryogenic suspensions of oil palm (Elaeis guineensis Jacq.). Five embryogenic callus lines were obtained from selected hybrid seeds and then proliferated as suspension cultures. Each clonal line obtained from a single genotype was subdivided into three independent subclonal lines. Once established, cultures proliferated for 12 months and genomic DNA was sampled at 4 months intervals for the estimation of global DNA methylation rates through high performance liquid chromatography (HPLC) quantitation of deoxynucleosides. Our results show that in vitro proliferation induces DNA hypermethylation in a time-dependent fashion. Moreover, this trend is statistically significant in several clonal lines and shared between subclonal lines originating from the same genotype. Interestingly, the only clonal line undergoing loss of genomic methylation in the course of proliferation has been found unable to generate somatic embryos. We discuss the possible implications of genome-wide DNA methylation changes in proliferating cells with a view to the maintenance of genomic and epigenomic stability.

Environmental regulation of sex determination in oil palm: current knowledge and insights from other species.

BACKGROUND: The African oil palm (Elaeis guineensis) is a monoecious species of the palm subfamily Arecoideae. It may be qualified as 'temporally dioecious' in that it produces functionally unisexual male and female inflorescences in an alternating cycle on the same plant, resulting in an allogamous mode of reproduction. The 'sex ratio' of an oil palm stand is influenced by both genetic and environmental factors. In particular, the enhancement of male inflorescence production in response to water stress has been well documented. SCOPE: This paper presents a review of our current understanding of the sex determination process in oil palm and discusses possible insights that can be gained from other species. Although some informative phenological studies have been carried out, nothing is as yet known about the genetic basis of sex determination in oil palm, nor the mechanisms by which this process is regulated. Nevertheless new genomics-based techniques, when combined with field studies and biochemical and molecular cytological-based approaches, should provide a new understanding of the complex processes governing oil palm sex determination in the foreseeable future. Current hypotheses and strategies for future research are discussed.

Epigenetic imbalance and the floral developmental abnormality of the in vitro-regenerated oil palm Elaeis guineensis.

BACKGROUND: The large-scale clonal propagation of oil palm (Elaeis guineensis) is being stalled by the occurrence of the mantled somaclonal variation. Indeed, this abnormality which presents a homeotic-like conversion of male floral organs into carpelloid structures, hampers oil production since the supernumerary female organs are either sterile or produce fruits with poor oil yields. SCOPE: In the last 15 years, the prevailing point of view on the origin of the mantled floral phenotype has evolved from a random mutation event triggered by in vitro culture to a hormone-dependent dysfunction of gene regulation processes. In this review, we retrace the history of the research on the mantled variation in the light of the parallel advances made in the understanding of plant development regulation in model systems and more specifically in the role of epigenetic mechanisms. An overview of the current state of oil palm genomic and transcriptomic resources, which are key to any comparison with model organisms, is given. We show that, while displaying original characteristics, the mantled phenotype of oil palm is morphologically, and possibly molecularly, related to MADS-box genes mutants described in model plants. We also discuss the occurrence of comparable floral phenotypes in other palm species. CONCLUSIONS: Beyond its primary interest in the search for discriminating markers against an economically crippling phenotype, the study of the mantled abnormality also provides a unique opportunity to investigate the regulation of reproductive development in a perennial tropical palm. On the basis of recent results, we propose that future efforts should concentrate on the epigenetic regulation targeting MADS-box genes and transposable elements of oil palm, since both types of sequences are most likely to be involved in the mantled variant phenotype.

Isolation and expression analysis of genes encoding MET, CMT, and DRM methyltransferases in oil palm (Elaeis guineensis Jacq.) in relation to the 'mantled' somaclonal variation.

In oil palm (Elaeis guineensis Jacq.), approximately 5% of somatic embryo-derived regenerants show homeotic changes during floral development, involving an apparent feminization of male parts in flowers of both sexes, called the 'mantled' phenotype. This variant phenotype is associated with a reduction in the level of global DNA methylation. To explore possible relationships between DNA methylation level and accumulation of DNA-(cytosine-5) methyltransferase (DNMT) transcripts, the full-length coding sequences corresponding to three different DNMT families in oil palm, namely the MET, CMT, and DRM classes, have been isolated and characterized. The corresponding genes were designated as EgMET1, EgCMT1, and EgDRM1, and encode predicted polypeptides of 1543, 925, and 591 amino acid residues, respectively. Expression of oil palm DNMTs was compared between normal and variant calli and inflorescence tissues using quantitative reverse-transcription PCR. A consistent increase in transcript levels of EgMET1 and EgCMT1 was found in variant fast-growing calli relative to nodular-compact calli. Nodular-compact calli give rise to about 5% of abnormal regenerants whereas fast-growing calli generate 95% of 'mantled' palms in their clonal offspring and were previously demonstrated as having markedly hypomethylated DNA. In immature abnormal inflorescences only EgMET1 transcript levels were increased, while no changes in relative abundance of the EgCMT1 or EgDRM1 transcripts were observed. Therefore, the genome-wide hypomethylation previously described in 'mantled' material cannot be explained by a decrease in expression levels of the de novo or maintenance DNMTs, a paradox which has been previously reported in tumour cells, where there is evidence for global hypomethylation of DNA.