A marker-based strategy for the assessment of epigenetic instability in oil palm

Plant tissues from the mantled somaclonal variant of oil palm have been found to display both large-scale perturbations of genomic methylation levels and sequence-specific DNA methylation pattern changes when compared to their true-to-type counterparts. Concomitantly, several markers showing phenotype-specific transcription patterns have been identified in our group. Some of them are likely to be indicators of the altered physiological state of abnormal cultures, others are possible targets of epigenetic misregulations of flower development. We now aim to establish the exact link between the expression of these candidate marker genes and the mantled somaclonal variation state, through the exploration of their global epigenetic behaviour. This will be achieved using three complementary approaches applied to normal and abnormal tissues: i) the evaluation of transcription levels through quantitative or semi-quantitative techniques (semi-quantitative RT-PCR, real-time PCR); ii) the determination of sequence-wide DNA methylation patterns (bisulfite sequencing) and iii) the identification of the chromatin regulatory environment of these genes (Chromatin Immuno-Precipitation: ChIP). These combined strategies will ultimately help to elucidate the epigenetic mechanisms which give rise to the mantled somaclonal variant. Also, we will be able to identify which of the candidate markers which are indeed consistently linked with the abnormal state. This work will provide the basis for a molecular detection test of the variant phenotype. (Résumé d'auteur

Identification of early molecular markers associated with the mantled phenotype in micropropagated oil palms by subtractive PCR and cDNA array analysis

Tissue culture of oil palm induces somaclonal variation. The main one, known as the mantled abnormality, displays homeotic floral modifications and is associated with a lower oil yield. To detect abnormal cell lines as early as possible during the tissue culture process, studies to identify molecular markers have been carried out. Given the epigenetic nature of the mantled phenotype, differential gene expression has been focused upon rather than genome structure. We investigated gene expression patterns at the transcriptome level for three developmental stages: embryogenic cell suspensions, somatic embryos and leafy shoots. In the first step, the technique of subtractive PCR (SSH) was used to construct six cDNA libraries from variant and normal plant materials allowing enrichment for cDNA corresponding to genes which are up- or down-regulated in association with clonal conformity. In the second step, transcript abundance was monitored by macroarray hybridization. A range of genes altered in their expression in abnormal tissues has been identified and a number of which have been validated by RT-PCR. This approach will provide insights into the biological processes involved in the generation of the mantle phenotype and the identified markers should lead to the development of a clonal conformity test. (Résumé d'auteur

A transcriptome approach to study epigentic somaclonal variation in oil palm (Elaeis guineeensis Jacq.)

Micropropagation by somatic embryogenesis in oil palm produces a small but significant proportion of individuals displaying homeotic floral modifications known as the "mantled" phenotype. The identification of molecular markers has been undertaken in order to discriminate as early as possible during the in vitro process those cell lines which are variant and also to better understand the mechanism leading to abnormal flower development. Since a DNA sequence mutation does not appear to be involved, but rather an epigenetic alteration, we chose to analyse differential gene expression patterns at the transcriptome level between variant and normal material. Four SSH libraries were constructed from oil palm cell suspensions and male inflorescences. Studies of transcript abundance by macroarray hybridization allowed the identification of a range of genes altered in their expression in abnormal tissues, several of which were subsequently validated by RT-PCR analysis. The relevance of the genes identified and the biological processes in which they may be involved will be discussed. (Texte intégral

A snapshot of the epigenetic mechanisms at work in "mantled" oil palm inflorescences

The "mantled" floral variation of oil palm is similar to floral B-class MADS-box genes mutant phenotypes, since the male organs appear to be converted into female ones. We propose to build upon the previous isolation of the EgDEF1 and EgGLO2 genes in oil palm and to undertake a comprehensive investigation of the epigenetic mechanisms controlling their expression and of their role in the onset and in the maintenance of the "mantled" somaclonal phenotype. We are currently investigating the DNA methylation patterns within the regulating and coding sequences of these genes, as well as the chromatin conformation of the surrounding genomic domains. Although the experiments are still in progress, our preliminary results suggest the existence of a differential epigenetic pattern between true-to-type and "mantled" inflorescences. The implication of this discovery, with respect to the "mantled" abnormality, will be discussed. (Texte intégral