Building up resources and knowledge to unravel transcriptomics dynamics underlying Eucalyptus globulusxylogenesis

The economic importance of some Eucalyptus species, including interspecific hybrids, has been extended from the traditional interest of pulp and paper production to the emergent areas of bio-fuels and bio-materials. New genomic resources and high throughput technologies have provided the Eucalyptus research international community with the opportunities to identify genomic regions of interest in order to comprehensively dissect, catalogue and characterize genes involved in the determination of wood formation and quality. Similar strategies can be now applied to identify key regulator genes and better understand the cellular mechanisms by which they modulate the complex molecular events occurring in xylogenesis.

The Geneglob^wq project (2006-2010) produced and used a set of genomic tools which, associated with Next Generation Sequencing (NGS) technologies, allowed us to expand the knowledge of the Eucalyptus genome by focusing on regions potentially involved in the determination of wood properties, namely pulp yield and lignin content. We first start to characterize two E. grandis BAC libraries [1] constructed by the Arizona Genome Institute (with DNA from the clone Brasuz S1 whose genome was sequenced recently by the DOE [http://www.jgi.doe.gov/sequencing/why/99176.html], and two E. globulus BAC libraries made available by RAIZ [http://www.raiz-iifp.pt/]. We than used 3D-pools of BAC libraries and BAC macroarrays to characterize genomic environment of several lignin and lignin-regulator genes (e.g.EguCCR, EguCAD2 and EguRAC1) both in E. grandis and E. globulus. The shotgun sequencing of selected BAC clones containing those genes generated a high amount of sequencing data that made it possible to map the E. globulus BAC sequenced clones against the E. grandis genome (8X coverage). These comparative analyses showed extended microlinearity between both genomes, at least in the studied regions. Additionally, we have sequenced and annotated the chloroplast genome of E. grandis (GeneBank Accession NC_014570) [1].

A global approach to unravel E. globulus transcriptome dynamics has also been included and structured in the Geneglob^wq project, aiming at the identification of genomic hotspots of transcription activity. Various E. globulus xylogenesis “models” have been considered comprising several paired, contrasting wood forming tissues (provided by RAIZ): i) xylem samples collected along the year (season variation); ii) juvenile and adult individuals of a single genotype; iii) contrasting genotypes for pulp yield. Samples from these tissues were used for transcriptome sequencing using Illumina Hi-Seq technology (mRNA-SEQ). The same E. globulus genotype used for both E. globulus BAC libraries (a parent tree used in controlled crosses by RAIZ) has been re-sequenced (pair-end 100bp), and provided the first draft of an E. globulus genome. This resequencing data was mapped against the E. grandis Brasuz S1 reference genome. Transcriptomic data were also blatted against the gene models annotated in E. grandis genome, to evaluate in silico the expression of each gene.

More recently, the microEGo project (2010-2012) started the identification and characterization of Eucalyptus globulus microRNAs and their target genes, involved in the regulation of wood formation. The E. globulus season variation xylogenesis “model” was used considered for this project as well as an E. globulus reaction wood “model”. The latter comprises reaction wood tissues (tension / opposite wood) formed in bent trees at different kinetic times of gravitropic stimulation and control wood (non-bent trees). Small RNA libraries have been generated from those tissues and sequenced using Illumina Hi-Seq technology (Small RNAs-SEQ). The sequencing data from both microEGo and Geneglob^wq projects together with a genome wide bioinformatics analysis of E. grandis reference genome and E. globulus genome are being used for identification of miRNA gene and putative their putative target-genes.

These projects will hopefully constitute an important piece in the assemblage of whole new categories of knowledge and genomic resources for the Eucalyptus community, providing insights into the nature of the molecular machinery involved in wood formation and most importantly in the identification of key players determining the variability of wood characteristics and its end-uses.