Loup Rimbaud
email: loup.rimbaud@supagro.inra.fr

Emmanuel Jacquot

Samuel Soubeyrand

Gaël Thébaud

Optimization of control strategies of plant epidemics through a simulation model validated by experiments

Keywords: infectious potential, latent period, Potyviridae, SEIR, stochastic spatiotemporal simulation model

come


Fast--growing tree species such as poplar and eucalypts represent one of the most appealing sources of renewable biomass feedstock for Northern/Western and Southern Europe as they produce high yields of lignocellulosic biomass. Because the chemical and structural composition of lignified secondary cell walls render woody feedstocks particularly recalcitrant to degradation, improved genetic material is needed to use these energy crops in an efficient manner. The overall goal of TREEFORJOULES was to identify genes regulating relevant cell wall properties before moving on to identify the specific desirable allelic variants for breeding. Here are described the achievements made in the four different workpackages: In WP1, New transcriptomic data (RNASeq & miRNA libraries) dedicated to wood formation were generated for both in eucalypts and poplar. In silico integration and mining of these data allowed the selection of candidate transcription factors (TF) and miRNAs differentially expressed in contrasted wood samples. Constructs (overexpression, dominant repression and/or RNAi…) have been generated for 19 candidate TF in Eucalyptus and 25 in poplar (including 2 miRNAs). Transgenic poplar (and Arabidopsis lines) have been generated and some of them fully characterized at the biochemistry level highlighting promising candidates whose modulation of expression improve SCW composition and saccharification potential. The behavior of such promising lines in field conditions would be worth evaluated in the near future. In addition, a cryopreservation protocol was implemented for long--term storage of Eucalyptus/poplar transgenic lines, and a fast and versatile transformation system (hairy--roots) was set up to allow medium throughput functional characterization of Eucalyptus genes in an homologous system. The effects of environmental stresses (nitrogen fertilization, drought, salinity, seasonal variation and cold) were characterized on four different eucalyptus and four poplar genotypes at the phenotypic (biomass production), molecular and biochemistry levels gathering important information for future plantations and/or for breeding. In WP2, PLS_R models for high--throughput NIR spectroscopic methods for wood property measurements including all key cell wall constituents have been obtained and used for both Eucalyptus and poplar species at felling age and also on very young trees. A micro steam--treatment system was constructed and a suitable method was developed to treat samples prior to saccharification. In WP3, The colinearity between the E. grandis and P trichocarpa genomes was restricted to only several small regions. The resolution of available genetic maps using high--throughput genotyping methods has been increased and wood--related QTLs have been localized. In Eucalyptus, Chromosome 10 contains both a major QTL for S/G ratio and 10 eQTLs in both parents suggesting a role for both regulatory and structural genes in regulating wood properties. A dedicated website http://tfj.lrsv.ups--tlse.fr/ has been set up with a link to a database to store, mine, and integrate the high--throughput genomic, genetic, and phenotypic data

Table of content

WP1. Identification and functional characterization of Candidate Genes (CG)________ 4

Task 1.1 in silico identification of CGs from a compendia of expression data_____________ 4 Subtask 1.1.1 Development of new transcriptomic resources ____________________________________________ 4 Subtask 1.1.2 Integration and data mining of transcriptomic resources_________________________________ 7 Task1.2 -Transcript profiling of selected CGs _________________________________________________ 9 Task1.3 Identification of transcriptional interactome_______________________________________ 13

WP1. Identification and functional characterization of Candidate Genes (CG)

The overall objective was to identify and characterize candidate genes (CGs) (focusing on transcription factors, miRNAs and their respective partners and/or targets) involved in the regulation of wood properties important for bioenergy in poplar (P) and eucalypts (E). Twelve RNA-seq libraries were prepared and sent to GenoToul platform for sequencing (HiSeq2000, Illumina). After cleaning and trimming the reads for quality, they were mapped against the E. grandis genome, in order to identify which loci where differentially expressed between tissue type and time of bending. We found 93 loci differentially expressed between tension and opposite E. globulus wood forming tissues (3 clones), 88 of these genes were assigned each to a single E. grandis genome model gene.

The major transcriptome remodeling occurred between tension and opposite wood, and to a lesser extent between bending times (Figure 1a). When we clustered samples by their transcriptomic profiles, we observed that samples were grouped in two major groups corresponding to the type of forming tissue (tension wood versus opposite wood (Figure 1b)) involving genes implicated in carbon reallocation between the two types of wood, and thus in the different strategies of secondary cell wall (SCW) biosynthesis. In the correlation circle pyrolysis products related with proteins are represented in green, lignins in red and polysaccharide in blue. Protein origin: aa1 (amino acid content) and H products (mainly protein but also from phydroxyphenyl lignin units origin). Lignin origin: G (guaiacyl units); S (syringyl units); Py_lignin (sum of H, G and S). From polysaccharide origin but main contributor cellulose: cH (hexosans); c (carbohydrates). hemicellulose origin: cP (pentosans). The ration cP/cH was also considered. b) Main plane (PC1-PC2) with the positional display of the differentiating xylem samples from tension (WT) and opposite wood (WO) kinetics (one to four weeks bending stress; S1-S4). PCA was performed using the ade4 library of the R package (Chessel et al.,2004).

To identify which of these genes were also post-regulated by miRNAs, P8 crossed the list of differentially expressed genes with the list of genes target by miRNAs (degradome analysis). We identified six candidate genes targeted by miRNAs, including one Beta-6tubulin known to be involved in cellulose microfibril orientation within the cell wall. P8 made available to the project 768 novel and conserved E. globulus miRNA candidates. Degradome libraries from tension wood (2) opposite wood (2) and control wood from non-bent trees (Carocha et al, in preparation).

P2 identified 5,869 putative miRNAs from tension wood (TW) and opposite wood (OW) tissues from 3 different poplar genotypes (P. deltoides x P. trichocarpa hybrids, P. trichocarpa N°101 174 and Populus tremula x P. alba INRA clone #717 1B4). From these predicted miRNAs, 213 were already described in mirbase corresponding to 46 families. Already described miRNAs in mirbase were well conserved between the different genotypes but a huge amount of unknown predicted miRNAs presented sequence variation between genotypes. In the ANR project "TROPIC" (2011)(2012)(2013)(2014)(2015) a Degradome approach has been developed for the clone 717-1B4 in order to experimentally 1) identify the targets of these miRNA and 2) validate them as miRNA actors (Figure 3) (Gourcilleau et al, 2012, Gourcilleau et al., in preparation). miR397a appeared up-regulated in TW from clone 101-174 and only detectable in TW tissues of P. deltoides x P. trichocarpa hybrids. As this miRNA was predicted to targets laccases, enzymes putatively involved in lignin polymerization, it has been selected for functional analysis in poplar (see Task1.4) with the aim to decrease lignification by interfering with lignin polymerization (Gourcilleau et al, in preparation).

P5a performed a transcriptome sequencing analysis of developing wood in Populus tremula x Populus tremuloides (Esch5) by Illumina High-Seq. 2000 (GenXPro, Frankfurt) and identified 19,139 different transcripts, including 707 transcription factors/transcription regulators. P5a also prepared miRNA from total RNA by size-fractionation using gel filtration.

In total, 191,214 different miRNA tags were generated by Illumina sequencing with HiSeq 2000 sequencer.

Subtask 1.1.2 Integration and data mining of transcriptomic resources P1 built within the 34J web site (see WP4; tfj.lrsv.ups-tlse.fr/) a database with restricted access to integrate and mine transcriptomic resources mainly for Eucalyptus. For poplar, whose genome has been sequenced long ago, several tools to mine transcriptomic data are publicly available.

The online database contains RNAseq and high throughput qPCR data generated within this project (P1 & P8, subtask 1.1.1; task 1.2) as well as large in-house collections of wood-associated ESTs and transcriptomic data already generated in parallel projects. It provides to users both online graphical representation and analytical tools (Figure 4). The representation of transcriptomic datasets (heat maps and eFP browser adapted for 34J purposes) as well as statistical tools (PCA, clustering, correlation analyses and gene network) have been developed. The data mining using this tool allowed the screening of candidate genes with specific expression patterns (see below task 1.2). Options for sorting out list of genes presenting similar profiles and drawing targeted gene correlation networks have been implemented. Because one of the major aims of this project was to take advantage of sharing resources developed on Eucalyptus and Poplar, all the tools have been developed to allow further integration of datasets available on both trees. Beyond its importance for the 34J project, this interactive database will be maintained and enriched by P1 bioinformaticians as perennial platform of analytical tools open for a network of collaborating teams working on the regulation of wood formation at the international level.

Task1.2 -Transcript profiling of selected CGs

Objective: To refine the number of CGs (selected in task 1.1), the most pertinent ones were selected, using highthroughput expression profiling tools in contrasted wood samples (juvenile vs mature, opposite vs tension, distinct wood tissues/cellular types…) from different E and P species and genotypes.

Eucalyptus

To allow accurate RT-qPCR analysis, we first selected 13 potential reference genes and validated 5 of them using 17 distinct experimental sets covering 17 organs and tissues, as well as various developmental and environmental conditions for 5 Eucalyptus species (published in [START_REF] Cassan-Wang | Reference Genes for High-Throughput Quantitative Reverse Transcription-PCR Analysis of Gene Expression in Organs and Tissues of Eucalyptus Grown in Various Environmental Conditions[END_REF]; collaboration between P1, P2 &P8). These genes were particularly useful for normalization of microfluidic experiments (large-scale qRT-PCR) used by several partners of the project but are also instrumental for the whole Eucalyptus community.

Thanks to the release of the sequence of the Eucalyptus grandis genome (Myburg et al., 2014) to which P1 and P8 participated for assembly and expert annotation), we performed an in depth analysis of 11 families of genes involved in lignin biosynthesis [START_REF] Carocha | Genome-wide analysis of the lignin toolbox of Eucalyptus grandis[END_REF] (P1 &P8) as well as 4 transcription factor families containing members known to be involved in secondary cell wall formation such as the MYB [START_REF] Soler | The Eucalyptus grandis R2R3-MYB transcription factor family: evidence for woody growth-related evolution and function[END_REF] (P1 &P8), NAC [START_REF] Hussey | Structural, evolutionary and functional analysis of the NAC domain protein family in Eucalyptus[END_REF] (P1), Aux/IAA [START_REF] Yu | Comprehensive Genome-Wide Analysis of the Aux/IAA Gene Family in Eucalyptus: Evidence for the Role of EgrIAA4 in Wood Formation[END_REF] (P1 &P8) and ARF [START_REF] Yu | Genome-wide characterization and expression profiling of the AUXIN RESPONSE FACTOR (ARF) gene family in Eucalyptus grandis[END_REF]) (P1 &P8). We performed comparative phylogenetic analyses including poplar genes as well as other sequenced genomes such as Arabidopsis, grapevine and rice. The genome-wide study of all the above-mentioned families enabled us to highlight some remarkable and new features. For instance, in the large MYB family, some subgroups are more expanded in woody plants than in herbaceous plants. We could also identify new subgroups containing only members from woody plants. Remarkably, 94% of the tandem duplicated R2R3-MYB genes belong to those woody-expanded and woody-preferential subgroups. In striking contrast, subgroups containing genes known involved in SCW formation are well conserved among woody and non-woody species and did not suffer tandem duplication events.

We then used RNA Seq data from a range of six organs and tissues (kindly provided by Prof Myburg, Pretoria University, South Africa) to highlight genes preferentially expressed in xylem. Combining the data from comparative phylogenetic analyses, bibliography and high throughput transcriptome analyses, we selected a subset of 250 genes to study more in depth their expression in a large range of organs and tissues, developmental stages and in response to different environmental conditions (thanks to plant material made available by P3 (FCBA), P4 (CIRAD), P7 (Altri Florestal) & P8 (IBET). One of the most remarkable results of our study is that all R2R3-MYB genes analyzed belonging to three of the woodysubgroups are preferentially expressed in cambium (Figure 5). Cambial activity is responsible for secondary-growth, one major feature of woody perennial plants. All these data were integrated in the 34J database (see task 1.1.2) and mined to select the best Eucalyptus candidate genes (CG) (Figure 6). Among the 250 genes tested, more than 60 CG were preferentially and/or highly expressed in xylem and/or cambium. This list included 25 MYB, 11 NAC, 8 Aux/IAA, 3 ARF. We established a ranking from 1 to 5 to restrict the number of CG for functional characterization by including several additional criteria such as: i) their relative level of expression in xylem/cambium, ii) their level of tissue specificity, iii) their novelty (gene or putative orthologs not studied before) -for instance in clades either « specific » of woody plants and/or expanded in Eucalyptus, (iv) their coexpression with lignin genes or other known SCW-related genes including transcription factors, (v) their responses to one or more abiotic stresses among those tested i.e. drought, cold, nitrogen status, mechanical stress.

We selected a total of 16 CG for functional characterization including 9 MYBs

Poplar

A list of more than 150 CGs was built based on bibliographic data and both in-house microarray and RNAseq expression data from tension wood (TW), opposite wood (OW) and straight wood (CW). P2 integrated these data into PathwayStudio ® database in order to select candidates genes based on their expression patterns in poplar tissues, mainly focusing on genes differentially expressed between TW as compared to opposite or straight wood and focusing on transcription factors (Figure 7). Some transcription factors have been chosen also based on known function in secondary cell wall (SCW) formation in Arabidopsis or Eucalyptus; however, in a more risky approach some genes with unknown function in SCW formation have also been selected.

Fig 7.

Example of biological knowledge for KNAT7 (top) and WLIM1 (bottom) from Pathway Studio® database in which sRNA data, degradome sequencing, poplar annotation, and expression data have been incorporated (view of RNAseq data on this example). We can notice KNAT7 interactors such as PtrWIN2B (NAC domain protein), BLH6, PtrMYW021 (MYB46 ortholog), diagrams showing expression values and one microRNA (miR1446) predicted (psRNA target prediction) to target KNAT7. NB: degradome sequencing analysis have not confirmed that miR1446 targeted KNAT7 in poplar. For WLIM1, degradome analysis and sRNA sequencing identified one miRNA (agpf_2444) targeting this gene.

Task1.3 Identification of transcriptional interactome

Objective: to identify protein partners of the most interesting TF P1 constructed and screened a Eucalyptus xylem yeast-two-hybrid library using EgMBY1, a SCW repressor previously characterized by P1 [START_REF] Legay | EgMYB1, an R2R3 MYB transcription factor from eucalyptus negatively regulates secondary cell wall formation in Arabidopsis and poplar[END_REF] as a bait.

Several candidates arose from this screening but the most abundant were a Histone linker (EgH1) and an ovate protein. We showed that EgH1 and EgOvate co-localize with EgMYB1 in the nucleus of transiently transformed cells. We further validated this interaction in planta using the powerful FRET-FLIM technology and confirmed the specificity of the interaction in planta (Figure 8. Soler et al, in preparation + Soler et al, com IUFRO, 2015). These two partners of EgMYB1 have been added to our list of 16 CGs (task 1.2) coming up to 18 CG and functionally characterized in Arabidopsis as well as in Eucalyptus hairy roots.

It is worth saying that the yeast-two-hybrid Eucalyptus xylem library is a unique tool available for the Eucalyptus community and we acquired the expertise to identify and validate further proteic partners of other genes involved in SCW formation.

Task 1.4 Functional characterization of a limited number of selected CGs

A new homologous stable transformation system for Eucalyptus As we did not succeed to obtain transgenic calli expressing in poplar the GFP tag fused to transcription factors, the grafting method allowing transgenic wood sectors initially planned could not be used and we decided instead to transform Arabidopsis and poplar plants for phenotyping. This change induced a big delay in the project and many transgenic lines generated could not be fully characterized at the end of the project. For Eucalyptus, transformation and regeneration are too long and fastidious, and are not appropriate for functional characterization of many candidate genes within relatively short research projects (3-4 years). To overcome this difficulty we developed a fast system allowing to high throughput characterization of candidate genes by producing transformed Eucalyptus hairy roots using Agrobacterium rhizogenes (Plasencia et al, com IUFRO 2015, paper in preparation). Composite plants, containing transformed hairy roots and non-transformed aerial stems were obtained in less than 3 months. We demonstrated that this homologous transformation system is suitable to study the function of SCW-related genes using as a proof of concept the down-regulation of the CCR gene encoding the first enzyme committed to the lignin specific pathway. The phenotype of the transformed roots with reduced lignin levels and irregular vessels shape was identical to that of stems of other transgenic plants downregulated for the CCR gene (Figure 9. Plasencia et al, com IUFRO, paper in preparation).

Constructs and transformation for Eucalyptus candidate genes

P1 selected 19 Eucalyptus CG: 2 ARF, 4 Aux/IAA, 1 NAC and 10 R2R3-MYB, 1 Histone linker, 1 ovate genes (see Table 1). Some of these genes were characterized for their activating or repressing activity in transient transactivation assays [START_REF] Yu | Genome-wide characterization and expression profiling of the AUXIN RESPONSE FACTOR (ARF) gene family in Eucalyptus grandis[END_REF][START_REF] Yu | Comprehensive Genome-Wide Analysis of the Aux/IAA Gene Family in Eucalyptus: Evidence for the Role of EgrIAA4 in Wood Formation[END_REF]. A total of 26 constructs were made (22 by P1 and 4 by P11) including overexpression of a native of mutated version (OEm) and/or dominant repression and transferred in Arabidopsis and/or poplar, and more recently in Eucalyptus hairy roots. In addition constructs overexpressing individually or in combination the two interacting partners of EgMYB1 (ovate and H1) were transformed in Arabidopsis and in Eucalyptus hairy roots.

Because of the high number of genes and constructs, P1 collaborated with P5a, P11 and P Up to five different lines were selected for each construct and transcript levels were assessed to further choose three them for in depth characterization of their wood-related phenotypes using

(1) histochemistry (Figure 10.) -Saccharification (P1) Some transgenic plants showed distinct clear wood-related phenotypes (Table 2) and the phenotypic characterization of one gene (EgrIAA4) is already published [START_REF] Yu | Genome-wide characterization and expression profiling of the AUXIN RESPONSE FACTOR (ARF) gene family in Eucalyptus grandis[END_REF][START_REF] Yu | Comprehensive Genome-Wide Analysis of the Aux/IAA Gene Family in Eucalyptus: Evidence for the Role of EgrIAA4 in Wood Formation[END_REF]) (Yu Hong PhD). Others are in preparation.

Although not all transgenics could be completely characterized before the end of the project, clearly some candidates seem very promising with for instance a lower lignin content and an improved saccharification efficiency, without showing detrimental phenotypes. Constructs and transformation for Poplar candidate genes P2 selected 12 candidate genes including one microRNA (miR397a) for functional analysis (see Table 3 below). Constructions were made to overexpress (OE) CG in their native form or fused to a dominant repression (DR) motif. We present below fully characterized OE transgenic lines from MYB156, MYB221, MYB090, miR397a and KNAT7. For the other transgenic lines greenhouse material have been produced, phenotypic alterations have been monitored but lignin analyses need to be done. Between 3 and 32 transgenic lines were produced and acclimatized depending on the transformation.

Table 3: Candidate genes selected by P2 for functional analysis.

Transformation ID Gene name JGI Name_v2.2 JGI name V3 MYB156 MYB156 POPTR_0009s13640 Potri.009G134000 MYB221 MYB221 POPTR_0004s18020 Potri.004G174400 MYB090 MYB090 POPTR_0015s05130 Potri.015G033600 MIR397 MiR397a NA NA A1 KNAT7 POPTR_0001s08550 Potri.001G112200 A2 WLIM1 POPTR_0001s30020 Potri.001G292900 A3 MYB37 POPTR_0003s13190 Potri.003G132000 A5 MYB104 POPTR_0015s09430 Potri.015G082700 A8 AtMYB48 POPTR_0009s03240 Potri.009G027300 A9 K15E6 (basic leucine zipper) POPTR_0008s11260 Potri.008G113400 A19 WRKY65 POPTR_0004s05920
Potri.004G060900 A35 F24B18.9 (zinc finger) POPTR_0013s14680

Potri.013G150500

Poplar MYB156 or MYB221 are a co-orthologs pair of the Eucalyptus EgMYB1 and the Arabidopsis AtMYB4 which have been shown to be repressor of lignification in these respective species. Respectively, 22 and 24 transgenic lines have been produced and finally 2 lines were selected for MYB156 and 3 lines for MYB221. These lines were grown for 3 months and analyzed for lignin content. We did not observe any growth defect in these lines except more sylleptic branches in MYB221 OE lines and cup-shaped leaves in MYB156 OE lines. A significant decrease in lignin content was observed in two MYB156 OE lines (Figure 11). Histological inspection of wood stem cross sections showed that secondary cell wall fibers of MYB156 OE (lines 10&15) are depleted in lignin and enriched in cellulose (less colored by safranin but more by astra blue). MYB221 OE lines showed more intense blue coloration in the perforation plate between vessels indicating a decreased lignin content of this cell wall. In conclusion, these 2 MYB transcription factors appear also to be repressors of lignification as for their orthologs EgMYB1 / AtMYB4. However, some functionalization occurred between the poplar paralogs as the overexpressing lines did not give the same phenotype, although effects of transgene insertion site or level of transgene expression cannot be completely ruled out. Chemical analysis revealed a slight reduction in lignin content relative to the control and histological investigations showed that ray parenchyma cell walls were poorly lignified (Figure 12; Lakhal et al., in preparation). were selected and further analyzed for lignin and cell wall sugars content and composition, and saccharification potential. Transcriptomics (RNAseq) and metabolomics analysis have also been done but results are currently being analyzed. All analyses have been done on 5-month-old plant in order to have sufficient amount of material for all the chemical analyses. No growth defect was observed. A slight reduction in total lignin of 9-10% was determined on the transgenic lines, line MIR397-A1 being the most affected followed by MIR397-A7, MIR397-A3 & MIR397-A25.

Thioacidolysis analyses showed a significant 12% increase in S/G ratio for line MIR397-A1.

Overexpression of pre-miR397a also slightly impacted the cell wall sugar composition.

Interestingly, all these cell wall modifications have a beneficial impact of the saccharification potential of the wood of these transgenic lines (Figure 13 &14; Ayangma et al., in preparation). Alterations in leaf size and leaf veins were also noted as well as the absence of nectaries on the most affected line KT1 (Figure 16.).

Fig. 16

Leaf phenotypes observed on KNAT7 over expressing lines. Severity of phenotypes ranges from KT1 to KT3.

Lignin content analysis was not significantly different between transgenic lines and controls but thioacidolysis analysis of lignin composition pointed 9% significant increase in S/G ratio for the most affected lines KT1 and KT2. Cell wall sugar analysis revealed a significant increase in glucose content and decrease in Mannose and Uronic acids, even the differences were quite low. A saccharification assay showed up to 22.9% significant increase in glucose release from the transgenic lines compared to the wild type poplar (Figure 17

Set up of a cryopreservation protocol for Eucalyptus and poplar transgenic lines

In order to enable securing valuable transgenic lines as soon as possible after their identification/selection, P4 setup a new cryopreservation protocol for in vitro axillary buds and shoot tips from a wide range of Eucalyptus and Populus species to be used for transgenics from P1, P2, P8, P11. It was shown to work with many broadleaved species, irrespectively of their cold hardiness (Table 4 below Table 4. Summary of our preliminary results on various plant material already in commercial plantation or used for upstream or basic studies

The cryopreservation service was opened for free in the frame of the project to the 34J partners by mid-2013 but the availability of transgenic lines for which cryopreservation was needed took time. Some previous Eucalyptus lines from P1 and all poplar lines provided were cryopreserved (Table 4). This service is continuing in the frame of the Xylobiotech platform ran by P2 and P4 (coordinated by P4; http://biotech.xyloforest.org/). The service includes actual survival ability checking to be sure that the frozen plant material is enough for many successful regrowth attempts.

Task 1.5 Environmental regulation of CGs and impact on biomass production and wood quality

Objective: To provide insights into the environmental effects on the expression of CGs (selected in task 1/1.2) and assess how environmental conditions impact the production of wood biomass and quality.

Subtask 1.5 Selection of Eucalyptus and poplar genotypes

Genotypes of interest for Spain based on their potential for bioenergy and growth under non-optimal or stress conditions (Figure 19 Overall, the four Eucalyptus genotypes responded in a similar way to the nitrogen treatments. They show a general decrease in growth and development parameters and xylem extractives contents under low nitrogen treatment and an increase of those parameters under high nitrogen treatment (Figure 20). These data negatively correlate with transcript levels of both sets of wood-related genes (Figure 21), as a general induction in transcript levels was observed under low nitrogen whereas high nitrogen conditions caused a reduction in transcript levels.

These results suggest that the genus Eucalyptus has a wider dynamic range to assimilate nitrogen than the genus Populus for the tested range (0 to 10 mM ammonium nitrate), since poplar genotypes did not show significant changes under high nitrogen treatment regarding the control condition (Figure 22). Values with significant differences with the control plants, according to a t-test (p< 0.05), are labelled with a star.

B--Drought assay (P11).

Eucalyptus and Populus plantlets were full watered (control) or watered to reach 50% of full capacity of water retention by the river sand substrate. Plantlets were maintained under drought stress until they showed clear stress symptoms.

Different growth and developmental parameters were evaluated in treated and control plants: The hybrids, P. x c "Oud" and P. t x a "TxA", showed greater repression in growth (height) than the two P. alba during the last week of drought treatment suggesting a relatively higher sensitivity to drought in the hybrids (Figure 23). Drought treatment caused a significant reduction in leaf area in the hybrids, while the P. alba genotypes showed the opposite trend, i. e. increases in leaf areas in response to drought, revealing important differences between these genotypes in the regulation of leaf development in response to drought stress. P. a. "J-1-3-18" showed the greatest reduction in the generation of internodes and total net growth during drought. "J-1-3-18" showed significant increases in the area of transversal sections of stem, including significant reductions in total area and area of the xylem. On the contrary, the other P. a. ("PO") showed reductions in these parameters. The two P. a. genotypes, not only showed opposite trends in parameters regarding the stem section areas, but also in transcript levels when compared to their controls.

"J-1-3-18" showed the greatest reduction among the four genotypes in transcript levels of the wood related genes and transcription factors, while "PO" showed an increment in the level of transcripts encoding transcription factors. These facts reveal clear differences between the two P. alba genotypes.

With regard to the Eucalyptus genus, drought caused a moderate decrease in the different growth and developmental parameters. However, no remarkable differences or a clear differential trend were observed among genotypes in response to the treatment both in growth/developmental parameters or in transcript levels. Values with significant differences with the control plants, according to a t-test (p< 0.05), are labelled with a star. Our assays have shown interspecific variability in the response to salt stress both for

C. Saline stress assay (P11/P13).

Acclimated plantlets of

Populus and Eucalyptus spp., both in relation to survival and production variables, as well as for functional, morphological and biochemical parameters. For poplar, we found significant differences between the species. P. alba genotypes proved to be very tolerant for both moderate and severe salt dose, with very high rates of survival and absence of leaf damages. Opposite, the two hybrids 'Oudemberg' (P. x canadensis) and 'TxA' (P. tremula x P. alba) showed higher sensitivity, higher percentage of dead plants, and a lot of leaf damages as necrosis, highlighting less capacity of adaptation of these genotypes to cope with salinity stress (Table 5 andTable 6).

These differences were also apparent in terms of transcript levels of wood-related genes (Figure 24). Both treatments (moderate and severe) caused a reduction in transcript levels for both functional group of genes (encoding enzymes involved in cellulose and lignin biosynthesis or transcriptional factors). However, the impact in both P. alba genotypes was clearly lower compared with the two hybrids (P. x canadensis and P.tremula x P. alba), with P. alba ' showing the lowest effect of salt treatments on transcript levels. Therefore, this phenotype at molecular level correlates with the higher performance and survival of these genotypes compared to the others.

Regarding Eucalyptus spp., all the tested genotypes showed a very high tolerance (100 % of survival) to grow under conditions of salt stress, both for moderate and severe dose, and with no symptoms of foliar damages. Despite the high percentage of survival displayed by all the Eucalyptus genotypes, a general reduction in growth and therefore in biomass production was observed for them when salt treatments were applied (Table 7). Concomitantly, a reduction in transcript levels for most of the tested genes were also observed in both stress conditions (Figure 25), although the impact was lower compared to poplar (compare scales of the X axis). The genotypes of the species E. urograndis and E. camaldulensis showed the highest values of woody and root biomass both in absence of salt and after treatments, being therefore more productive compared to E. globulus genotypes (Table 1.5.3). However, the most productive genotype, i.e. E. urograndis, was the one which suffered the more relevant decrease in production under saline treatments, followed by E. globulus ('Odiel'), and being E. camaldulensis and especially E. globulus 'Anselmo' the ones with lower biomass reductions. However, no remarkable differences were observed at an overall level in the transcript levels. In conclusion, we observe less tolerance to salt stress of the most productive genotypes with respect to those with lower amount of biomass in absence of salt. Regarding Populus spp., a similar patter was observed, where the genotypes of P. alba, which were less productive than the hybrids in the control treatment, were the only ones which showed a tolerance behavior to deal with salinity stress, being both hybrids P. x canadensis and P. tremula x P. alba much more sensitive, despite being more productive in absence of salt. Cold treatments were performed in a cold room (4-7°C) keeping the same photoperiod as in the greenhouse. Three samples were collected per genotype, each with 3 plants. The samples were collected between the 3 and 4 hours after sunrise: t = 0, samples collected in the greenhouse; t = 24 h, collected in the cold room; t = 4 weeks, collected in the cold room. Plants were grown to a height of 120-130 cm (3-4 months). Seasonal variation experiments were made with adult trees growing under natural conditions. Three samples were collected per genotype, each with 1 plant (E. globulus) or 3 plants (P. alba). Sampling was done between 3-4 hours after sunrise (morning samples) and 9-10h after sunrise (afternoon samples), in March, June, September and December. We used 3 genotypes of Eucalyptus globulus: MB43, GB3 and GM248, all growing in field plots owned by the company ALTRI in Óbidos, Portugal. For

Populus, we used one genotype, P. alba, grown in Madrid, Spain.

In cold experiments, samples were used for total RNA extraction and further analysis of gene expression by qRT-PCR (samples t = 0, t = 24h and t = 4 weeks) and for analysis of wood (samples t = 0 and t = 4 weeks). In the cold experiments, extraction of total RNA was made from the upper two thirds of the stem were used (in the sample t = 24h the lower third was eliminated).

In seasonal variation experiments, samples were also used for total RNA extraction, analysis of gene expression by qRT-PCR, and for analysis of wood. 2-3 branches per tree were collected (2 years-old branches). The srapped material was immediately frozen in liquid N2 and stored at -80°C.

For the analysis of wood in the cold experiments with Eucalyptus and poplar, the lower third of the stem was used. Bark was separated from the xylem, and the pith was removed from the xylem. For the analysis of wood in the seasonal variation experiments (made only for the Eucalyptus clones) 6 branches from 6 different individuals were sampled. Bark was separated from the xylem, and the pith was removed from the xylem. The sliced material was immediately frozen in liquid N2 and stored at -80°C. Subsequently the material was grinded in an IKA® A11 basic apparatus keeping it frozen, until obtaining a fine powder. Cortex was ground by hand in a mortar and the obtained powder was lyophilized overnight in Cryodos TELSTAR apparatus keeping it frozen. Cold-treated samples of poplar and eucalyptus, and seasonal samples of eucalyptus were sent for wood analysis to P9 (IICT, Portugal).

-LRSV (France, P1): Eucalyptus seedlings (Eucalyptus gundal, genotype 121) were obtained from FCBA (P4). Plants were grown in a phytotron until approximately 60 cm height, using a long day photoperiod (16h of day / 8h of night), 80% of humidity. In normal conditions, temperatures varied between 25°C (day) and 22°C (night). During cold acclimation, temperatures varied between 8°C (day) and 4°C (night). For each plant, the lower part of main stem was collected immediately after dawn at T0 (0 days of cold exposure), T1 (2 days of cold exposure), T2 (15 days of cold exposure) and T3 (46 days of cold exposure). For biochemical analyses, the bark was removed. Six biological replicates from independent plants were harvested for each time point, immediately frozen in liquid nitrogen. Subsequently the material was grinded using a RETSCH 400M and stored at -80°C. For the analysis of wood biochemistry, powder was lyophilized 48h using a Christ Alpha 1-4 LSC freeze dryer. Klason and thioacidolysis were performed as described in Méchin et al., 2014. Extractive quantification, analytical pyrolysis and NIRS were performed by partner P9 (IICT, Portugal).

D2. Regulation of gene expression by cold temperature

Candidate genes differentially expressed in response to cold and along the year: The PhD student, Daniel Conde Rodríguez, did a stay in 2013 in the P1 (LRSV, France) to carry out the gene expression analyses by qRT-PCR using the high throughput Fluidigm BioMarkTM HD machine. We analyzed the expression of the CGs selected by the groups involved in this working package as potential regulators of wood properties relevant for bioenergy production.

The partners P1 (LRSV, France) and P8 (IBET, Portugal), and P2 (INRA, France) and P5

(VTi, Germany), provided us with the primers for eucalyptus and poplar respectively. We analyzed the performance of the selected CGs in response to cold and along the four seasons of the year both in poplar and Eucalyptus.

In poplar, in response to cold, the following genes were found to be deregulated in the 4 analyzed genotypes: P. alba J-3-18, P. PO0-10-20, P. x canadienses Oudemberg (P. deltoides S.513-60 x P. nigra S57-3) and P. tremula x P. alba INRA 717 1B4 (Table 8).

Table 8. CG list in response to cold for various genotypes of poplar

In poplar and Eucalyptus, comparing the expression in December vs. June, the following genes were found to be deregulated in the P. alba and the 3 analyzed Eucalyptus genotypes E.

globulus MB43, GB3 and GM248 (Table 9) -Candidate genes differentially expressed in response to cold in Eucalyptus:

Because of a technical problem with the Fluidigm system, the Eucalyptus cold experiment could not be analyzed. A similar experiment using >60 seedlings of a single eucalyptus clone submitted to 7 weeks of cold acclimation has been performed later by P1 (LRSV, France).

Gene expression profiles were successfully analyzed by using Fluidigm BioMarkTM HD on similar candidate genes. More than 120 gene expression profiles were assessed by classical Real time QPCR (ABI7900 Applied Biosysteme) as well as using the Fluidigm BioMarkTM HD. Selected candidate genes were coding for enzymes related to SCW deposition, or transcription factors belonging to MYB, NAC bZIP, bHLH or WRKY families. They were selected according to their role in SCW deposition already described in literature (Hussey et al., 2013;[START_REF] Myburg | The genome of Eucalyptus grandis[END_REF] or their putative involvement in xylem tissue patterning [START_REF] Myburg | The genome of Eucalyptus grandis[END_REF]Soler et al., 2014[START_REF] Hussey | Structural, evolutionary and functional analysis of the NAC domain protein family in Eucalyptus[END_REF]. From this analysis, we detected 67 genes up-regulated including 30 transcription factors (ratio T3/T0>2) and 28 were downregulated including 4 transcription factors (ratio T3/T0<0.5) after 46 days of cold () (Figure 26). Together these results suggest that the lignin pathway was induced in response to cold temperature and pointed out transcription factors putatively involved in this regulation. The best transcription factor candidates have been targeted for functional analyses to decipher their role in wood formation. Gene expression profiles were in accordance with biochemical analyses of wood composition (P9, IICT).

D3. Changes in wood composition in response to cold and along the year:

Poplar and Eucalyptus wood samples from the cold experiment were analyzed by P9 (IICT, Portugal). Eucalyptus wood samples from the seasonal variation experiment were analyzed byP8 (IBET, Portugal) and P9 (IICT, Portugal). Wood after exposure of seedlings to four weeks of cold were compared (P12). Specifically, an increase in the amount of extractives with ethanol after exposure to cold, both in xylem and bark, was detected in all the genotypes of poplar and Eucalyptus examined (Figure 27). Estimations of lignin content by the Klason method revealed that no significant changes was detected after four weeks of cold acclimation in poplar or eucalyptus, neither in xylem nor in bark. Given these results, P1 (LRSV, France) analyzed xylem samples from eucalyptus submitted to a longer period of cold acclimation (seven weeks) and a significant increase of lignin content was finally detected (Figure 27

Conclusions

The main objectives of WP1 have been achieved beyond what was expected:

 For both poplar and eucalypts: new transcriptomic resources have been generated (both RNA Seq and miRNA libraries). Genes and miRNAs differentially expressed between wood samples contrasting by their SCW structure an composition have been identified.

These data have been made available to select candidate genes, although some arose late in the project (miRNAs) and will be a valuable source of candidate for further projects.

 Thanks to the participation of P1 and P8 to the Eucalyptus grandis genome annotation, several key gene families for secondary cell wall formation have been characterized at the phylogenetic and expressional (developmental and in response to abiotic stress) levels  An interactive database within a dedicated website (restricted access to 34J Partners) has been constructed to integrate and mine transcriptomic resources and transcript profiling mainly for Eucalyptus for which public tools are not available in contrast to Poplar.

 A Eucalyptus xylem 2-yeast hybrid library had been constructed, a unique tool which will be useful for the entire community, allowing identification of proteic Interactants of EgMYB1 a repressor of the SCW formation  A new versatile, fast and powerful transformation system (eucalyptus hairy roots) has been set up for Eucalyptus, and shown to be suitable to analyze SCW-related genes.

 19 Eucalyptus Candidate TFs with a putative role in SCW formation have been selected

for functional characterization using overexpression and dominant repression constructs, and transformations have been made either in Arabidopsis and/or poplar, and/or in Eucalyptus hairy roots.

 25 Poplar candidate genes including two miRNAs have been selected for functional characterization in poplar.

 several promising candidates have been identified (KNAT7 OE lines 1 & 2, MYB156 OE lines 10 & 15, MIR397-A1, A3 & A7) for a future field trial evaluation.

 A cryopreservation protocol has been set up, and used for poplar transgenic lines generated in the project  The behavior of different eucalyptus and poplar clones has been evaluated in response to different environmental cues; drought, salinity, nitrogen, cold, seasonal variations highlighting complex responses depending on the genus, genotype, and stress applied.

WP2--CHARACTERIZATION OF WOOD & BIO--ENERGY POTENTIALS Task 2.1 Development and application of high throughput methodologies

The objective of this task was to develop high-throughput, non-destructive methodologies to replace costly, time consuming, and destructive phenotyping methods for poplar and eucalypts.

Selected wood samples, 72 Populus deltoides x trichocarpa (P2), 34 Eucalyptus urophylla

x grandis (P3), and 24 Eucalyptus globulus (P9) evenly distributed within the range of NIR spectral variations were assessed for extractives (P2, P3, P9), Klason lignin (P5, P9) total lignin (P9) and sugar composition by HPLC (borate complex anion exchange chromatography) methods (P5) and used further to develop high throughput PLS-R models based on near infrared spectroscopy (NIR) (P2, P3, P9). Analytical pyrolysis was used for lignin composition analysis (P9) and for discrimination of samples (P5). A set of 26 samples (E. globulus, P9) and 36

samples (E. urophylla x E. grandis, E. grandis, P3) covering the predicted range of variation for ethanol extractives (1 -6%) were selected and sent to P2 for the analysis of the extractives composition.

Table 1a. PLS-R results of the cross-validation (CV) for Eucalyptus and poplar (P9)

Table 1b. PLS-R results of the cross-validation (CV) for poplar from P2.

In general the developed PLS-R models showed good/very good statistics with high coefficient of determination of the cross validation (R CV 2 ) and low root mean square errors of cross validation (RMSECV). Furthermore the ratio of performance to deviation (RPD) which gives the predictive ability of the models were close to or above 2.5 meaning that the models are well suited for screening in breeding programs. In the case of S/G ratio for Poplar the RPD was lower (1.9) but the RMSECV was similar to the one obtained for Eucalyptus globulus the lower RPD for poplar was mainly due to the small range of variation of the S/G for Poplar.

Based on these models, P3 predicted the chemical properties on 1150 samples corresponding to the progenies of E. urophylla x E. grandis mating used for genetic mapping (WP3, P3); P9 predicted the chemical composition of 189 samples form an E. globulus family (WP3, P3); P2 predicted the chemical composition on 1460 samples a Populus deltoides x trichocarpa mapping population.

Additionally P9 received 650 wood samples of very young plantlets often with less than a year (Eucalyptus and poplar) submitted to different types of stress (WP1) provided by P1; P11; P12 and P13. A number of additional PLS-R models (Table 2) were developed for these analysis since these samples could not be assessed using the PLS-R models for the same specie/genera but older. Only a small fraction of the samples were assessed by wet chemistry (Table 2) allowing a considerable saving in time, samples (NIR is a non-destructive analysis) and at a fraction of the cost.

Table 2. PLS-R results of the cross-validation (CV) for Eucalyptus and poplar plantlets.

The PLS-R models for plantlets were also good or very good with high coefficient of determination of the cross validation (RCV2) and low random mean square errors of cross validation (RMSECV) as well as RPD close to or above 2.5.

P2 developed FT-IR calibrations for klason lignin and S/G ratio in 33 plantlets of poplar mir397 lines from WP1. Despite the low number of samples, these calibrations were already pretty accurate with RMSECV of 0.59 and 0.07 and R² of 0.68 and 0.73 for klason lignin and S/G ratio respectively. These calibrations are currently being extended to other lines in order to provide a high throughput method for screening transgenic lines in poplar.

A round-robin test for lignin content determination was agreed between partners.

Extractive-free milled wood samples of E. globulus, E urophylla and poplar GS were prepared by P9, P3 and P2 respectively, representative aliquots of each sample were distributed between P9, P3, P2, P6 and P5.

The results of the round-robin test showed small differences results between partners except for P6 especially so for total lignin which was 5 points higher than the remaining partners, no explanation was provided as for these discrepancies (Table 3).

For chemical analyses (P5), a total of 428 transgenic samples were received from four different partner organizations comprising 51 poplar hybrids (P. deltoids x P. trichocarpa), 61

Eucalyptus hybrids (E. urophylla x E. grandis), and 316 poplars (P. tremula, P. tremuloides, P. x canescens, P. alba).

Table 3 Round Robin test results

Task 2.2 Bioenergy potentials, saccharification, and bio-oil

Networking activities:

For chemical analyses, 127 transgenic samples were received from three different partners comprising 50 poplar hybrids (P. deltoids x P. trichocarpa), 24 Eucalyptus hybrids (E.

urophylla x E. grandis), and 38 poplars (P. tremula, P. tremuloides, P. x canescens, P. alba).

Selected contrasting samples of E. globulus, E. urophylla x E. grandis and P. deltoids x P. trichocarpa (two high, two low and two intermediate) regarding lignin amount for bioenergy potential (5 grams milled samples) and bio-oil production were sent to P5.

Lignin content and sugar composition

The samples were subjected to a two-step acid hydrolysis for determination of the Klason lignin content. Klason lignin contents of the poplar hybrids varied between 19.4 and 22.5 wt%, the Eucalyptus species varied between 23.7 and 33 wt% and the poplars varied between 15.8 and 25.5 wt%. Monomeric sugars resembling the carbohydrate composition of the cell wall were also determined by acid hydrolysis with subsequent borate anion exchange chromatography.

Micro steam-treatment

A micro steam-treatment system was constructed (Fig. 2) and a suitable method was developed and optimized (210 °C for 15 min, see Fig. 3) to give comparable results with a procedure at laboratory scale. The samples were further enzymatically hydrolyzed and yielded approximately 65 wt% of fermentable sugars. The resulting data from pyrolysis have been chemometrically evaluated by multivariate data treatment (see Fig. 5). The calculations include a multistep procedure consisting of (1) peak alignment, (2) transformation (3) principal component analysis and ( 4) partial least square regression analysis. Preliminary results show that samples with different gene constructs could be discriminated based on some lignin and carbohydrate peaks (see Fig. 6). Further evaluation and data interpretation is still ongoing. Preparative Mini--Pyrolysis

In order to obtain mass balances and investigate products from bio-oil production, a mini pyrolyzer was used (see Figure 6a). The whole system could be weight before and after the experiment. Approximately 100 mg were pyrolyzed, so that oil yields and char yields could be determined gravimetrically. In addition, the bio-oil composition could be analyzed by gas chromatography.

Fig. 6a

Mini pyrolyzer for bio-oil production Yields of bio-oil and char are presented in Figure 6b. In comparison to standard beech wood, the oil yields are slightly lower (2-5 % absolute) and could be caused by the lower lignin content. Figure 6c demonstrates great differences in bio-oil yield (45-50 wt%) from P5, P3, P2 samples. Samples from the wild type yields typically more bio-oil which can be related to the lignin content. Furthermore, lignin and carbohydrate contents could be predicted (r2 = 0.91) with partial least square regression (PLS) analysis. Also the organic carbon content showed in some cases a correlation with the lignin content (r2 = 0.44 -0.96).

Task 2.3 Micro-phenotyping of transgenic wood zones through microscopic and microspectrophotometric investigation methods

For microphenotyping (P5), samples from four partner institutions were taken from various transgenic poplar (Populus tremula x tremuloides) and Eucalyptus trees (Eucalyptus urograndis).

The samples were analyzed by transmission electron microscopy (TEM) and cellular UV-microspectrophotometry. The different transgenic lines of poplar trees developed xylem portions with varying degrees of lignification of their fibers as revealed by UV-spectroscopy. For example, some lines with distinctly lower lignin contents than controls were composed of fibers with less lignin in all layers. In extreme, secondary walls and middle lamella regions appeared nearly unlignified. On the other hand, we found transgenic lines with relatively high lignin contents.

Electron microscopy added information on the cell wall architecture of these topochemically varying xylem fibers. Wall thicknesses and the formation of G-layers may also play a certain role because ratios between secondary wall regions with less lignin and middle lamella regions with more lignin affect the overall lignin content as determined with chemical methods. Vessel walls with their higher lignin content as compared with surrounding fibers seem less affected by these genetic modifications.  These models were used and shown to be suitable for high throughput screening in breeding mapping populations for both poplar and Eucalyptus (WP3)  NIR PLS-R models for very-young trees (less than one-year-old eucalypts and poplar) were developed for extractives content (dichloromethane, ethanol water and total extractives), Lignin content (Klason and total lignin) and Lignin composition (S/G). This was challenging task due to the low range of variation as well as the limited quantities of samples available which was an important achievement not only for WP2 but for future projects.

 A micro steam-treatment system was constructed and a suitable method was developed and optimized to give comparable results with a procedure at laboratory scale prior to saccharification tests.

 The Bio-oil potential was shown to be variable between samples and was in general slightly lower compared in poplar and eucalyptus samples as compared to beech wood, due to lower lignin content  Microphenotyping method using UV micro-spectrometry was able to reveal lignin content variations in transgenic poplars This problems seems related to the exceptional difficulty to purify large DNA pieces from Azura, which is likely the result of its extreme phenotype (most frost tolerant Eucalyptus individual known) since many specific DNA extraction protocols (designed for Eucalyptus as well as other kind of very difficult materials like ancient oak fragments and forest soil) were probed. However, these data allowed solving inconstancies from previous targeted shotgun sequencing approaches focused on particular TF gene families (eg DREB/CBF genes involved in abiotic stress resistance). This sequencing attempt will be pursued through specific programs currently setup with Australian partners to study the genome of eucalyptus species of the tasmanian central highlands (mainly E. gunnii and E. dalrympleana).

Re-sequencing of the two parental genome E. urophylla and E. grandis was also done and data were aligned on the reference genome of E. grandis (BRASUZ1). A total of 6,667,808 polymorphic SNPs were found in at least one parent. A homemade bioinformatic pipeline allowed selection of 6000 SNPs to design a SNP array (2618 heterozygous SNPs in E. urophylla, 2524 heterozygous SNPs in E. grandis, and 858 SNPs heterozygous in both parents). The designed 6K Infinium iSelect HD Custom Genotyping BeadChip was used to genotype 1025 individuals of the mapping population. Finally, 4353 SNPs (72.6%) were usable for genetic mapping and allowed the construction of the first high-resolution genetic maps in Eucalyptus [START_REF] Bartholome | Plasticity of primary and secondary growth dynamics in Eucalyptus hybrids: a quantitative genetics and QTL mapping perspective[END_REF][START_REF] Bartholome | High-resolution genetic maps of Eucalyptus improve Eucalyptus grandis genome assembly[END_REF] (P3). The mean coverage of these high-resolution genetic maps compared to the reference genome was 97% for the E. urophylla parent and 98% for E. grandis.

The total lengths of the genetic maps were 886 cM for E. urophylla and 822 cM for E. grandis.

These maps have been used to improve the first version for the reference genome of E. grandis (version 2 proposed by P3) [START_REF] Bartholome | High-resolution genetic maps of Eucalyptus improve Eucalyptus grandis genome assembly[END_REF].

Figure from [START_REF] Bartholome | High-resolution genetic maps of Eucalyptus improve Eucalyptus grandis genome assembly[END_REF] The genetic maps constructed during the Tree for Joules project constitute invaluable genetic resources to study the recombination rate in Eucalyptus. A collaboration was engaged on this field with an Australian group (university of Tasmania). Such investigations on the recombination rate should be useful to a better understanding of the genetic architecture of wood quality traits in Eucalyptus.

Task 3.2 Comparative architecture of wood quality traits in Eucalyptus and Populus

Subtask 3.2 QTL mapping for each mapping pedigree

The Populus mapping pedigree used for the QTL analysis is a F1 full-sib family issued from an interspecific cross Populus deltoides '73028-62' x P. trichocarpa '101-74' x (330 ind.).

Two parental maps were constructed with microsatellites, SNP, AFLP and RAPD markers: the female framework map include 337 markers spanning 4028 cM on 20 linkage groups; the male map include 705 markers spanning 5320 cM and 22 linkage groups.

QTL detection was performed using phenotypic data from 147 F1 progenies (see WP2), and Rqtl. Nine traits were selected for QTL detection based on the quality of their prediction, the heritability of the trait and correlation between traits: total lignin, soluble lignin, Klason lignin (evaluated at Vti and at INRA), S/G ratio, extractives (%), cellulose, hemicellulose, total sugars from saccharification. A total of 35 QTLs (LOD>2) where detected (17 QTLs with False Discovery Rate <10%; Table 1); No QTL for cellulose content were detected. These QTLs explain 6 to 161 % of the variation. Twenty QTLs where detected on the P. trichocarpa map and 15 QTLs on the P. deltoides map. In Eucalyptus, using the mapping population E. urophylla x E. grandis used to construct low density genetic maps, P4 made a QTL analysis for cellulose and holocellulose contents. We detected one QTL for cellulose content on the LG6 of the E. urophylla parent explaining 9% of the phenotypic explained value (PEV), and one QTL for holocellulose content on the LG2 of E.

grandis with 10% PEV. The carbon isotope composition ( 13 C) of cellulose (extracted from wood) was also analyzed. This trait is kwon as a proxy of water used efficiency. As reported in Bartholomé et al. (2015), we detected several QTLs for 13 C (P93 and P97) in different linkage group for both the two parents (E. u and E. g). Major QTLs were detected on the LG4 of E.

urophylla and in a genomic region were QTLs for 13 C measured from leaves were also identified (RS2).

Figure from [START_REF] Bartholome | High-resolution genetic maps of Eucalyptus improve Eucalyptus grandis genome assembly[END_REF] Wood cores were sampled on the 1152 E. urophylla x E. grandis hybrids. Each sample was transformed into powder using robot facilities of GenoBois platform. Then, NIRs profile was assessed for each individual and NIRs calibrations (from WP2) were used to estimate different chemical properties of wood: lignin content, lignin composition (S/G ratio), sugar contents (Arabinose, Mannose, Galactose, Xylose). A QTL analysis was done for each wood property. A total of 13 QTLs regions were detected in E. grandis and 19 QTLs regions in E. urophylla (cf table 2 below). PEV was higher for lignin trait for which the quality of prediction was higher than those for sugar contents (cf WP2). In E. grandis, linkage groups 2, 3, 5, 8, 10, 11 harbored QTLs and

LGs 1,2,3,5,6,8,10,11 for E. urophylla. Six LGs were common between the two parents. This result is relevant with the polygenic determinism of such quantitative traits. -Simple co-localization between QTLs and candidate genes selected for functional studies in the WP1 were examined. For example, the QTL explaining the highest variability for -Pathway analysis integrate bibliographic data to evaluate if genes of a given list have functionally connections between them. As GO enrichment tools, these kind of tools is mainly used to analyse gene expression data, but also any kind of list of genes. A pathway has been constructed for genes under lignin QTLs.

In Eucalyptus, a web-based application "EucaMaps" was developed by P3 [START_REF] Bartholome | High-resolution genetic maps of Eucalyptus improve Eucalyptus grandis genome assembly[END_REF] available at http://w3.pierroton.inra.fr/eucamaps/. EucaMaps allows QTL comparisons between 6

Eucalyptus species and 1 Corymbia species for 267 referenced QTLs for different quantitative traits as wood quality.

Task 3.3 A case study: Dissection of a major QTL for lignin

The objective of this task was to characterize a S/G QTL detected in [START_REF] Gion | Comprehensive genetic dissection of wood properties in a widely-grown tropical tree: Eucalyptus[END_REF] on the LG6 (previous nomenclature) which corresponds to the chromosome 10 of the reference genome and LG10 of the high resolution genetic maps. First the high density mapping of the chromosome 10 was done thanks to the design of the SNPs array. The linkage group 10 was constructed using 139 SNPs and 162 SNPs in E. urophylla and E. grandis respectively. The high number of individuals genotyped (1152) allowed to precise the total genetic distance of the LG10 in the two parents: 71.9 cM in E. urophylla and 63.6 cM in E. grandis. The average interval between tow SNPs of the LG10 was 0.52 cM in E. urophylla and 0.39 cM in E. grandis. A QTL analysis for S/G was done using this high-density linkage group. As indicated on Table 2, we validated the QTL for S/G ratio detected in [START_REF] Gion | Comprehensive genetic dissection of wood properties in a widely-grown tropical tree: Eucalyptus[END_REF], using the new dataset from the project. Indeed, a major QTL was detected for the two parents: i/ a S/G QTL was detected in E.

grandis at 50 cM with a low phenotypic explained value (PEV) of 1.6%, with the nearest SNP "2-sca10_3363_0", ii/ A QTL was detected in E. urophylla at 60 cM with a major effect of S/G variation (20% of PEV), with the nearest SNP "1-sca10_293_0". The SNP "2-sca10_3363_0 of E.

grandis matched with the gene model Eucgr.J02217 of the reference genome which correspond to AT3G27810, a myb domain protein 21. In E. urophylla, SNP 1-sca10_293_0 was localized into the gene Eucgr.J02321 corresponding to AT5G14180, a MPL1 gene. This positional candidate gene approach (underlying a major QTL) brought supplemental information (in terms of putative candidate genes) to decipher the functional variability that control the variation of wood quality traits.

In parallel, an expressional candidate gene approach was also followed using a factorial mating design of E. urophylla, which enabled to characterize quantitative trait nucleotides (QTNs) in two genes underlying the S/G QTL [START_REF] Mandrou | Nucleotide diversity in lignification genes and QTNs for lignin quality in a multi-parental population of Eucalyptus urophylla[END_REF]. CCR and EgROP1 genes explained different part of the S/G variation, which suggested that both the two genes are involved in the genetic control of S/G variation in E. urophylla. We tested the effect of the two genes (CCR and EgROP1) into a background of E. globulus (an elite full-sib family of E. globulus from P7 Altri Florestal with 200 individuals). Using two marker microsatellites designed into the genes we were able revealing the polymorphism within the progeny. For CCR we observed two classes of genotypes in the progeny meaning that only one parent was heterozygous for the marker, whereas for EgROP1 four classes were observed revealing that the two parents were heterozygous. Five chemical traits predicted in WP2 (Klason lignin, S/G ratio, total extractive) were analyzed using a simple model of variance analysis considering a marker effect. EgROP1

showed a significant effect (at 1%) on both the Klason lignin and S/G ratio. This result indicated that the EgROP1 is a good candidate gene for S/G ratio in both E. urophylla and E. globulus.

Using EucaMaps web-based application [START_REF] Bartholome | High-resolution genetic maps of Eucalyptus improve Eucalyptus grandis genome assembly[END_REF], we can observe that this region of chromosome 10 was already reported to be linked to lignin content in E. globulus [START_REF] Freeman | QTL influencing growth and wood properties in Eucalyptus globulus[END_REF], to cellulose content and pulp yield in E. globulus [START_REF] Thamarus | Identification of quantitative trait loci for wood and fibre properties in two full-sib properties of Eucalyptus globulus[END_REF], and microfibrille angle in E. nitens [START_REF] Thumma | Quantitative trait locus (QTL) analysis of growth and vegetative propagation traits in Eucalyptus nitens full-sib families[END_REF].

P3 collected 220 developing xylem samples on three years-old trees of the E. urophylla x E. grandis population. The 220 individuals were selected in order to maximize the recombinant information within the full-sib family. P8 extracted RNA, from which synthesized the cDNAs (P8).

Expression analyses of 95 genes (selected form WP1) involved in cell wall biosynthesis for the 220 offspring were performed by qRT-PCR using a Fluidigm BioMark HD System, by P1 and P8.

Expression data were then send to P3 for QTL analyses. For each gene, a QTL analysis was done using the expression of the gene as a quantitative trait. Finally, we identified eQTLs all along the genome for the two parental genotypes: i/ a total of 45 eQTLs were detected for the E. grandis parent. 10 eQTLs were localized on the chromosome 10 with 4 cis-and 6 trans-eQTLs. ii/ 42 eQTLs were detected for the E. urophylla parent, from which 10 eQTLs were localized on the chromosome 10 (5 cis-eQTLs and 5 trans-eQTLs). These results suggested that the genetic variability of several structural genes and regulation genes of the chromosome 10 should be able to influence the wood properties.

Conclusions

 Consensus orthologous sequences (COS) markers between E. grandis and P trichocarpa revealed only several small collinear regions between the two genomes.

 Existing genetic maps have been densified both for populus and Eucalyptus parents of mapping pedigree, using SNPs and/or DART markers  High resolution maps obtained for E. grandis and E. urophylla using a SNP array enabling improvement the first version of the E. grandis genome sequence.

 These maps are also invaluable genetic resources to study the combination rate in Eucalyptus  New genetic maps (Low density) have been established using DART markers for the E.

  silico identification of CGs from a compendia of expression data Objective: In this task, we aimed to analyze in silico the transcriptomic data available publicly or within the consortium as well as the new resources generated during the project to select a reservoir of regulatory CGs potentially involved in secondary wall (SW) formation in E & P Subtask 1.1.1 Development of new transcriptomic resources P1 & P8 performed transcriptome analysis of developing wood of E. globulus clones GB3, MB3, and GM2-58, collected on upper side (tension wood, TW) and bottom side (opposite wood, OW) of trees bent for one week and 3 and 4 weeks (Carocha et al., in preparation, see Figure below). Plant material and experimental trials were made available by P7 (Altri Florestal).

Fig 1

 1 Fig 1. a) Identification of pairwise significant genes between conditions (P<0.05); b) Clustering analysis of samples by their transcriptomic profile: S1WO -1 week of bending and opposite side; S1WT -1 week of bending and tension side; S34WO -pool of 3 and 4 weeks of bending and opposite side; S34WT -pool of 3 and 4 week of bending and tension side

Fig 2 .

 2 Fig 2. Principal components analysis (PCA) of the synthetic chemical variables. a) Correlation circle showing the position of nine selected variables from synthetic pyrolysis products on the PC1-PC2 plane.In the correlation circle pyrolysis products related with proteins are represented in green, lignins in red and polysaccharide in blue. Protein origin: aa1 (amino acid content) and H products (mainly protein but also from phydroxyphenyl lignin units origin). Lignin origin: G (guaiacyl units); S (syringyl units); Py_lignin (sum of H, G and S). From polysaccharide origin but main contributor cellulose: cH (hexosans); c (carbohydrates). hemicellulose origin: cP (pentosans). The ration cP/cH was also considered. b) Main plane (PC1-PC2) with the positional display of the differentiating xylem samples from tension (WT) and opposite wood (WO) kinetics (one to four weeks bending stress; S1-S4). PCA was performed using the ade4 library of the R package(Chessel et al.,2004).

Fig 3 .

 3 Fig 3. Comparison of miRNA prediction and degradome sequencing analysis. TW: tension wood; OW: opposite wood.

Fig 4 .

 4 Fig 4. Screenshot of the 34J website. Database information are accessible through a set of tabs providing, for each gene, sequences, orthologs, phylogenetic analyses and expression profiles. The combination of heat maps and eFP Browser graphical representations allow easy analyses of gene expression profiles. Statistical tools to highlight relevant variables and correlate gene expression patterns have been implemented.

Fig 5 .

 5 Fig 5. Heat map of the transcript abundance patterns assessed using microfluidic qPCR of Eucalyptus genes in 19 different tissues from Eucalyptus globulus, from Eucalyptus Gundal hybrids (Eucalyptus gunnii × Eucalyptus dalrympleana) and from Urograndis hybrids (Eucalyptus urophylla × Eucalyptus grandis). Genes and samples were hierarchically clustered according to their transcript abundance (expressed in relation to the mean of all samples and log 2 -transformed), showing two cluster of genes, one including genes highly expressed in xylem and stems under different conditions, whereas the other including genes preferentially expressed in cambium. Gene names are shown to the left of the heat map, whereas the short name of the phylogenetic subgroup is included to the right; and according to Soler et al. (2014, New Phytol), woody preferential genes are highlighted in orange whereas woody expanded genes are highlighted in yellow. Ph, phloem; CMT, cambium from mature (25-yr-old trees); CYT, cambium from young (7-yr-old) trees; XCt, control (straight, not bended) xylem; X, xylem; XYT, young xylem; XMTs, mature xylem; OX, opposite xylem; TX, tension xylem; PS, primary stem; SS, secondary stem; XN-, xylem with low nitrogen fertilization; XNA, xylem with high nitrogen fertilization; FB, floral buds; FC, fruit capsule; R, lateral roots; ST, shoot tip; ML, mature leaves; YL, young leaves; Egl, E. globulus; Egn, E. gunnii × E. dalrympleana; Eug, E. urophylla × E. grandis.

(Fig 6 .

 6 Fig 6. The schema showing the procedure to identify new regulators involved in wood formation

Fig 8 .

 8 Fig 8. Yeast two hybrid interaction and co-localization of EgMYB1 with a Histone linker (EgH1.3) protein. A. Specific interaction of EgMYB1 with EgH1.3 in yeast cells. Specificity of the interaction was assessed with another MYB gene from Arabidopsis not expressed in xylem (AtMYB30) and with another MYB gene from Eucalytpus highly expressed in xylem (EgrMYB137), showing in both cases negative results. B. Colocalization of EgMYB1 and EgH1.3, transiently expressed in tobacco leaves, in the nuclei of epidermal cells.

Fig 9 .

 9 Fig 9. Development and validation of a fast system for functional characterization of candidate genes in a homolog system of Eucalyptus using hairy roots. A. Eucalyptus plants transformed with A. rhizogenes harbouring a plasmid with 35S:GFP were detected under fluorescence 21 days after infection. B. Composite plants resulting of the A. rhizogenes transformation were hardened in pots with inert substrate and are ready to be harvested after 60-90 days after infection. C. Suitability of this system was assessed by transforming Eucalyptus with the antisense construct of the CCR gene, showing hairy roots section stained with phloroglucinol reagent with thinner xylem ell walls and lower lignin content. D. Deeper analysis using Scanning Electron Microscopy demonstrated thinner xylem cell walls with a phenotype resembling to irregular xylem, as previously observed (Piquemal et al. 1998).

Fig 10 .

 10 Fig 10. Stem section of poplar and Arabidopsis plants harboring the Dominant Repression construct of EgrMYB137. A. Section of stems from WT and EgrMYB137-DR poplar plants stained with phloroglucinol, showing that xylem cells from transgenic lines have lower lignin content, as indicated by fainter staining. B. Section of inflorescence stems from WT and EgrMYB137-DR Arabidopsis plants stained with phloroglucinol, showing thinner xylem cell walls with lower lignin in the transgenic lines.

( 2 )

 2 different methods to characterize the SCW content and composition including -Wet chemistry: Klason lignin, and thioacidolysis (P1 for poplar and in collaboration with Prof C Lapierre INRA Versailles for Arabidopsis) -NIRS for poplar samples (P9, José Rodrigues), -Analytical Pyrolysis by P9 for poplar samples and in collaboration with Dr E Pesquet (Umea Plant Science Center, Sweden) for Arabidopsis.

Fig 11 .

 11 Fig 11. Wood anatomy and lignin content of most affected transgenic lines over expressing either MYB156 or MYB221. WT: wild type poplar.

Fig 12 .

 12 Fig 12. Wood anatomy and lignin content of MYB090 OE lines and wild type poplar (WT)

Fig 13 .

 13 Fig 13. Cell wall sugar composition of MIR397-OE lines.

Fig 14 .

 14 Fig 14. Saccharification potential of MIR397-OE lines.

Fig 15 .

 15 Fig 15. Growth characteristics or KNAT7 overexpressing lines.

  ). The new protocol prove faster (cheaper) to use and enabling higher and faster plantlet regrowth (Figure 18. Harvengt et al, 2015 -IUFRO Tree Biotechnology meeting, Florence, Italy; paper in preparation).

Fig 18 .

 18 Fig 18. Results from cryopreservation with the new protocol: (A) = regrowth (withish parts) of dissected Eucalyptus gunnii x dalrympleana shoot tip one week after thawing from cryopreserved sample; (B) kinetics of regrowth of one hybrid poplar axillary bud (GUS transgenic line from P2, INRA 717-1-B4 clone) at 1, 5 and 10 days after thawing. The scale bar is representing 1 mm (A) and 3 mm (B).

Fig 19 .

 19 Fig 19. Eucalyptus and poplar genotypes selected and treatments applied

Fig 20 .

 20 Fig 20. Effects of nitrogen nutrition in growth and development parameters and xylem extractives contents in Eucalyptus genotypes. Values represent the log ratio of data from plants under the nitrogen treatment and data from plants under control conditions [Log 2 (low or high nitrogen/control)]. A. Data were obtained from stem regions developed during the treatment period and compared to equivalent regions of plants in control growth condition. B. Extractives contents were measured in xylem from stem regions between nodes 11 to 21 (counting from the apex).Low nitrogen (0 mM NH 4 NO 3 ), high nitrogen (10 mM NH 4 NO 3 ), control (1 mM NH 4 NO 3 ). Values with significant differences with the control plants, according to a t-test (p< 0.05), are labelled with a star.

Fig 21 .

 21 Fig 21. Effects of nitrogen nutrition on expression of genes related to wood formation in Eucalyptus genotypes. Values represent the log ratio of data from plants under the nitrogen treatment and data from plants under control conditions [Log 2 (low or high nitrogen/control)]. Data were obtained from stem regions developed during the treatment period and compared to equivalent regions of plants in control growth condition. Top panels show changes in transcript levels of genes encoding enzymes involved in cellulose and lignin synthesis, and bottom panels show idem for transcription factors related to wood formation. Low nitrogen (0 mM NH 4 NO 3 ), high nitrogen (10 mM NH 4 NO 3 ), control (1 mM NH 4 NO 3 ). Values with significant differences with the control plants, according to a t-test (p< 0.05), are labelled with a star.

  stem length, number of internodes, leaf surface, dry weight, histological analysis of xylem morphology and development. Changes in extractive content, S/G ratios and Klason lignin were determined by Partner P9 (IICT) as described in Task 2. Transcript levels for wood-related genes encoding enzymes involved in cellulose and lignin synthesis and several transcription factors were quantified by qRTPCR using a Fluidigm BioMark HD System.

Fig 23 .

 23 Fig 23. Effects of the drought stress in growth and development parameters, xylem extractives contents and expression of genes related to wood formation in Populus genotypes. Values represent the log ratio of data from plants under the stress and data from plants under control conditions [Log 2 (Dro/WW)]. A. Data were obtained from stem regions developed during the treatment period and compared to equivalent regions of plants in control growth condition. B. Extractive contents were measured in xylem from stem regions between nodes 11 to 21 (counting from the apex). C. The top panel shows changes in transcript levels of genes encoding enzymes involved in cellulose and lignin synthesis, and the bottom panel shows idem for transcription factors related to wood formation.Values with significant differences with the control plants, according to a t-test (p< 0.05), are labelled with a star.

  Eucalyptus spp and Populus spp, were subjected to two saline treatments. These consisted of two concentrations of NaCl (50 and 125 mM) with electric conductivity of 7 and 14 dSm-1 respectively, which are considered optimum for testing response differences in this genus. The concentration of salt was gradually increased, watering manually twice a week over the trial period. Once the final concentrations had been reached, exposure to salt stress lasted twelve weeks in the case of the Eucalyptus as opposed to six weeks in the case of the poplar as the growth rate in the later was much greater. A control treatment was included (water alone or water + half concentration of Hocking nutrient solution once a week). The effect of the saline stress was evaluated in genotypes of both genera by measuring different parameters: a) survival and leaf scorch expression, b) growth rate relative to height and diameter and volume c) production in volume (mm3), d) dry biomass, e) differentiating between the aboveground woody biomass fraction and the root biomass, f) the number of sylleptic branches produced, g) Rate of net photosynthesis (A, µmol.m-2.s-1) and stomatal conductance (gs, mol.m-2.s-1), h) specific leaf area (SLA, m2.kg-1) inferred as the area of a leaf disc divided by its oven-dry mass per area, i) Chlorophyll content index (ICC), j) Isotope discrimination (∆,‰) and k) macro and microelements content. Changes in extractive content, S/G ratios and Klason lignin were determined by Partner P9 (IICT) as described in Task 2. Transcript levels for wood-related genes encoding enzymes involved in cellulose and lignin synthesis and several transcription factors were quantified by qRTPCR using a Fluidigm BioMark HD System.

Table 5 .Fig 24 .Fig 25 .

 52425 Fig 24. Effects of exposure to six weeks of saline stress on expression of genes related to wood formation in Populus genotypes. Values represent the log ratio of data from plants under the salt treatment (50mM or 125 mM NaCl) and data from plants under control conditions [Log 2 (salt stressed/control)]. Data were obtained from stem regions developed during the treatment period and compared to equivalent regions of plants in control growth condition. A and B, changes in transcript levels of genes encoding enzymes involved in cellulose and lignin synthesis C and D, changes in transcript levels of genes encoding transcription factors related to wood formation. The NaCl concentrations in the watering solutions were 50 mM (A and C) or 125 mM (B and D). Values with significant differences with the control plants, according to a t-test (p< 0.05), are labelled with a star. Table 7. Average values and standard deviation for different genotypes of Eucalyptus spp. under salt treatments (control, 50 and 125 mM of NaCl) in relation to their aboveground woody biomass (WB), root biomass (RB) and the ratio (WB/RB)

Fig 27 .

 27 Fig 27. Temperature affects SCW composition of xylem cells in eucalyptus. Analyses were performed on xylem without bark. For lignin and SG ratio, results presented in this figure correspond to klason and thioacidolysis but were confirmed by analytical pyrolysis. Stars means a significant increase (T test, n=6, p<0.05) (A) Ethanol Extractive (% of cell wall mass) (B) Lignin content (% of cell wall mass) (C) S/G ratio

Fig 2 .Fig. 3 :

 23 Fig 2. Equipment for micro-steam treatment

Fig. 4 :

 4 Fig. 4: Typical chromatogram of volatile components from poplar wood

Fig. 5 :Fig. 6 :

 56 Fig. 5: Typical workflow from sample to multivariate data analysis

Fig. 6b

 6b Fig. 6b Variation of bio-oil yields from INRA samples

Figure 7 :

 7 Figure 7: Comparison of poplar fibers analyzed by electron microscopy and UV scanning microspectroscopy with constant wavelength 280nm (resolution 0,25um2)

  total sugars (produced by saccharification; 16%) co-localized with two genes studied in WP1: on coding for a GATA zinc finger protein and the other for a basic leucine zipper protein. The unclear relationship between the trait and the underlying gene function may be explain by a colocalization occurring only by chance. The two following methodologies used then could -The GO enrichment tools could be used to test if some functional categories are statistically over-represented in QTL regions. One of the most significant result with this strategy was for lignin QTLs found in chromosome 1 and 6 (in the present study and also in Novaes et al[START_REF] Freeman | QTL influencing growth and wood properties in Eucalyptus globulus[END_REF] Yin et al 2010). The GO enrichment analysis show significant enrichment in the molecular function "Cinnamoyl coA reductase", well known enzyme from the lignin biosynthesis pathway. Additionally, this molecular function point at a cluster of genes duplicated in tandem on chromosome 1.

  gunni and gundal parents of a cold-tolerant mapping population  Shortgun sequencing of the Eucalyptus gunnii genome has been performed but only led to a very fragmented assembly  35 QTLs were detected in a Populus mapping pedigree including total, Klason and soluble lignin, S/G ratio, extractives (%), hemicellulose, total sugars from saccharification. No QTL for cellulose content was detected  13 and 19 QTLs were detected in E. grandis and E.urophylla, respectively for wood quality traits. A major QTL for S/G ratio was detected confirming the importance of the region whose effect is stable in different environments  MetaQTLs analysis was not feasible for Populus because of the limited connections between mpas and the scarcity of wood-related traits studies  For Eucalyptus, "EucaMaps" a web-based application was developed allowing QTLs comparisons between 6 species and 267 referenced wood quality-related QTLs  The position of the major QTL for S/G ratio was refined on chr 10, both expression and physical putative candidate genes were identified  45 and 42 eQTLs were found for E. grandis and E. urophylla respectively, of which 10 were located on Chr 10 suggesting the involvement of several structural and regulation genes underlying wood properties on this chromosome  on the web page of Adjacent Government http://www.adjacentgovernment.co.uk/stakeholders/treeforjoules-securing-bioenergy-supplyfrom-trees

Table 1 .

 1 Table of constructs (overexpression (OE), dominant repression (DR), promoter GUS fusion) of selected Eucalyptus candidate TFs and status of transformation in either Arabidopsis and/or in poplar, and/or in Eucalyptus hairy roots.

		Accession		Construction	Transformation
	Gene name	number			Arabidopsis	Poplar	Eucalyptus roots
	MYB
	1 EgMYB1	Eucgr.G01774	DR	35S EAR	X
	EgMYB1	Eucgr.G01774	OE	35S	Legay et al. 2010 Legay et al. 2010 (P1)
	EgMYB1	Eucgr.G01774	Promoter Prom::GFP--GUS	X		X
	2 EgMYB2	Eucgr.G03385	DR	35S EAR	X	X
	3 EgrMYB20	Eucgr.B00866	OE	35S	X	expression clone sent to P5a
	4 EgrMYB36	Eucgr.C00721	DR	35S EAR
	EgrMYB36	Eucgr.C00721	OE	35S
	5 EgrMYB64	Eucgr.D02390	OE	35S		ENTRY clone sent to P11	X
	EgrMYB64	Eucgr.D02390	Promoter Prom::GFP--GUS			X
	6 EgrMYB68	Eucgr.E01031	OE	35S		expression clone sent to P5a
	EgrMYB68	Eucgr.E01031	Promoter Prom::GFP--GUS	X		X
	7 EgrMYB80	Eucgr.F02756	OE	35S		expression clone sent to P5a X
	EgrMYB80	Eucgr.F02756	Promoter Prom::GFP--GUS			X
	8 EgrMYB88	Eucgr.F04423	OE	35S	X	X
	EgrMYB88	Eucgr.F04423	DR	35S EAR	X	X
	9 EgrMYB135	Eucgr.K02297	OE	35S
	EgrMYB135	Eucgr.K02297	Promoter Prom::GFP--GUS			X
	10 EgrMYB137	Eucgr.K02806	OE	35S		X Not able to regenerate	X
	EgrMYB137	Eucgr.K02806	DR	35S EAR	X	X
	EgrMYB137	Eucgr.K02806	Promoter Prom::GFP--GUS			X
	Auxin signaling
	11 EgrmIAA4	Eucgr.H04336	OE m	35S	X	ENTRY clone sent to P11
	12 EgrmIAA9	Eucgr.H02407	OE m	35S	X
	13 EgrmIAA13	Eucgr.H02914	OE m	35S	X	ENTRY clone sent to P11
	14 EgrIAA20	Eucgr.K00561	OE	35S	X
	15 EgrmARF10	Eucgr.J00923	OE m	35S	X
	16 EgrARF19.2	Eucgr.C03293	OE	35S	X
	EgrARF19.2	Eucgr.C03293	DR	35S EAR	X
	Others
	17 EgHISTONE	Eucgr.I02364	OE	35S	X
	EgHISTONE	Eucgr.I02364	Promoter Prom::GFP--GUS	X		X
	18 EgOVATE	Eucgr.J01842	OE	35S	X
	EgOVATE	Eucgr.J01842	DR	35S EAR	X
	EgOVATE	Eucgr.J01842	Promoter Prom::GFP--GUS	X		X
	EgMYB1 + EgH1		OE	35S	X
	EgMYB1 + EgOv		OE	35S	X
	NACs
	19 EgrNAC64	Eucgr.E03226	OE	35S		ENTRY clone sent to P11

Table 2 .

 2 Biochemical data for poplar transgenic lines harboring Eucalyptus MYB genes. Klason lignin and total reducing sugars were experimentally measured by wet chemistry methods. Lignin content, S/G, H/G and cP/cH (pentoses vs hexoses carbohydrates) ratio were measured by pyrolysis coupled to GC-MS (P9). Total lignin, Klason lignin and S/G ratio were also predicted using NIRS. Statistical analyses were performed by T test (* < 0.05, ** < 0.01, *** < 0.001).

		Klason	Reducing	Pyrolysis	S/G	H/G	cP/cH	Total	Klason	S/G
		lignin	sugars	lignin	pyrolysis	pyrolysis	pyrolysis	lignin	lignin	(NIRS)
		(%)	(mg/ml)					(NIRS)	(NIRS)
	WT	19.88 ±	1.64 ±	21.43 ±	0.72 ±	0.085 ±	24.58 ±	21,99	19,12 ±	0,74 ±
		0.99	0.20	1.22	0.01	0.006	1.22	± 0,2	0,36	0,01
	EMPTY	19.29 ±	1.55 ±	21.7 ± 14	0.72 ±	0.077 ±	25.54 ±	22,19	19,5 ±	0,74 ±
		0.72	0.02		0.02	0.005	0.91	± 0,44	0,7	0,04
	EgrMYB1-	167 ±	2.50 ±	20.98 ±	0.72 ±	0.095 ±	22,22 ±	20,8 ±	18,47 ±	0,70 ±
	DR	1.7 **	0.68 *	1.05	0.04	0.01 **	1,92 **	0,57	0,56	0,03 *
								***	***
	EgrMYB2-	17.79 ±	2.21 ±	22.36 ±	0.71 ±	0,082 ±	23,89 ±	20,9 ±	19,36 ±	0,73 ±
	DR	0.64 **	0.61	0.75	0.02	0,005	0,97 *	0,82	0,7	0,04
	EgrMYB137-	15.69 ±	2.97 ±	20.62 ±	0.79 ±	0,089 ±	22,76 ±	22,0 ±	18,1 ±	0,80 ±
	DR	1.4 ***	0.77 **	1.07	0.05 **	0,007 *	1,63 **	0,42	0,87	0,03 ***
								***	***
	EgrMYB88-	18.26 ±	1.95 ±	22.21 ±	0.70 ±	0,082 ±	24,63 ±	22,1 ±	19,57 ±	0,71 ±
	DR	0.96 *	0.44	0.54	0.03	0,005	1,12	0,32	0,42	0,04 *
	EgrMYB88-	17.74 ±	2.05 ±	22.47 ±	0.77 ±	0,081 ±	24,56 ±	22,2 ±	19,30 ±	0,78 ±
	OE	0.78 *	0.42	0.68	0.05	0,01	1,2	0,22	0,3	0,02

Table 9 .

 9 Genes deregulated in December vs. June in Populus alba and 3 analyzed Eucalyptus genotypes

E. globulus MB43, GB3 and GM248.

Table 1 .

 1 Characteristics of QTL detected

	Traits	LG	a	Position	LOD	b	Adjacent marker	R2	c	IC
				(cM)						(Mpb)	d
	Hemicellulose	1T		30	4.08**	rscaf_1_42427246	11.99	12.2
		7T		50	2.97*		scaf_1105_3008	8.89	3.3
		8T		120	2.69		rscaf_8_15205004	8.08	5.6
		9T		105	2.67		scaf_9_7873548	8.02	1.8
		14T	135	2.55		rscaf_14_10971571	7.68	1.8
		17T	220	2.03		scaf_17_6159651	66		1
		2D		130	2.77**	rE1M1.6.D	8.31	19.5
		3D		250	37*		rscaf_3_16531703	9.44	4
		14D	0	26		P168.D	6.55	12
	Total Sugars	2D		160	2.99**	E2M4.0.D	8.95	3.5
	(from saccharification)	3D		170	2.58		rORPM30.D	7.76	5.4
		4D		125	2.72*		rE7M4.2.D	87		2.7
		8D		140	5.61**	rscaf_8_8098956	163	3.7
		10D	45	3.06**	Q14300.D	93		3.4
		16D	15	24		rGCPM1547.T	6.49	2
	S/G	2T		80	5 .05**	rPMGC422.T	14.62	1.8
		5T		155	3 .31**	E2M32.T	9.86	12.2
		10T	290	3 .21**	rE2M3.T	9.56	8
		17T	70	2 .31		rscaf_202_21595	6.99	17.9
		18T	20	2 .91*	rscaf_18_13934893	8.72	2.6
		17D	145	3.00**	E4M1.2.D	8.97	20.9

a linkage group, D : P. deltoides map; T: P. trichocarpa map.

b LOD peak associated with the QTL; ** FDR 5%; *FDR 10%. c percentage of variation explained by the QTL. d size of confidence intervals in Mbp; calculated as in Monclus et al 2012

(BMC Plant Biology 2012, 12:173 doi:10186/1471-2229-12-173)

Table 2 .

 2 QTL results for wood quality traits in E. urophylla and E. grandis First of all, QTL position and confidence intervals were projected from the genetic maps onto the genome P. trichocarpa reference genome thanks to genetic markers with sequence information (as described in Monclus et al[START_REF] Cassan-Wang | Reference Genes for High-Throughput Quantitative Reverse Transcription-PCR Analysis of Gene Expression in Organs and Tissues of Eucalyptus Grown in Various Environmental Conditions[END_REF] BMC Plant Biology 2012, 12:173). QTLs from the present study and different published and unpublished studies were used(El-Malki et al., submitted; Yin et al 2010; Novaes et al. 2009; Zhang et al. 2006).

WP3. COMPARATIVE ARCHITECTURE OF WOOD PROPERTIES BETWEEN EUCALYPTUS AND POPLAR

The objective of WP3 was to identify and characterize genomic regions in eucalypts (E) and poplar (P) that control wood properties relevant for bioenergy.

Task 3. 1 Comparative Genomics between Eucalyptus and Populus

Consensus orthologous sequences (COS) based on sequence similarity have been identified retrieving a total of 3918 orthologous genes from the Phytozome database using the following criteria: 1 to 1 orthology relationship between A. thaliana (Ath), E. grandis (E) and P. trichocarpa (P) gene sets. The Ath genome has been used to enhance stringency. This criteria lead to the selection of highly conserved genes but no analysis of their function has been performed yet. Genome positions on E and P genomes of these orthologous genes revealed the presence of only several small collinear regions (P2).

SNP discovery in the two parents of the Populus mapping pedigree have been performed using Illumina NGS, and after filtering quality, more than 1 000 000 SNPs are available for selection for genotyping. Additionally, from a sideground project, a 6K SNP has been used to genotype ~300 F1 progenies of the mapping pedigree and about 10% of SNP are polymorphic and the resulting genetic map has been anchored to the reference Populus trichocarpa genome (P2).

DARTs technology was used to genotype 180 hybrids from the E. urophylla x E. grandis cross (P3) and 180 progenies from the E. gunnii x E. gundal cross (P4 and P3). Low-density genetic maps have been done for each parent (4 genotypes). The alignment of the low-density genetic maps was done with the reference genome. For E. gunnii and E. gundal, 123 and 137 DARTs markers were used to construct two genetic maps with a total length of 1811 cM and 1733 cM and a percentage of coverage of the reference genome estimated to 95% to 93%, respectively. These maps are the first genetic maps developed for Eucalyptus gunnii species (Article in prep). The mapping population will constitute the reference population for P4 for studying the genetic architecture of quantitative traits of interest.

The shotgun sequencing was done by P4 for the parent E. gunnii (a E. gunnii cv 'Azura'; 80 Gb Illumina Paired-end reads of 100 bp inserts, 40 Gb Mate-Pairs of 4 kb inserts and 6 Gb PacBio long reads). The genetic maps were used for improving the assembly but prove difficult since many DART sequence were not available. All kind of reads mapped to 75% of the E grandis genome. Pair ends reads as well as PacBio data could be assembled but the mate pair sequences could not be integrated, leading to an overall small scaffold size (N50 of ca 50 kb).

A major QTL for S/G ratio was detected on LG10 for the two parents. This QTL region was already shown as involved in S/G ratio [START_REF] Gion | Comprehensive genetic dissection of wood properties in a widely-grown tropical tree: Eucalyptus[END_REF], and our result suggested a stable effect of this region on S/G ratio between several environments. These QTLs results will be detailed and discussed in a new publication (in prep). The importance of the targeted region on

LG10 of E. urophylla was confirmed by the results.

Subtask 3.2.2 Meta QTL analysis and comparison with others QTL results

A tool for QTL meta-analysis was investigate Biomercator v4 (Sosnowski et al (2012) http://prodinra.inra.fr/record/255679). A workshop between WP3 participants (P2, P3) has been organized in Bordeaux the 30 th , May 2013. For Populus genus, the tool has been prove to be difficult to use because (i) Populus maps show limited connections between them and (ii) QTL studies for wood related traits are still scarce.

Different approaches were tested to reveal pertinent candidate gene / QTL co-location.

WP4. Project coordination & dissemination

Coordination

The exchanges between all the partners were numerous, easy and friendly, they included formal We have invited at the 3 rd and the 5 th meetings, experts in the field of sacharification (Dr L Gomez, York university) and of Eucalyptus genetics and genomics (A Myburg, University of Pretoria, South Africa) to give a seminar and to benefit from their expertise and advice.

Dissemination

The high numbers of peer-reviewed publications, oral communications, posters, PhD thesis and master thesis including those involving more than two partners, illustrate that the project was successful and highly collaborative.


Florence Garcia-Launay

Virginie Rouillon

Justine Faure

Alexandre Fonseca

Life Cycle Assessment of pig production systems of the Noir de Bigorre pork chain

Keywords: 00028683AF Themes: Production Systems -Management, natural resources and sustainability Presentation: Oral Communication Life Cycle Assessment, Sustainability

Outdoor pig production systems relying on local pig breeds may cope with environmental and socio-economic challenges of animal production. They produce high quality products with added economic value and mainly rely on local feed resources. Within the European TREASURE 1 project, we aimed at evaluating the sustainability of these systems. We conducted the Life Cycle Assessment (LCA) of the Noir de Bigorre (NDB) pig production systems located in South West of France. The environmental impacts were calculated at farm gate and expressed per kg live pig and per ha land use. From surveys on 25 farms of the NDB pork chain and data collected by the chain, we estimated the flows and average live weights of animals produced as well as the quantities of feeds distributed to the animals. Formulas of the complete feeds were collected from manufacturers. Feed ingredients impacts came from the ECOALIM dataset of the AGRIBALYSE® database. Climate Change (CC), Acidification (AC), Eutrophication (EU), Cumulative Energy Demand and Land Occupation impacts were in the range of the impacts of outdoor systems previously studied. CC impact per kg live pig was higher than in intensive systems due to the higher amount of feed needed to reach the slaughter weight. AC and EU impacts per ha of land were relatively low. NDB pig farming systems exhibit LCA impacts typical of pig extensive and outdoor systems. Further studies within the European TREASURE project will also give insights on the economic and societal dimensions of sustainability of these systems.

1 "This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 634476 (Project acronym: TREASURE). The content of this paper reflects only the author's view and the European Union Agency is not responsible for any use that may be made of the information it contains."