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Whole genome characterizations of crop plants based on ancient DNA have provided unique keys for a better understanding of the evolutionary origins of modern cultivars, the pace and mode of selection underlying their adaptation to new environments and the production of phenotypes of interest. Although forests are among the most biologically rich ecosystems on earth and represent a fundamental resource for human societies, no ancient genome sequences have been generated for trees. This contrasts with the generation of multiple ancient reference genomes for important crops. Here, we sequenced the first ancient tree genomes using two white oak wood remains from Germany dating to the Last Little Ice Age (15th century CE, 7.3× and 4.0×) and one from France dating to the Bronze Age (1700 BCE, 3.4×). We assessed the underlying species and identified one medieval remains as a hybrid between two common oak species (Quercus robur and Q. petraea) and the other two remains as Q. robur. We found that diversity at the global genome level had not changed over time. However, exploratory analyses suggested that a reduction of diversity took place at different time periods. Finally, we determined the timing of leaf unfolding for ancient trees for the first time. The study extends the application of ancient wood beyond the classical proxies of dendroclimatology, dendrochronology, dendroarchaeology and dendroecology, thereby enhancing resolution of inferences on the responses of forest ecosystems to past environmental changes, epidemics and silvicultural practices.

The pace of tree microevolution during Anthropocene warming is largely unknown. We used a retrospective approach to monitor genomic changes in oak trees since the Little Ice Age (LIA). Allelic frequency changes were assessed from whole-genome pooled sequences for four age-structured cohorts of sessile oak (Quercus petraea) dating back to 1680, in each of three different oak forests in France. The genetic covariances of allelic frequency changes increased between successive time periods, highlighting genome-wide effects of linked selection. We found imprints of convergent linked selection in the three forests during the late LIA, and a shift of selection during more recent time periods. The changes in allelic covariances within and between forests mirrored the documented changes in the occurrence of extreme events (droughts and frosts) over the last three hundred years. The genomic regions with the highest covariances were enriched in genes involved in plant responses to pathogens and abiotic stresses (temperature and drought). These responses are consistent with the reported sequence of frost (or drought) and disease damage ultimately leading to the oak dieback after extreme events. Our results therefore provide evidence of selection operating on long-lived species during recent climatic changes.

Additional file 8 Suppl. Fig. 6. Proportion of linear model (LM, top row) and neural network (NN, bottom row) predictions with a R2 above (left column) or below (right column) the 95% confidence interval computed from the predictions with the random sets of genes for each gene set (there is no neural network model computed for the Complete BLUP full set).

Additional file 5 Suppl. Fig. 3. Relationship between Spearman’s correlations between module-trait (y-axis) and gene significance-kME (x-axis).

Additional file 10 Suppl. Fig. 8. Predictions scores on test sets for increasing numbers of the peripheral genes. Violin and boxplots of prediction R2 for the LM Ridge algorithm and for increasing sizes of the peripheral genes set (in brown) and the peripheral NG genes set (in orange), used for the predictions (in percent of the full set).

Additional file 4 Suppl. Fig. 2. Traits hierarchical ascendant clustering dendrogram. Clustering was performed from the correlations between traits with Ward method ("Ward.D2") by the R package pvclust. Approximately Unbiased (au, in red) and Bootstrap Probability (bp, in green) p-values indicated the degree of belief associated with clusters. Highly supported modules are framed by a red square, grouping (a) the mean sample diameter with the two circumference traits, (b) the S/G ratios with glucose composition, (c) the two C5/C6 together, and (d) the H/G ratios.

Additional file 3 Suppl. Fig. 1. PCA score plots on gene expression data. Each plot represents the distribution of the individuals on the 2 first axes of the PCA (representing 17,7% of the variation), colored by class of various experimental factors (Xylem and cambium scraper, extractor and extraction method, population, sequencing column, line and plate, the growth rate at harvest, sampling date, time, temperature, solar radiation, humidity and wind speed). Cofactors related to weather are presented in the 6 lower plots.

Additional file 7 Suppl. Fig. 5. Histograms of the centrality scores for the genes selected by Boruta at different p-values thresholds. Repartition of selected genes within the following gene sets is hilighted, with core in blue, peripheral NG in orange, peripheral in brown and other (NA) in black. Four p-value thhresolds for Boruta selections were considered: 0.01, 0.05, 0.1 and 0.2.

Additional file 6 Suppl. Fig. 4. Histograms of the centrality scores without (top) or with (bottom) the grey group. Core, peripheral and peripheral without grey sets are represented respectively by the blue, dark orange and orange bars. Random sets are distributed across the histogram and do not appear on this figure. Distribution of genes clustered in the grey module is represented by the grey bars, white bars are for other genes.

International audience; The pulse of the tree (diurnal cycle of stem radius fluctuations) has been widely studied as a way of analyzing tree responses to the environment, including the phenotypic plasticity of tree-water relationships in particular. However, the genetic basis of this daily phenotype and its interplay with the environment remain largely unexplored. We characterized the genetic and environmental determinants of this response, by monitoring daily stem radius fluctuation (dSRF) on 210 trees from a Eucalyptus urophylla x E. grandis full-sib family over 2 years. The dSRF signal was broken down into hydraulic capacitance, assessed as the daily amplitude of shrinkage (DA), and net growth, estimated as the change in maximum radius between two consecutive days (Delta R). The environmental determinants of these two traits were clearly different: DA was positively correlated with atmospheric variables relating to water demand, while Delta R was associated with soil water content. The heritability for these two traits ranged from low to moderate over time, revealing a time-dependent or environment-dependent complex genetic determinism. We identified 686 and 384 daily quantitative trait loci (QTL) representing 32 and 31 QTL regions for DA and Delta R, respectively. The identification of gene networks underlying the 27 major genomics regions for both traits generated additional hypotheses concerning the biological mechanisms involved in response to water demand and supply. This study highlights that environmentally induced changes in daily stem radius fluctuation are genetically controlled in trees and suggests that these daily responses integrated over time shape the genetic architecture of mature traits.

Additional file 9 Suppl. Fig. 7. Difference of prediction scores between algorithms (top) and sets (bottom). On the top panel, the difference between LM and NN prediction scores for the core (in blue), random (in grey), peripheral (in brown), peripheral (in orange) and Boruta gene sets (in green). On the bottom panel, the LM differences are in red and the NN differences in turquoise and the color filling the bar represents the difference between core and peripheral genes in brown, core and peripheral NG in orange and between the random sets in grey. For the random pairs, error bars represent the first and third quartiles of the differences between pairs of randomized sets and the bar corresponds to the median.

Recent literature on the differential role of genes within networks, including the omnigenic model, distinguishes core from peripheral genes in the layout underlying phenotypes. Cores are typically few, each of them highly contributes to phenotypic variation, but because they are so few, they altogether only explain a small part of trait heritability. In contrast, peripherals, each of small influence, are so numerous that they finally lead phenotypic variation. We collected and sequenced RNA from 459 European black poplars and built co-expression networks to define core and peripheral genes as the most and least connected ones. We computed the role of each of these gene sets in the prediction of phenotypes and showed that cores contribute additively to phenotypes, consistent with a downstream position in a biological cascade, while peripherals interact to influence phenotypes, consistent with an upstream position. Quantitative and population genetics analyses further revealed that cores are more expressed than peripherals but they tend to vary less and to be more differentiated between populations suggesting that they are more constrained by natural selection. Our work is the first attempt to integrate core and peripheral terminologies from co-expression networks and omnigenic theory. In the end, we showed, that there seems to be a strong overlap between them, with core genes from co-expression networks likely being a mixture of integrative hubs with a direct effect on phenotype in agreement with the omnigenic theory, and master regulators which control the overall metabolic flow towards the phenotype.

International audience; Somatic embryogenesis techniques have been developed for most coniferous species, but only using very juvenile material. To extend the techniques' scope, better integrated understanding of the key biological, physiological and molecular characteristics of embryogenic state is required. Therefore, embryonal masses (EMs) and non-embryogenic calli (NECs) have been compared during proliferation at multiple levels. EMs and NECs originating from a single somatic embryo (isogenic lines) of each of three unrelated genotypes were used in the analyses, which included comparison of the lines' anatomy by transmission light microscopy, transcriptomes by RNAseq Illumina sequencing, proteomes by free-gel analysis, contents of endogenous phytohormones (indole-3-acetic acid, cytokinins and ABA) by LC-MS analysis, and soluble sugar contents by HPLC. EMs were characterized by upregulation (relative to levels in NECs) of transcripts, proteins, transcription factors and active cytokinins associated with cell differentiation accompanied by histological, carbohydrate content and genetic markers of cell division. In contrast, NECs were characterized by upregulation (relative to levels in EMs) of transcripts, proteins and products associated with responses to stimuli (ABA, degradation forms of cytokinins, phenols), oxidative stress (reactive oxygen species) and carbohydrate storage (starch). Sub-Network Enrichment Analyses that highlighted functions and interactions of transcripts and proteins that significantly differed between EMs and NECs corroborated these findings. The study shows the utility of a novel approach involving integrated multi-scale transcriptomic, proteomic, biochemical, histological and anatomical analyses to obtain insights into molecular events associated with embryogenesis and more specifically to the embryogenic state of cell in Douglas-fir.

Key message This review highlights some of the discoveries and applications made possible by “omics” technologies over the last 10 years and provides perspectives for pioneering research to increase our understanding of tree biology. Context A decade after the first forest tree genome sequence was released into the public domain, the rapidly evolving genomics and bioinformatics toolbox has advanced our understanding of the structure, functioning, and evolution of forest tree genomes. Aims and methods This review highlights some of the discoveries and applications that “omics” technologies have made possible for forest trees over the past 10 years. Results In this review, we start by our current understanding of genome evolution and intricacies of gene regulation for reproduction, development, and responses to biotic and abiotic stresses. We then skim over advances in interactome analysis and epigenomics, the knowledge of the extent of genetic variation within and between species, revealing micro- and macro-evolutionary processes and species history, together with the complex architecture of quantitative traits. We finally end with applications in genetic resource conservation and breeding. Conclusion The knowledge gained through the use of these technologies has a huge potential impact for adapting forests to the main challenges they will have to face: changing demand from ecosystem services with potentially conflicting strategies in terms of conservation and use, as well as climate changes and associated threats. Genomics will undoubtedly play a major role over the next decade and beyond, not only to further understand the mechanisms underlying adaptation and evolution but also to develop and implement innovative management and policy actions to preserve the adaptability of natural forests and intensively managed plantations.

Somatic embryogenesis techniques have been developed for most coniferous species, but only using very juvenile material. To extend the techniques’ scope, better integrated understanding of the key biological, physiological and molecular characteristics of embryogenic state is required. Therefore, embryonal masses (EMs) and non-embryogenic calli (NECs) have been compared during proliferation at multiple levels. EMs and NECs originating from a single somatic embryo (isogenic lines) of each of three unrelated genotypes were used in the analyses, which included comparison of the lines’ anatomy by transmission light microscopy, transcriptomes by RNAseq Illumina sequencing, proteomes by free-gel analysis, contents of endogenous phytohormones (indole-3-acetic acid, cytokinins and ABA) by LC-MS analysis, and soluble sugar contents by HPLC. EMs were characterized by upregulation (relative to levels in NECs) of transcripts, proteins, transcription factors and active cytokinins associated with cell differentiation accompanied by histological, carbohydrate content and genetic markers of cell division. In contrast, NECs were characterized by upregulation (relative to levels in EMs) of transcripts, proteins and products associated with responses to stimuli (ABA, degradation forms of cytokinins, phenols), oxidative stress (reactive oxygen species) and carbohydrate storage (starch). Sub-Network Enrichment Analyses that highlighted functions and interactions of transcripts and proteins that significantly differed between EMs and NECs corroborated these findings. The study shows the utility of a novel approach involving integrated multi-scale transcriptomic, proteomic, biochemical, histological and anatomical analyses to obtain insights into molecular events associated with embryogenesis and more specifically to the embryogenic state of cell in Douglas-fir.

GnpIS is an information system designed to integrate and link genomic, genetic and environmental data into a single environment dedicated to plant (crops and forest trees) and fungi data. GnpIS is regularly improved with new functionalities answering specific needs raised by scientists and released several times a year. We propose to illustrate the integrated genome annotation system we set up with a focus on the interoperability between genomic and genetic data (e.g. Markers, QTL) present in GnpIS-core, through the use case Quercus robur (the pedunculate oak), a large, complex and highly heterozygous genome.This genome annotation system relies on GMOD interfaces such as WebApollo/JBrowse and Intermine to make these data available under a user-friendly environment. All annotations and analysis results (Transposable Elements (TEs), genes, ncRNA ...) and functional annotation (protein-coding genes) were obtained using powerful and robust pipelines: (i) REPET used to detect, classify and annotate TEs representing 50% of the genome; (ii) Eugene which integrates ab initio and similarity gene finding softwares to predict gene models; (iii) ncRNA were annotated using different tools to annotate lncRNA, miRNA, rRNA, tRNA (iv) A functional annotation pipeline mainly based on Interproscan and comparative genomics was performed on the 25,808 highly confident predicted proteins. This system allows experts to analyze their protein families of interest and curate/validate gene structure.All together these resources provide a framework to study the two key evolutionary processes that explain the remarkable diversity found within the Quercus genus: local adaptation and speciation.

Figure S3. Histology of non-embryogenic cells (NECs) clusters from 1ry and/or 2ry lines of genotypes TD17 and SD4. A / Histology of TD17 groups of loosely arranged NECs (arrow) in the vicinity of small somatic embryos (arrowhead); B / histology of TD17 compact NEC cluster accumulating phenolics (blue and brown cells, arrowheads) and starch (arrow); C / Lugol staining of TD17 NEC cluster showing starch (arrows) and phenolic compounds (small dark granules marked with arrowhead); D / TD17–1 NEC cluster with dividing cells (arrowheads); E /SD4–2 NEC cluster (arrow) within polyembryogenic center besides well-arranged embryonal heads (EH), note phenolic content (in dark blue-grey) of cells separating NEC from suspensor cells (in light blue); F / SD4–6 meristemoid-like NEC cluster; note phenolic content of cells between two meristemoid-like structures (in blue-grey). Scale bar: A, E = 200 μm; B, F = 100 μm; C, D = 50 μm. (DOCX 417 kb)

Figure S1. Macroscopic aspect of embryonal masses (EMs) from two Douglas-fir genotypes (SD4, TD17). These embryonal masses were obtained after 1ry somatic embryogenesis from zygotic embryo and two cycles (2ry and 3ry) of repetitive somatic embryogenesis from somatic embryos. Note the granular aspect of most embryonal masses indicating the occurrence of large polyembryogenic centers (arrowheads) and/or singulated early somatic embryos that are sometimes protruding from the embryonal masses surface. (DOCX 687 kb)

Figure S2. Structure of embryonal masses from primary and secondary lines of genotype TD17. A / TD17; B / TD17–1. Trypan blue staining of squashes of fresh EMs; m – meristem of polyembryogenic centers, s – suspensor. Scale bar = 200 μm. (DOCX 178 kb)

Table S3. Total protein content (mean ± SD, n = 4) in proliferating 2ry and 3ry embryogenic lines of Douglas-fir. (DOCX 45 kb)

Table S2. Total protein content (mean ± SD, n = 4) in proliferating 1ry and 2ry embryogenic lines of Douglas-fir. (DOCX 45 kb)

Table S1. A summary of macromorphological (EM colour and morphotype) and histo-cytological traits (occurrence of polyembryogenic centers (PECs), singulated SEs and NECs) of 1ry (SD4, TD17), 2ry (SD4–2, SD4–6, SD4–8; TD17–1) and 3ry (SD4–8-1, SD4–8-2, SD4–8-3) embryogenic lines of Douglas-fir. (DOCX 16 kb)

Table S4. Functional classification according to gene ontology (GO) of significant proteins identified after two cycles of repetitive somatic embryogenesis in Douglas-fir. (DOCX 72 kb)

REALLY INTERESTING NEW GENE (RING) proteins play important roles in the regulation of many processes by recognizing target proteins for ubiquitination. Previously, we have shown that the expression of PtaRHE1, encoding a Populus tremula × Populus alba RING-H2 protein with E3 ubiquitin ligase activity, is associated with tissues undergoing secondary growth. To further elucidate the role of PtaRHE1 in vascular tissues, we have undertaken a reverse genetic analysis in poplar. Within stem secondary vascular tissues, PtaRHE1 and its corresponding protein are expressed predominantly in the phloem. The downregulation of PtaRHE1 in poplar by artificial miRNA triggers alterations in phloem fibre patterning, characterized by an increased portion of secondary phloem fibres that have a reduced cell wall thickness and a change in lignin composition, with lower levels of syringyl units as compared with wild-type plants. Following an RNA-seq analysis, a biological network involving hormone stress signalling, as well as developmental processes, could be delineated. Several candidate genes possibly associated with the altered phloem fibre phenotype observed in amiRPtaRHE1 poplar were identified. Altogether, our data suggest a regulatory role for PtaRHE1 in secondary phloem fibre development.

RG-I and AGP, but not XG, are associated to the building of the peculiar mechanical properties of tension wood. Hardwood trees produce tension wood (TW) with specific mechanical properties to cope with environmental cues. Poplar TW fibers have an additional cell wall layer, the G-layer responsible for TW mechanical properties. We investigated, in two poplar hybrid species, the molecules potentially involved in the building of TW mechanical properties. First, we evaluated the distribution of the different classes of non-cellulosic polysaccharides during xylem fiber differentiation, using immunolocalization. In parallel, G-layers were isolated and their polysaccharide composition determined. These complementary approaches provided information on the occurrence of non-cellulosic polysaccharides during G-fiber differentiation. We found no evidence of the presence of xyloglucan (XG) in poplar G-layers, whereas arabinogalactan proteins (AGP) and rhamnogalacturonan type I pectins (RG-I) were abundant, with an apparent progressive loss of RG-I side chains during G-layer maturation. Similarly, the intensity of immunolabeling signals specific for glucomannans and glucuronoxylans varies during G-layer maturation. RG-I and AGP are best candidate matrix components to be responsible for TW mechanical properties.

National audience; The large, complex and highly heterozygous genome of pedunculate oak (Quercus robur) was sequenced using a whole-genome shotgun approach [1] . Roche 454 GS-FLX sequence reads were assembled into contigs and combined with Illumina reads from paired-end, mate-pair libraries and true synthetic long reads to build a total of 8,827 scaffolds (1.46 Gb total size; N50=821 kb). Both haplotypes were merged into an haploid version and 12 pseudomolecules were established using a high-density linkage map [2] combined with a syntenome approach using the peach genome sequence.The structural (Transposable Elements (TEs), genes, ncRNA) and functional annotation of automatically predicted genes relies on powerful and robust pipelines: (i) REPET package [3] [4] was first used to de novo detect, classify and annotate TEs representing about 50% of the genome; (ii) Eugene was trained and launched to integrate ab initio and similarity gene finding software to finally predict 43,240 genes including 29,665 highly confident gene models; (iii) ncRNA were predicted using feelcn (lncRNA), similarities against databases and small RNAseq data analysis (miRNA), RNAmmer (rRNA), tRNAscan-SE (tRNA) and Infernal package (other non-coding RNA) (iv) A functional annotation pipeline mainly based on Interproscan to search for patterns/motifs and Blast based comparative genomics was launched onto the 43,240 predicted proteins. The assignation of a provisional definition for predicted protein according to the results of the most reliable tools and their occurrence in Oak annotation was produced (D. Goodstein method, personal communication). We will present here these pipelines and the results of this annotation.We also set up an integrated genome annotation system (dedicated to oak) based on GMOD web interfaces such as WebApollo/JBrowse and Intermine to make these data available under a user-friendly environment. This system allowed experts to analyze their respective protein families of interest and curate/validate gene structure. We will also present the interoperability between these genomic data and genetic data produced in Quercus (SNPs, linkage maps, QTLs) available in GnpIS [5] an information System for plants. All together these resources provide a framework to study the two key evolutionary processes that explain the remarkable diversity found within the Quercus genus: local adaptation and speciation.

Wood formation is a complex biological process, involving five major developmental steps, including (1) cell division from a secondary meristem called the vascular cambium, (2) cell expansion (cell elongation and radial enlargement), (3) secondary cell wall deposition, (4) programmed cell death, and (5) heartwood formation. Thanks to the development of genomic studies in woody species, as well as genetic engineering, recent progress has been made in the understanding of the molecular mechanisms underlying wood formation. In this review, we will focus on two different aspects, the lignification process and the control ofmicrofibril angle in the cell wall of wood fibres, as they are both key features of wood material properties., La formation du bois est un processus de développement complexe, impliquant cinq étapes majeures : (1) la division des cellules à partir d’un méristème secondaire, appelé le cambium; (2) l’élongation des cellules ; (3) le dépôt d’une paroi secondaire épaisse ; (4) la mort programmée des cellules ; et (5) leur imprégnation par des composés phénoliques conduisant à la formation du bois de coeur. Grâce au développement des techniques de génomique et aux travaux menés sur la caractérisation d’arbres transgéniques, certains mécanismes moléculaires impliqués dans la formation du bois commencent à être mieux connus. Dans cette revue, deux aspects importants pour la qualité du bois seront approfondis : la lignification et le contrôle de l’angle des microfibrilles de cellulose dans la paroi des fibres de bois.

*Summary A molecular marker compound, derived from lignin by the thioacidolysis degradative method, for structures produced when ferulic acid is incorporated into lignin in angiosperms (poplar, Arabidopsis, tobacco), has been structurally identified as 1,2,2-trithioethyl ethylguaiacol [1-(4-hydroxy-3-methoxyphenyl)-1,2,2-tris(ethylthio)ethane]. Its truncated side chain and distinctive oxidation state suggest that it derives from ferulic acid that has undergone bis-8-O-4 (cross) coupling during lignification, as validated by model studies. A diagnostic contour for such structures is found in two-dimensional 13 C‐ 1 H correlated (HSQC) NMR spectra of lignins isolated from cinnamoyl CoA reductase (CCR)-deficient poplar. As low levels of the marker are also released from normal (i.e. non-transgenic) plants in which ferulic acid may be present during lignification, notably in grasses, the marker is only an indicator for CCR deficiency in general, but is a reliable marker in woody angiosperms such as poplar. Its derivation, together with evidence for 4-O-etherified ferulic acid, strongly implies that ferulic acid is incorporated into angiosperm lignins. Its endwise radical coupling reactions suggest that ferulic acid should be considered an authentic lignin precursor. Moreover, ferulic acid incorporation provides a new mechanism for producing branch points in the polymer. The findings sharply contradict those reported in a recent study on CCR-deficient Arabidopsis.

International audience; In Eukaryotes, LIM proteins act as developmental regulators in basic cellular processes such as regulating the transcription or organizing the cytoskeleton. The LIM domain protein family in plants has mainly been studied in sunflower and tobacco plants, where several of its members exhibit a specific pattern of expression in pollen. In this paper, we finely characterized in poplar six transcripts encoding these proteins. In Populus trichocarpa genome, the 12 LIM gene models identified all appear to be duplicated genes. In addition, we describe several new LIM domain proteins deduced from Arabidopsis and rice genomes, raising the number of LIM gene models to six for both species. Plant LIM genes have a core structure of four introns with highly conserved coding regions. We also identified new LIM domain proteins in several other species, and a phylogenetic analysis of plant LIM proteins reveals that they have undergone one or several duplication events during the evolution. We gathered several LIM protein members within new monophyletic groups. We propose to classify the plant LIM proteins into four groups: aLIM1, bLIM1, gLIM2, and dLIM2, subdivided according to their specificity to a taxonomic class and/or to their tissue-specific expression. Our investigation of the structure of the LIM domain proteins revealed that they contain many conserved motifs potentially involved in their function.

International audience; The 1.5 Gbp/2C genome of pedunculate oak (Quercus robur) has been sequenced. A strategy was established for dealing with the challenges imposed by the sequencing of such a large, complex and highly heterozygous genome by a whole-genome shotgun (WGS) approach, without the use of costly and time-consuming methods, such as fosmid or BAC clone-based hierarchical sequencing methods. The sequencing strategy combined short and long reads. Over 49 million reads provided by Roche 454 GS-FLX technology were assembled into contigs and combined with shorter Illumina sequence reads from paired-end and mate-pair libraries of different insert sizes, to build scaffolds. Errors were corrected and gaps filled with Illumina paired-end reads and contaminants detected, resulting in a total of 17,910 scaffolds (>2 kb) corresponding to 1.34 Gb. Fifty per cent of the assembly was accounted for by 1468 scaffolds (N50 of 260 kb). Initial comparison with the phylogenetically related Prunus persica gene model indicated that genes for 84.6% of the proteins present in peach (mean protein coverage of 90.5%) were present in our assembly. The second and third steps in this project are genome annotation and the assignment of scaffolds to the oak genetic linkage map. In accordance with the Bermuda and Fort Lauderdale agreements and the more recent Toronto Statement, the oak genome data have been released into public sequence repositories in advance of publication. In this presubmission paper, the oak genome consortium describes its principal lines of work and future directions for analyses of the nature, function and evolution of the oak genome.

Background Many northern-hemisphere forests are dominated by oaks. These species extend over diverse environmental conditions and are thus interesting models for studies of plant adaptation and speciation. The genomic toolbox is an important asset for exploring the functional variation associated with natural selection. Results The assembly of previously available and newly developed long and short sequence reads for two sympatric oak species, Quercus robur and Quercus petraea, generated a comprehensive catalog of transcripts for oak. The functional annotation of 91 k contigs demonstrated the presence of a large proportion of plant genes in this unigene set. Comparisons with SwissProt accessions and five plant gene models revealed orthologous relationships, making it possible to decipher the evolution of the oak genome. In particular, it was possible to align 9.5 thousand oak coding sequences with the equivalent sequences on peach chromosomes. Finally, RNA-seq data shed new light on the gene networks underlying vegetative bud dormancy release, a key stage in development allowing plants to adapt their phenology to the environment. Conclusion In addition to providing a vast array of expressed genes, this study generated essential information about oak genome evolution and the regulation of genes associated with vegetative bud phenology, an important adaptive traits in trees. This resource contributes to the annotation of the oak genome sequence and will provide support for forward genetics approaches aiming to link genotypes with adaptive phenotypes. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1331-9) contains supplementary material, which is available to authorized users.

International audience; Wood is a complex and highly variable tissue, the formation of which is developmentally and environmentally regulated. In reaction to gravitropic stimuli, angiosperm trees differentiate tension wood, a wood with specific anatomical, chemical and mechanical features. In poplar the most significant of these features is an additional layer that forms in the secondary wall of tension wood fibres. This layer is mainly constituted of cellulose microfibrils oriented nearly parallel to the fibre axis. Tension wood formation can be induced easily and strongly by bending the stem of a tree. Located at the upper side of the bent stem, tension wood can be compared with the wood located on its lower side. Therefore tension wood represents an excellent model for studying the formation of xylem cell walls. This review summarizes results recently obtained in the field of genomics on tension wood. In addition, we present an example of how the application of functional genomics to tension wood can help decipher the molecular mechanisms responsible for cell wall characteristics such as the orientation of cellulose microfibrils.

Hybrid poplars (Populus tremula ×Populus tremuloides) have been genetically engineered viaAgrobacterium tumefaciens, to express a syntheticcry3Aa gene derived from the native Bacillusthuringiensis subsp. tenebrionis cry3Aa gene.The presence and the expression of the transgene have been verified in fourtransgenic poplar lines, using Southern, northern and western analyses. Thetransgenic poplar's toxicity towards the phytophagous beetleChrysomela tremulae (Coleoptera, Chrysomelidae) has beenassessed on six month-old greenhouse-grown selected plants in laboratoryconditions. Laboratory experiments consisted of feeding tests of fresh detachedleaves on C. tremulae at all developmental stages. Ourresults indicate that the transgenic poplar leaves, expressing a Cry3Aa proteinamount in a range of 0.05–0.0025% of total soluble protein, weredefinitely deleterious for C. tremulae, regardless of thedevelopmental stage.

International audience; Molecular marker compounds, derived from lignin by the thioacidolysis degradative method, for cinnamyl alcohol dehydrogenase (CAD) deficiency in angiosperms have been structurally identified as indene derivatives. They are shown to derive from hydroxycinnamyl aldehydes that have undergone 8-O-4-cross-coupling during lignification. As such, they are valuable markers for ascertaining plant responses to various levels of CAD down-regulation. Their derivation illustrates that hydroxycinnamyl aldehydes incorporate into angiosperm lignins by endwise coupling reactions in much the same way as normal monolignols do, suggesting that the hydroxycinnamyl aldehydes should be considered authentic lignin precursors.

A rapid and efficient micropropagation method has been established for six European poplar cultivars of economic interest – four Populus × interamericana and two Populus × euramericana. Using a three-step procedure, we were able to regenerate plantlets from callus and acclimate them within 4 months. In the first step, callogenesis was induced when explants were cultured for 25 days on culture medium supplemented with 10 µM α-naphthaleneacetic acid and 5 µM N6(2-isopentenyl)adenine. Bud regeneration followed by shoot elongation was then obtained from callus tissue by combining the cytokinin-like compound thidiazuron with the surfactant Pluronic F-68 at concentrations adjusted for each cultivar. The usefulness of this procedure in the area of genetic engineering is discussed.

International audience; Systematic sequencing is the method of choice for generating genomic resources for molecular marker development and candidate gene identification in non-model species. We generated 47,357 Sanger ESTs and 2.2M Roche-454 reads from five cDNA libraries for European beech (Fagus sylvatica L.). This tree species of high ecological and economic value in Europe is among the most representative trees of deciduous broadleaf forests. The sequences generated were assembled into 21,057 contigs with MIRA software. Functional annotations were obtained for 85% of these contigs, from the proteomes of four plant species, Swissprot accessions and the Gene Ontology database. We were able to identify 28,079 in silico SNPs for future marker development. Moreover, RNAseq and qPCR approaches identified genes and gene networks regulated differentially between two critical phenological stages preceding vegetative bud burst (the quiescent and swelling buds stages). According to climatic model based projection, some European beech populations may be endangered, particularly at the southern and eastern edges of the European distribution range, which are strongly affected by current climate change. This first genomic resource for the genus Fagus should facilitate the identification of key genes for beech adaptation and management strategies for preserving beech adaptability.

We thank the technical staff for managing and sampling both field trials, Ingeborg Stals and Dieter Depuydt for help in SSF analyses, and René Custers for requesting regulatory permission for the Belgian field trial.; Lignin is one of the main factors determining recalcitrance to enzymatic processing of lignocellulosic biomass. Poplars (Populus tremula x Populus alba) down-regulated for cinnamoyl-CoA reductase (CCR), the enzyme catalyzing the first step in the monolignol-specific branch of the lignin biosynthetic pathway, were grown in field trials in Belgium and France under short-rotation coppice culture. Wood samples were classified according to the intensity of the red xylem coloration typically associated with CCR down-regulation. Saccharification assays under different pretreatment conditions (none, two alkaline, and one acid pretreatment) and simultaneous saccharification and fermentation assays showed that wood from the most affected transgenic trees had up to 161% increased ethanol yield. Fermentations of combined material from the complete set of 20-mo-old CCR-down-regulated trees, including bark and less efficiently down-regulated trees, still yielded ∼ 20% more ethanol on a weight basis. However, strong down-regulation of CCR also affected biomass yield. We conclude that CCR down-regulation may become a successful strategy to improve biomass processing if the variability in down-regulation and the yield penalty can be overcome.

Over the last decade, we have witnessed impressive advances in tree molecular biology and the consolidation of tree genomics. We have essentially moved from a small portfolio of genes focusing on a specifi c genetic trait to large databases including thousands of genes and their respective expression profi les. In 2006, we saw the publication of the fi rst genomic sequence of a tree, the model tree species Populus trichocarpa . Though, not surprisingly, much progress has been made with Populus, impressive research results have also been realized in more recalcitrant coniferous species such as pines and spruces.Despite the rapid advances in tree genomics, tree genetic engineering (GE) proved to be a bottleneck requiring the development of whole-tree regeneration protocols using in vitro culture and an effective method of DNA transfer. The introduction of simple single gene traits such as insect resistance was the early target oftree genetic engineers. Today more tree species are compatible with GE and at a higher throughput, making functional genomics approaches possible to improve our understanding of gene functions.In this chapter we will provide an historical overview of the advances made in GE of trees. We will also explore the various applications of tree GE to improving response to biotic and abiotic stresses, which is becoming more important in an ever-changing environment. Improvement of specifi c traits for tree domestication will also be covered. Lastly, we will briefl y discuss issues related to the regulation of GM trees, particularly concerning genetic containment and environmental risk assessment.

Background In temperate regions, the time lag between vegetative bud burst and bud set determines the duration of the growing season of trees (i.e. the duration of wood biomass production). Dormancy, the period during which the plant is not growing, allows trees to avoid cold injury resulting from exposure to low temperatures. An understanding of the molecular machinery controlling the shift between these two phenological states is of key importance in the context of climatic change. The objective of this study was to identify genes upregulated during endo- and ecodormancy, the two main stages of bud dormancy. Sessile oak is a widely distributed European white oak species. A forcing test on young trees was first carried out to identify the period most likely to correspond to these two stages. Total RNA was then extracted from apical buds displaying endo- and ecodormancy. This RNA was used for the generation of cDNA libraries, and in-depth transcriptome characterization was performed with 454 FLX pyrosequencing technology. Results Pyrosequencing produced a total of 495,915 reads. The data were cleaned, duplicated reads removed, and sequences were mapped onto the oak UniGene data. Digital gene expression analysis was performed, with both R statistics and the R-Bioconductor packages (edgeR and DESeq), on 6,471 contigs with read numbers ≥ 5 within any contigs. The number of sequences displaying significant differences in expression level (read abundance) between endo- and ecodormancy conditions ranged from 75 to 161, depending on the algorithm used. 13 genes displaying significant differences between conditions were selected for further analysis, and 11 of these genes, including those for glutathione-S-transferase (GST) and dehydrin xero2 (XERO2) were validated by quantitative PCR. Conclusions The identification and functional annotation of differentially expressed genes involved in the “response to abscisic acid”, “response to cold stress” and “response to oxidative stress” categories constitutes a major step towards characterization of the molecular network underlying vegetative bud dormancy, an important life history trait of long-lived organisms.

Summary Lignin is a polymeric constituent of the cell wall that needs to be removed during the paper making process. Bi-specific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) catalyses the O-methylation of caffeic acid and 5-hydroxyferulic acid to ferulic acid and sinapic acid, respectively. These compounds are intermediates in the biosynthesis of the lignin precursors. Therefore, COMTs are potential target enzymes for reducing the amount, or modifying the composition, of lignin in plants. Different antisense and sense constructs have been expressed of a gene encoding a COMT from poplar (Populus trichocarpa x P. deltoides) in a P. tremula x P. alba clone under the control of the cauliflower mosaic virus 35S promoter. From all analysed transformants, four lines transformed with an antisense construct had a reduced COMT activity. Two showed a 50% reduction of COMT activity, which altered only slightly the monomeric composition. In the two other transformants, the COMT activity was reduced by 95%. In the latter case, the syringyl/ guaiacyl ratio (S/G) was reduced by sixfold (due to a decrease of S and an increase of G), as analysed by thioacidolysis. A new component of lignin, the 5-hydroxyguaiacyl residue, was detected among the thioacidolysis products. Moreover, in contrast to the white/yellow colour of wild-type wood, the xylem of the transgenic lines with a 95% reduction of COMT activity was pale rose. A similar phenotype was observed in brown-midrib mutants of maize and sorghum, known for their altered lignification. Although the lignin composition was consistently modified, the lignin content of the transgenic poplars was similar to that of the controls.

Waterlogging causes stressful conditions for perennial species. The temporary overabundance of water in waterlogged soil can induce hypoxia in the rhizosphere, leading to root death, tree decline and even dieback. Two closely related members of the European white oak complex, pedunculate (Quercus robur L.) and sessile (Quercus petraea Matt. Liebl.) oaks, have different ecological characteristics, especially regarding their adaptation to soil waterlogging. The tolerance of waterlogging observed in pedunculate oak is driven principally by its ability to produce adaptive structures, hypertrophied lenticels and adventitious roots, and to switch rapidly its metabolism to the fermentative pathway. This study had two objectives: (i) to identify genes important for adaptation to waterlogging and (ii) to gain insight into the molecular mechanisms involved in hypertrophied lenticel formation in pedunculate oak. We subjected seedlings of the two species to hypoxia by maintaining the water level 2 cm above the collar. The immersed part of the stem (i.e., containing hypertrophied lenticels in pedunculate oak) was sampled after 9 days of waterlogging stress and its gene expression was investigated by RNA-seq. Genes displaying differential expression between the two species were identified with the DESeq R package and a false discovery rate of 0.001. We found that 3705 contigs were differentially regulated between the two species. Twenty-two differentially expressed genes were validated by real-time quantitative polymerase chain reaction. The suberin biosynthesis pathway was found to be upregulated in pedunculate oak, consistent with molecular mechanisms analogous to those operating in the radial oxygen loss barrier in waterlogging-tolerant species.

Background Hybrid poplars species are candidates for biomass production but breeding efforts are needed to combine productivity and water use efficiency in improved cultivars. The understanding of the genetic architecture of growth in poplar by a Quantitative Trait Loci (QTL) approach can help us to elucidate the molecular basis of such integrative traits but identifying candidate genes underlying these QTLs remains difficult. Nevertheless, the increase of genomic information together with the accessibility to a reference genome sequence (Populus trichocarpa Nisqually-1) allow to bridge QTL information on genetic maps and physical location of candidate genes on the genome. The objective of the study is to identify QTLs controlling productivity, architecture and leaf traits in a P. deltoides x P. trichocarpa F1 progeny and to identify candidate genes underlying QTLs based on the anchoring of genetic maps on the genome and the gene ontology information linked to genome annotation. The strategy to explore genome annotation was to use Gene Ontology enrichment tools to test if some functional categories are statistically over-represented in QTL regions. Results Four leaf traits and 7 growth traits were measured on 330 F1 P. deltoides x P. trichocarpa progeny. A total of 77 QTLs controlling 11 traits were identified explaining from 1.8 to 17.2% of the variation of traits. For 58 QTLs, confidence intervals could be projected on the genome. An extended functional annotation was built based on data retrieved from the plant genome database Phytozome and from an inference of function using homology between Populus and the model plant Arabidopsis. Genes located within QTL confidence intervals were retrieved and enrichments in gene ontology (GO) terms were determined using different methods. Significant enrichments were found for all traits. Particularly relevant biological processes GO terms were identified for QTLs controlling number of sylleptic branches: intervals were enriched in GO terms of biological process like ‘ripening’ and ‘adventitious roots development’. Conclusion Beyond the simple identification of QTLs, this study is the first to use a global approach of GO terms enrichment analysis to fully explore gene function under QTLs confidence intervals in plants. This global approach may lead to identification of new candidate genes for traits of interest.

Plant LIM domain proteins may act as transcriptional activators of lignin biosynthesis and/or as actin binding and bundling proteins. Plant LIM genes have evolved in phylogenetic subgroups differing in their expression profiles: in the whole plant or specifically in pollen. However, several poplar PtLIM genes belong to uncharacterized monophyletic subgroups and the expression patterns of the LIM gene family in a woody plant have not been studied., In this work, the expression pattern of the twelve duplicated poplar PtLIM genes has been investigated by semi quantitative RT-PCR in different vegetative and reproductive tissues. As in other plant species, poplar PtLIM genes were widely expressed in the tree or in particular tissues. Especially, PtXLIM1a, PtXLIM1b and PtWLIM1b genes were preferentially expressed in the secondary xylem, suggesting a specific function in wood formation. Moreover, the expression of these genes and of the PtPLIM2a gene was increased in tension wood. Western-blot analysis confirmed the preferential expression of PtXLIM1a protein during xylem differentiation and tension wood formation. Genes classified within the pollen specific PLIM2 and PLIM2-like subgroups were all strongly expressed in pollen but also in cottony hairs. Interestingly, pairs of duplicated PtLIM genes exhibited different expression patterns indicating subfunctionalisations in specific tissues., The strong expression of several LIM genes in cottony hairs and germinating pollen, as well as in xylem fibers suggests an involvement of plant LIM domain proteins in the control of cell expansion. Comparisons of expression profiles of poplar LIM genes with the published functions of closely related plant LIM genes suggest conserved functions in the areas of lignin biosynthesis, pollen tube growth and mechanical stress response. Based on these results, we propose a novel nomenclature of poplar LIM domain proteins.

For the last 20 years, as a complement to biochemical approaches, lignin-modified transgenic trees have proven to be invaluable tools for deciphering the lignin biosynthetic pathway and increasing our knowledge about the role of lignins in wood-specific properties important for tree physiology. In addition, their evaluation in the field has been very useful for the identification of possible routes to improve wood properties for specific industrial uses, initially pulp and paper making and now biofuel production. In this chapter, we review our present knowledge gained through numerous studies analysing transgenic trees, with emphasis on field trial evaluations, for assessing the effects of lignin modification on wood properties and tree physiology. All the data presented here led to the conclusion that, prior to use in forestry for pulp and paper or bioenergy production, the fitness of lignin-modified transgenic trees required careful evaluation in a variety of field environments. Although limited in number, environmental studies failed to indicate any strong effects of lignin-modified transgenic trees on the ecosystem. Finally, this review helps to identify some promising options where biotech trees could be used to further optimize the production of wood for human uses.

Interest in the genomics of Eucalyptus has skyrocketed thanks to the recent sequencing of the genome of Eucalyptus grandis and to a growing number of large-scale transcriptomic studies. Quantitative reverse transcription-PCR (RT-PCR) is the method of choice for gene expression analysis and can now also be used as a high-throughput method. The selection of appropriate internal controls is becoming of utmost importance to ensure accurate expression results in Eucalyptus. To this end, we selected 21 candidate reference genes and used high-throughput microfluidic dynamic arrays to assess their expression among a large panel of developmental and environmental conditions with a special focus on wood-forming tissues. We analyzed the expression stability of these genes by using three distinct statistical algorithms (geNorm, NormFinder and ΔCt), and used principal component analysis to compare methods and rankings. We showed that the most stable genes identified depended not only on the panel of biological samples considered but also on the statistical method used. We then developed a comprehensive integration of the rankings generated by the three methods and identified the optimal reference genes for 17 distinct experimental sets covering 13 organs and tissues, as well as various developmental and environmental conditions. The expression patterns of Eucalyptus master genes EgMYB1 and EgMYB2 experimentally validated our selection. Our findings provide an important resource for the selection of appropriate reference genes for accurate and reliable normalization of gene expression data in the organs and tissues of Eucalyptus trees grown in a range of conditions including abiotic stresses.