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Genetically improved forest reproductive materials are now widely accessible in many European countries due to decades of continuous breeding efforts. Tree breeding does not only contribute to higher-value end products but allows an increase in the rate of carbon capture and sequestration, helping to mitigate the effects of climate change. The usefulness of breeding programmes depends on (i) the relevance of the set of selected traits and their relative weights (growth, drought tolerance, phenology, etc.); (ii) the explicit management of targeted and “neutral” diversity; (iii) the genetic gain achieved; and (iv) the efficiency of transferring diversity and gain to the plantation. Several biological factors limit both operational breeding and mass reproduction. To fully realise the potential of tree breeding, the introduction of new technologies and concepts is pivotal for overcoming these constraints. We reviewed several European breeding programmes, examining their current status and factors that are likely to influence tree breeding in the coming decades. The synthesis was based on case studies developed for the European Union-funded B4EST project, which focused on eight economically important tree species with breeding histories and intensities ranging from low-input breeding (stone pine, Douglas-fir and ash) to more complex programmes (eucalyptus, maritime pine, Norway spruce, poplar, and Scots pine). Tree breeding for these species is managed in a variety of ways due to differences in species’ biology, breeding objectives, and economic value. Most programmes are managed by governmental institutes with full or partial public support because of the relatively late return on investment. Eucalyptus is the only tree species whose breeding is entirely sponsored and managed by a private company. Several new technologies have emerged for both phenotyping and genotyping. They have the potential to speed up breeding processes and make genetic evaluations more accurate, thereby reducing costs and increasing genetic gains per unit of time. In addition, genotyping has allowed the explicit control of genetic diversity in selected populations with great precision. The continuing advances in tree genomics are expected to revolutionise tree breeding by moving it towards genomic-based selection, a perspective that requires new types of skills that are not always available in the institutions hosting the programmes. We therefore recognise the importance of promoting coordination and collaboration between the many groups involved in breeding. Climate change is expected to bring in new pests and diseases and increase the frequency of extreme weather events such as late frosts and prolonged droughts. Such stresses will cause slow growth and mortality, reducing forest productivity and resilience. Most of these threats are difficult to predict, and the time-consuming nature of conventional breeding does not allow for an adequate and timely reaction. We anticipate that most breeding programmes will need to revise their selection criteria and objectives to place greater emphasis on adaptive performance, tolerance to multiple environmental stresses, stability in different environments, and conservation of genetic diversity. Testing breeding materials in a variety of environments, including potentially contrasting climates, will become increasingly important. Climate change may also force the incorporation of new genetic resources that provide new useful adaptations, which may involve the use of new, previously unexplored gene pools or hybridisation, with the enormous challenge of incorporating useful alleles without adding along an unfavourable genetic background. Decision-support tools to help landowners and foresters select the best-performing forest reproductive material in each specific environment could also help reduce the impact of climate change.

The analysis of the wild-type and the mutant forms of cinnamyl alcohol dehyrdogenase isoform 2 (CAD2) when subjected to site-directed mutagenesis (S212D) and overexpression, reveal that the phosphate moiety of NADPH is identified through S212 and the structural characteristics for NADPH specificity. The enzymatic binding positions of the coniferyl alcohol substrate undergo physical interaction with the 2'-phosphate binding region. The single mutation S212D is responsible for reducing the catalytic performance of CAD2 where NADP+ is the coenzyme.

Abstract Background Eucalyptus is an important genus in industrial plantations throughout the world and is grown for use as timber, pulp, paper and charcoal. Several breeding programmes have been launched worldwide to concomitantly improve growth performance and wood properties (WPs). In this study, an interspecific cross between Eucalyptus urophylla and E. grandis was used to identify major genomic regions (Quantitative Trait Loci, QTL) controlling the variability of WPs. Results Linkage maps were generated for both parent species. A total of 117 QTLs were detected for a series of wood and end-use related traits, including chemical, technological, physical, mechanical and anatomical properties. The QTLs were mainly clustered into five linkage groups. In terms of distribution of QTL effects, our result agrees with the typical L-shape reported in most QTL studies, i.e. most WP QTLs had limited effects and only a few (13) had major effects (phenotypic variance explained > 15%). The co-locations of QTLs for different WPs as well as QTLs and candidate genes are discussed in terms of phenotypic correlations between traits, and of the function of the candidate genes. The major wood property QTL harbours a gene encoding a Cinnamoyl CoA reductase (CCR), a structural enzyme of the monolignol-specific biosynthesis pathway. Conclusions Given the number of traits analysed, this study provides a comprehensive understanding of the genetic architecture of wood properties in this Eucalyptus full-sib pedigree. At the dawn of Eucalyptus genome sequence, it will provide a framework to identify the nature of genes underlying these important quantitative traits.

Abstract Background Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing. Results We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1) digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes. Conclusions The two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae, including genome sequencing, gene isolation, functional and comparative genomics. Because they have been constructed using the same tree (E. grandis BRASUZ1) whose full genome is being sequenced, they should prove instrumental for assembly and gap filling of the upcoming Eucalyptus reference genome sequence.

Abstract Background Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the final steps in the biosynthesis of monolignols, the monomeric units of the phenolic lignin polymers which confer rigidity, imperviousness and resistance to biodegradation to cell walls. We have previously shown that the Eucalyptus gunnii CCR and CAD2 promoters direct similar expression patterns in vascular tissues suggesting that monolignol production is controlled, at least in part, by the coordinated transcriptional regulation of these two genes. Although consensus motifs for MYB transcription factors occur in most gene promoters of the whole phenylpropanoid pathway, functional evidence for their contribution to promoter activity has only been demonstrated for a few of them. Here, in the lignin-specific branch, we studied the functional role of MYB elements as well as other cis-elements identified in the regulatory regions of EgCAD2 and EgCCR promoters, in the transcriptional activity of these gene promoters. Results By using promoter deletion analysis and in vivo footprinting, we identified an 80 bp regulatory region in the Eucalyptus gunnii EgCAD2 promoter that contains two MYB elements, each arranged in a distinct module with newly identified cis-elements. A directed mutagenesis approach was used to introduce block mutations in all putative cis-elements of the EgCAD2 promoter and in those of the 50 bp regulatory region previously delineated in the EgCCR promoter. We showed that the conserved MYB elements in EgCAD2 and EgCCR promoters are crucial both for the formation of DNA-protein complexes in EMSA experiments and for the transcriptional activation of EgCAD2 and EgCCR promoters in vascular tissues in planta. In addition, a new regulatory cis-element that modulates the balance between two DNA-protein complexes in vitro was found to be important for EgCAD2 expression in the cambial zone. Conclusions Our assignment of functional roles to the identified cis-elements clearly demonstrates the importance of MYB cis-elements in the transcriptional regulation of two genes of the lignin-specific pathway and support the hypothesis that MYB elements serve as a common means for the coordinated regulation of genes in the entire lignin biosynthetic pathway.

Abstract Background Renowned for their fast growth, valuable wood properties and wide adaptability, Eucalyptus species are amongst the most planted hardwoods in the world, yet they are still at the early stages of domestication because conventional breeding is slow and costly. Thus, there is huge potential for marker-assisted breeding programs to improve traits such as wood properties. To this end, the sequencing, analysis and annotation of a large collection of expressed sequences tags (ESTs) from genes involved in wood formation in Eucalyptus would provide a valuable resource. Results We report here the normalization and sequencing of a cDNA library from developing Eucalyptus secondary xylem, as well as the construction and sequencing of two subtractive libraries (juvenile versus mature wood and vice versa). A total of 9,222 high quality sequences were collected from about 10,000 cDNA clones. The EST assembly generated a set of 3,857 wood-related unigenes including 2,461 contigs (Cg) and 1,396 singletons (Sg) that we named 'EUCAWOOD'. About 65% of the EUCAWOOD sequences produced matches with poplar, grapevine, Arabidopsis and rice protein sequence databases. BlastX searches of the Uniref100 protein database allowed us to allocate gene ontology (GO) and protein family terms to the EUCAWOOD unigenes. This annotation of the EUCAWOOD set revealed key functional categories involved in xylogenesis. For instance, 422 sequences matched various gene families involved in biosynthesis and assembly of primary and secondary cell walls. Interestingly, 141 sequences were annotated as transcription factors, some of them being orthologs of regulators known to be involved in xylogenesis. The EUCAWOOD dataset was also mined for genomic simple sequence repeat markers, yielding a total of 639 putative microsatellites. Finally, a publicly accessible database was created, supporting multiple queries on the EUCAWOOD dataset. Conclusion In this work, we have identified a large set of wood-related Eucalyptus unigenes called EUCAWOOD, thus creating a valuable resource for functional genomics studies of wood formation and molecular breeding in this economically important genus. This set of publicly available annotated sequences will be instrumental for candidate gene approaches, custom array development and marker-assisted selection programs aimed at improving and modulating wood properties.

Abstract Background Several members of the R2R3-MYB family of transcription factors act as regulators of lignin and phenylpropanoid metabolism during wood formation in angiosperm and gymnosperm plants. The angiosperm Arabidopsis has over one hundred R2R3-MYBs genes; however, only a few members of this family have been discovered in gymnosperms. Results We isolated and characterised full-length cDNAs encoding R2R3-MYB genes from the gymnosperms white spruce, Picea glauca (13 sequences), and loblolly pine, Pinus taeda L. (five sequences). Sequence similarities and phylogenetic analyses placed the spruce and pine sequences in diverse subgroups of the large R2R3-MYB family, although several of the sequences clustered closely together. We searched the highly variable C-terminal region of diverse plant MYBs for conserved amino acid sequences and identified 20 motifs in the spruce MYBs, nine of which have not previously been reported and three of which are specific to conifers. The number and length of the introns in spruce MYB genes varied significantly, but their positions were well conserved relative to angiosperm MYB genes. Quantitative RTPCR of MYB genes transcript abundance in root and stem tissues revealed diverse expression patterns; three MYB genes were preferentially expressed in secondary xylem, whereas others were preferentially expressed in phloem or were ubiquitous. The MYB genes expressed in xylem, and three others, were up-regulated in the compression wood of leaning trees within 76 hours of induction. Conclusion Our survey of 18 conifer R2R3-MYB genes clearly showed a gene family structure similar to that of Arabidopsis. Three of the sequences are likely to play a role in lignin metabolism and/or wood formation in gymnosperm trees, including a close homolog of the loblolly pine PtMYB4, shown to regulate lignin biosynthesis in transgenic tobacco.

A multi-tiered transcriptional network regulates xylem differentiation and secondary cell wall (SCW) formation in plants, with evidence of both conserved and lineage-specific SCW network architecture. We aimed to elucidate the roles of selected R2R3-MYB transcription factors (TFs) linked to Eucalyptus wood formation by identifying genome-wide TF binding sites and direct target genes through an improved DAP-seq protocol combined with machine learning for target gene assignment (DAP-seq-ML). We applied this to five TFs including a well-studied SCW master regulator (EgrMYB2; homolog of AtMYB83), a repressor of lignification (EgrMYB1; homolog of AtMYB4), a TF affecting SCW thickness and vessel density (EgrMYB137; homolog of PtrMYB074) and two TFs with unclear roles in SCW regulation (EgrMYB135 and EgrMYB122). Each DAP-seq TF peak set (average 12,613 peaks) was enriched for canonical R2R3-MYB binding motifs. To improve the reliability of target gene assignment to peaks, a random forest classifier was developed from Arabidopsis DAP-seq, RNA-seq, chromatin, and conserved noncoding sequence data which demonstrated significantly higher precision and recall to the baseline method of assigning genes to proximal peaks. EgrMYB1, EgrMYB2 and EgrMYB137 predicted targets showed clear enrichment for SCW-related biological processes. As validation, EgrMYB137 overexpression in transgenic Eucalyptus hairy roots increased xylem lignification, while its dominant repression in transgenic Arabidopsis and Populus reduced xylem lignification, stunted growth, and caused downregulation of SCW genes. EgrMYB137 targets overlapped significantly with those of EgrMYB2, suggesting partial functional redundancy. Our results show that DAP-seq-ML identified biologically relevant R2R3-MYB targets supported by the finding that EgrMYB137 promotes SCW lignification in planta., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Wood (secondary xylem) formation is regulated by auxin, which plays a pivotal role as an integrator of developmental and environmental cues. However, our current knowledge of auxin-signaling during wood formation is incomplete. Our previous genome-wide analysis of Aux / IAAs in Eucalyptus grandis showed the presence of the non-canonical paralog member EgrIAA20 that is preferentially expressed in cambium. We analyzed its cellular localization using a GFP fusion protein and its transcriptional activity using transactivation assays, and demonstrated its nuclear localization and strong auxin response repressor activity. In addition, we functionally tested the role of EgrIAA20 by constitutive overexpression in Arabidopsis to investigate for phenotypic changes in secondary xylem formation. Transgenic Arabidopsis plants overexpressing EgrIAA20 were smaller and displayed impaired development of secondary fibers, but not of other wood cell types. The inhibition in fiber development specifically affected their cell wall lignification. We performed yeast-two-hybrid assays to identify EgrIAA20 protein partners during wood formation in Eucalyptus , and identified EgrIAA9A, whose ortholog PtoIAA9 in poplar is also known to be involved in wood formation. Altogether, we showed that EgrIAA20 is an important auxin signaling component specifically involved in controlling the lignification of wood fibers.

Key Message: Our Induced Somatic Sector Analysis and protein-protein interaction experiments demonstrate that Eucalyptus grandis IAA13 regulates xylem fibre and vessel development, potentially via EgrIAA13 modules involving ARF2, ARF5, ARF6 and ARF19. Auxin is a crucial phytohormone regulating multiple aspects of plant growth and differentiation, including regulation of vascular cambium activity, xylogenesis and its responsiveness towards gravitropic stress. Although the regulation of these biological processes greatly depends on auxin and regulators of the auxin signalling pathway, many of their specific functions remain unclear. Therefore, the present study aims to functionally characterise Eucalyptus grandis AUX/INDOLE-3-ACETIC ACID 13 (EgrIAA13), a member of the auxin signalling pathway. In Eucalyptus and Populus, EgrIAA13 and its orthologs are preferentially expressed in the xylogenic tissues and downregulated in tension wood. Therefore, to further investigate EgrIAA13 and its function during xylogenesis, we conducted subcellular localisation and Induced Somatic Sector Analysis experiments using overexpression and RNAi knockdown constructs of EgrIAA13 to create transgenic tissue sectors on growing stems of Eucalyptus and Populus. Since Aux/IAAs interact with Auxin Responsive Factors (ARFs), in silico predictions of IAA13-ARF interactions were explored and experimentally validated via yeast-2-hybrid experiments. Our results demonstrate that EgrIAA13 localises to the nucleus and that downregulation of EgrIAA13 impedes Eucalyptus xylem fibre and vessel development. We also observed that EgrIAA13 interacts with Eucalyptus ARF2, ARF5, ARF6 and ARF19A. Based on these results, we conclude that EgrIAA13 is a regulator of Eucalyptus xylogenesis and postulate that the observed phenotypes are likely to result from alterations in the auxin-responsive transcriptome via IAA13-ARF modules such as EgrIAA13-EgrARF5. Our results provide the first insights into the regulatory role of EgrIAA13 during xylogenesis., (© 2022. Crown.)

Eucalypts are the most planted hardwoods worldwide. The availability of the Eucalyptus grandis genome highlighted many genes awaiting functional characterization, lagging behind because of the lack of efficient genetic transformation protocols. In order to efficiently generate knock-out mutants to study the function of eucalypts genes, we implemented the powerful CRISPR/Cas9 gene editing technology with the hairy roots transformation system. As proofs-of-concept, we targeted two wood-related genes: Cinnamoyl-CoA Reductase1 ( CCR1 ), a key lignin biosynthetic gene and IAA9A an auxin dependent transcription factor of Aux/IAA family. Almost all transgenic hairy roots were edited but the allele-editing rates and spectra varied greatly depending on the gene targeted. Most edition events generated truncated proteins, the prevalent edition types were small deletions but large deletions were also quite frequent. By using a combination of FT-IR spectroscopy and multivariate analysis (partial least square analysis (PLS-DA)), we showed that the CCR1 -edited lines, which were clearly separated from the controls. The most discriminant wave-numbers were attributed to lignin. Histochemical analyses further confirmed the decreased lignification and the presence of collapsed vessels in CCR1 -edited lines, which are characteristics of CCR1 deficiency. Although the efficiency of editing could be improved, the method described here is already a powerful tool to functionally characterize eucalypts genes for both basic research and industry purposes., Competing Interests: The authors declare no conflict of interest.

Eucalypts are the most planted trees worldwide, but most of them are frost sensitive. Overexpressing transcription factors for CRT-repeat binding factors ( CBFs ) in transgenic Eucalyptus confer cold resistance both in leaves and stems. While wood plays crucial roles in trees and is affected by environmental cues, its potential role in adaptation to cold stress has been neglected. Here, we addressed this question by investigating the changes occurring in wood in response to the overexpression of two CBFs , taking advantage of available transgenic Eucalyptus lines. We performed histological, biochemical, and transcriptomic analyses on xylem samples. CBF ectopic expression led to a reduction of both primary and secondary growth, and triggered changes in xylem architecture with smaller and more frequent vessels and fibers exhibiting reduced lumens. In addition, lignin content and syringyl/guaiacyl (S/G) ratio increased. Consistently, many genes of the phenylpropanoid and lignin branch pathway were upregulated. Most of the features of xylem remodeling induced by CBF overexpression are reminiscent of those observed after long exposure of Eucalyptus trees to chilling temperatures. Altogether, these results suggest that CBF plays a central role in the cross-talk between response to cold and wood formation and that the remodeling of wood is part of the adaptive strategies to face cold stress.

Willow ( Salix spp. L.) species are fast-growing trees and shrubs that have attracted emergent attention for their potential as feedstocks for bioenergy and biofuel production, as well as for pharmaceutical and phytoremediation applications. This economic and environmental potential has propelled the creation of several genetic and genomic resources for Salix spp. Furthermore, the recent availability of an annotated genome for Salix purpurea has pinpointed novel candidate genes underlying economically relevant traits. However, functional studies have been stalled by the lack of rapid and efficient coupled regeneration-transformation systems for Salix purpurea and Salix spp. in general. In this report, we describe a fast and highly efficient hairy root transformation protocol for S. purpurea. It was effective for different explant sources and S. purpurea genotypes, with efficiencies between 63.4% and 98.7%, and the screening of the transformed hairy roots was easily carried out using the fluorescent marker DsRed. To test the applicability of this hairy root transformation system for gene functional analysis, we transformed hairy roots with the vector pGWAY- SpDRM2 , where the gene SpDRM2 encoding a putative Domain Rearranged Methyltransferase (DRM) was placed under the control of the CaMV 35S constitutive promoter. Indeed, the transgenic hairy roots obtained exhibited significantly increased expression of SpDRM2 as compared to controls, demonstrating that this protocol is suitable for the medium/high-throughput functional characterization of candidate genes in S. purpurea and other recalcitrant Salix spp., (Copyright © 2019 Gomes, Dupas, Pagano, Grima-Pettenati and Paiva.)

Although lignin is essential to ensure the correct growth and development of land plants, it may be an obstacle to the production of lignocellulosics-based biofuels, and reduces the nutritional quality of crops used for human consumption or livestock feed. The need to tailor the lignocellulosic biomass for more efficient biofuel production or for improved plant digestibility has fostered considerable advances in our understanding of the lignin biosynthetic pathway and its regulation. Most of the described regulators are transcriptional activators of lignin biosynthesis, but considerably less attention has been devoted to the repressors of this pathway. We provide a comprehensive overview of the molecular factors that negatively impact on the lignification process at both the transcriptional and post-transcriptional levels., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Wood production in fast-growing Eucalyptus grandis trees is highly dependent on both potassium (K) fertilization and water availability but the molecular processes underlying wood formation in response to the combined effects of these two limiting factors remain unknown. E. grandis trees were submitted to four combinations of K-fertilization and water supply. Weighted gene co-expression network analysis and MixOmics-based co-regulation networks were used to integrate xylem transcriptome, metabolome and complex wood traits. Functional characterization of a candidate gene was performed in transgenic E. grandis hairy roots. This integrated network-based approach enabled us to identify meaningful biological processes and regulators impacted by K-fertilization and/or water limitation. It revealed that modules of co-regulated genes and metabolites strongly correlated to wood complex traits are in the heart of a complex trade-off between biomass production and stress responses. Nested in these modules, potential new cell-wall regulators were identified, as further confirmed by the functional characterization of EgMYB137. These findings provide new insights into the regulatory mechanisms of wood formation under stressful conditions, pointing out both known and new regulators co-opted by K-fertilization and/or water limitation that may potentially promote adaptive wood traits., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Re-engineering of transcriptional networks regulating secondary cell wall formation may allow the improvement of plant biomass in widely grown plantation crops such as Eucalyptus. However, there is currently a scarcity of freely available standardized biological parts (e.g., Phytobricks) compatible with Type IIS assembly approaches from forest trees, and there is a need to accelerate transcriptional network inference in nonmodel biomass crops. Here we describe the design and synthesis of a versatile three-panel biological parts collection of 221 secondary cell wall-related Eucalyptus grandis transcription factor coding sequences and 65 promoters that are compatible with GATEWAY, Golden Gate, MoClo, and GoldenBraid DNA assembly methods and generally conform to accepted Phytobrick syntaxes. This freely available resource is intended to accelerate synthetic biology applications in multiple plant biomass crops and enable reconstruction of secondary cell wall transcriptional networks using high-throughput assays such as DNA affinity purification sequencing (DAP-seq) and enhanced yeast one-hybrid (eY1H) screening.

The development of lysigenous aerenchyma starts with cell expansion and degradation of pectin from the middle lamella, leading to cell wall modification, and culminating with cell separation. Here we report that nutritional starvation of sugarcane induced gene expression along sections of the first 5 cm of the root and between treatments. We selected two candidate genes: a RAV transcription factor, from the ethylene response factors superfamily, and an endopolygalacturonase (EPG), a glycosyl hydrolase related to homogalacturonan hydrolysis from the middle lamella. epg1 and rav1 transcriptional patterns suggest they are essential genes at the initial steps of pectin degradation during aerenchyma development in sugarcane. Due to the high complexity of the sugarcane genome, rav1 and epg1 were sequenced from 17 bacterial artificial chromosome clones containing hom(e)ologous genomic regions, and the sequences were compared with those of Sorghum bicolor. We used one hom(e)olog sequence from each gene for transactivation assays in tobacco. rav1 was shown to bind to the epg1 promoter, repressing β-glucuronidase activity. RAV repression upon epg1 transcription is the first reported link between ethylene regulation and pectin hydrolysis during aerenchyma formation. Our findings may help to elucidate cell wall degradation in sugarcane and therefore contribute to second-generation bioethanol production.

Oaks are an important part of our natural and cultural heritage. Not only are they ubiquitous in our most common landscapes 1 but they have also supplied human societies with invaluable services, including food and shelter, since prehistoric times 2 . With 450 species spread throughout Asia, Europe and America 3 , oaks constitute a critical global renewable resource. The longevity of oaks (several hundred years) probably underlies their emblematic cultural and historical importance. Such long-lived sessile organisms must persist in the face of a wide range of abiotic and biotic threats over their lifespans. We investigated the genomic features associated with such a long lifespan by sequencing, assembling and annotating the oak genome. We then used the growing number of whole-genome sequences for plants (including tree and herbaceous species) to investigate the parallel evolution of genomic characteristics potentially underpinning tree longevity. A further consequence of the long lifespan of trees is their accumulation of somatic mutations during mitotic divisions of stem cells present in the shoot apical meristems. Empirical 4 and modelling 5 approaches have shown that intra-organismal genetic heterogeneity can be selected for 6 and provides direct fitness benefits in the arms race with short-lived pests and pathogens through a patchwork of intra-organismal phenotypes 7 . However, there is no clear proof that large-statured trees consist of a genetic mosaic of clonally distinct cell lineages within and between branches. Through this case study of oak, we demonstrate the accumulation and transmission of somatic mutations and the expansion of disease-resistance gene families in trees.