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Although eucalypts are the most planted hardwood trees worldwide, the majority of them are frost sensitive. The recent creation of frost-tolerant hybrids such as Eucalyptus gundal plants (E. gunnii × E. dalrympleana hybrids), now enables the development of industrial plantations in northern countries. Our objective was to evaluate the impact of cold on the wood structure and composition of these hybrids, and on the biosynthetic and regulatory processes controlling their secondary cell-wall (SCW) formation. We used an integrated approach combining histology, biochemical characterization and transcriptomic profiling as well as gene co-expression analyses to investigate xylem tissues from Eucalyptus hybrids exposed to cold conditions. Chilling temperatures triggered the deposition of thicker and more lignified xylem cell walls as well as regulation at the transcriptional level of SCW genes. Most genes involved in lignin biosynthesis, except those specifically dedicated to syringyl unit biosynthesis, were up-regulated. The construction of a co-expression network enabled the identification of both known and potential new SCW transcription factors, induced by cold stress. These regulators at the crossroads between cold signalling and SCW formation are promising candidates for functional studies since they may contribute to the tolerance of E. gunnii × E. dalrympleana hybrids to cold.

Pines, eucalypts and cork oak are ecologically and economically important species for wood, paper and cork industries. The identification of candidate genes and understanding of transcriptional regulation mechanisms related to wood and cork formation are considered as very important in breeding and conservation programmes for these species. The assessment of high-quality RNAs from different species, genotypes and tissues is required for transcriptomics studies. Most of the available protocols of total RNA isolation are suitable for needle or leaf tissue, including that reported by Chang et al. Here, an improved protocol for total RNA extraction from secondary tissues based on the protocol by Chang et al. is presented. The total RNA samples isolated from secondary tissues of Pinus sp., Eucalyptus globulus Labill. and Quercus suber L. showed good yield and quality. In the extracted RNA, the small RNA fraction was preserved, being suitable for different downstream approaches, such as expression analysis of coding and non-coding genes. It is considered that this protocol could be useful to other researchers who are working in the transcriptomics field of woody species.

The R2R3-MYB family, one of the largest transcription factor families in higher plants, controls a wide variety of plant-specific processes including, notably, phenylpropanoid metabolism and secondary cell wall formation. We performed a genome-wide analysis of this superfamily in Eucalyptus, one of the most planted hardwood trees world-wide. A total of 141 predicted R2R3-MYB sequences identified in the Eucalyptus grandis genome sequence were subjected to comparative phylogenetic analyses with Arabidopsis thaliana, Oryza sativa, Populus trichocarpa and Vitis vinifera. We analysed features such as gene structure, conserved motifs and genome location. Transcript abundance patterns were assessed by RNAseq and validated by high-throughput quantitative PCR. We found some R2R3-MYB subgroups with expanded membership in E. grandis, V. vinifera and P. trichocarpa, and others preferentially found in woody species, suggesting diversification of specific functions in woody plants. By contrast, subgroups containing key genes regulating lignin biosynthesis and secondary cell wall formation are more conserved across all of the species analysed. In Eucalyptus, R2R3-MYB tandem gene duplications seem to disproportionately affect woody-preferential and woody-expanded subgroups. Interestingly, some of the genes belonging to woody-preferential subgroups show higher expression in the cambial region, suggesting a putative role in the regulation of secondary growth.

The Portuguese Landrace of Eucalyptus globulus is of unknown origin, with the earliest plantings of this tree species dating back to the early 19th century. In Portugal it is currently a major seed source for plantations and is also used in breeding programs. Eucalyptus globulus is native to south-eastern Australia. The substantial genetic differentiation of chloroplast and nuclear DNA markers between different native geographic races of this species allowed us to uncover the Australian origins of the Portuguese Landrace and to study its genetic diversity. To achieve this, we sequenced a highly polymorphic region of chloroplast DNA from 47 Portuguese Landrace individuals, and genotyped 34 of these using seven nuclear microsatellites. We compared these individuals to those in a database comprising chloroplast DNA sequence profiles from 292 native trees and seven nuclear microsatellites from 372 native trees. The majority of the Portuguese Landrace samples had closest affinities, in both marker systems, to native trees from south-eastern Tasmania, but some had affinities to trees from south-eastern Victoria. The discrepancies in the affinities indicated by chloroplast versus nuclear DNA markers could be explained by inter-race hybridisation after introduction. The genetic diversity in the Portuguese Landrace was less than that found in native E. globulus at the species level, but was similar to the average diversity found in native races of the species. This study demonstrates the power of using independent marker systems to identify the origins and diversity of domesticated populations, by comparison with variation in native stands.

Lignin, a major component of secondary cell walls, hinders the optimal processing of wood for industrial uses. The recent availability of the Eucalyptus grandis genome sequence allows comprehensive analysis of the genes encoding the 11 protein families specific to the lignin branch of the phenylpropanoid pathway and identification of those mainly involved in xylem developmental lignification. We performed genome-wide identification of putative members of the lignin gene families, followed by comparative phylogenetic studies focusing on bona fide clades inferred from genes functionally characterized in other species. RNA-seq and microfluid real-time quantitative PCR (RT-qPCR) expression data were used to investigate the developmental and environmental responsive expression patterns of the genes. The phylogenetic analysis revealed that 38 E. grandis genes are located in bona fide lignification clades. Four multigene families (shikimate O-hydroxycinnamoyltransferase (HCT), p-coumarate 3-hydroxylase (C3H), caffeate/5-hydroxyferulate O-methyltransferase (COMT) and phenylalanine ammonia-lyase (PAL)) are expanded by tandem gene duplication compared with other plant species. Seventeen of the 38 genes exhibited strong, preferential expression in highly lignified tissues, probably representing the E. grandis core lignification toolbox. The identification of major genes involved in lignin biosynthesis in E. grandis, the most widely planted hardwood crop world-wide, provides the foundation for the development of biotechnology approaches to develop tree varieties with enhanced processing qualities.

International audience; Eucalypts are the world's most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

MicroRNAs are endogenous molecules that act by silencing targeted messenger RNAs, and which have an important regulatory role in many physiological processes in both plants and animals. Here, we propose a pipeline that makes use of CRAVELA, a single-genome microRNA finding tool originally developed for microRNA discovery in animals, and an NGS data analysis algorithm that provides a novel scoring function to evaluate the expression profile of candidates, taking advantage of the expected relative abundance of RNA fragments originating from the mature sequence, compared to other portions of the microRNA precursor. This approach was tested in Eucalyptus spp. for which, despite their economic importance, no microRNAs have been documented. The outcome of our approach was a short list of candidates, including both conserved and non-conserved sequences. Experimental validation showed amplification in 6 out of 8 candidates chosen from the best-scoring non-conserved sequences.

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.

Background RAIZ is a Portuguese private non-profit research institute owned by the Pulp & Paper Portucel Soporcel Group (http://www.raiz-iifp.pt). RAIZ E. globulus genetic improvement program is managed in order to generate trees with increased economic value, through gains in forest productivity and wood properties. Molecular markers have been used for clonal identification and genetic diversity management in the program since 1990. Moreover, RAIZ has been engaged in a longer term genomics project aiming to identify quantitative trait loci (QTL) for wood properties. The ability to detect QTL depends on sample size, genetic background, environment and genetic interactions. Most importantly, the ability to use detected QTL depends on their adequate map location and the identification of the molecular variation behind them. This in turn is determined by linkage map quality, the choice of phenotypes and the precision of phenotyping. There are many reports in the literature on QTL detection for economically important traits in Eucalyptus, but very few present data on QTL verification (at the statistical and/or biological levels). We illustrate the importance of this issue for pulp yield related traits by comparing available results from QTL mapping studies in the literature with those obtained from a QTL detection and verification experiment pursued by RAIZ. Methods

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.

International audience; Tension wood formed in response to gravitational force is a striking example of the plasticity of angiosperm wood. In this study our goal was to characterize the early changes in gene expression during tension wood formation in Eucalyptus.Using cDNA array technology, transcript profiling of 231 genes preferentially expressed in differentiating Eucalyptus xylem was followed from 6 h to 1 wk of a tension time course of artificially bent Eucalyptus trees.196 genes were differentially regulated between control and bent trees, some exhibiting distinctive expression patterns related to changes in secondary cell wall structure and composition. For instance, expression of a cellulose synthase gene was well correlated with the appearance of the G-layers. Cluster correlation analysis revealed differential regulation of lignin biosynthetic genes and may also be used to help infer the function of unknown gene products.Eucalyptus wood transcriptome analysis during tension wood formation not only provided new clues into the transcriptional regulatory network of genes preferentially expressed in xylem, but also highlighted candidate genes responsible for the genetic and environmentally induced variation of wood quality traits.