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Detailed knowledge of the tissue specificity of gene expression is of central importance not only for our understanding of developmental processes during wood formation, but also is a prerequisite for the deliberate manipulation of xylogenesis candidate genes. Today, much of our knowledge about specific gene expression is based on annual model plants in part because perennial tree systems are often seen as too cumbersome for detailed promoter studies. Here we address this issue in a novel way with the investigation of a number of well characterised cellulose synthase (CesA) genes in the stems of perennial tree species using the in vivo Induced Somatic Sector Analysis (ISSA) system. Three primary and four secondary cell wall associated CesA gene promoters from Eucalyptus grandis and Arabidopsis thaliana were previously isolated and cloned in front of the GUS reporter gene then, using Agrobacterium-mediated transformation, transformed into cambial initials in stems of eucalypt and poplar plants. A CAMV35S promoter::GUS vector was also used as a control. After a period of growth, stems were harvested and GUS-staining patterns were analysed and interpreted. Results from this work show that in eucalypts the staining patterns are consistent with observations in other species whereas in poplar conflicting results were observed. These findings and the general utility of ISSA for these studies are discussed.

BackgroundEucalyptus species, hybrids and clones are attacked byvarious fungal and bacterial pathogens and pests duringtheir life-time. Global climate changes are predicted tocreate favourable environments for such pathogens andpests and increase incidence of host jumping from othercrops, resulting in increased losses to the forestry indus-try [1]. The use of tolerant or resistant plant varieties aspart of an integrated disease management strategy isrecognised as a desirable means to curb disease inci-dence. Vertical resistanc e mediated by resistance ( R )genes, may be easily overcome by a pathogen and isthus not adequate on plantation species such as Euca-lyptus , which would be exposed to various pathogensduring its life-time. Broad spectrum resistance on theother hand, would be desirable to provide resistanceagainst multiple challenges [2].The aim of this study is to investigate mechanismsinvolved in host resistance with an emphasis on broad-spectrum resistance. The availability of the completegenome sequence of Eucalyptus grandis (http://www.eucagen.org) and the transcriptome sequence of aE. grandis X E. urophylla (GU) hybrid [3] has providedresources to investigate defense responses in the naturalhost. When a pathogen attacks a plant, the plantlaunches a sophisticated defense response involving phy-tohormones such as salicylic acid (SA), methyl jasmo-nate (MeJA) and ethylene (ET). These responses arefinely tuned and tailored to the invader [4]. Down-stream of the signalling cascade is the production ofpathogenesis related ( PR ) genes and antimicrobial geneswhich serve to limit the pathogen and afford protection.PR genes, such as PR-1 and PR-5, are known markers ofthe salicylic acid defense pathway, while PR-3 , PR-4 andthe lipoxygenase ( LOX ) genes are known markers of theMeJA and ET signalling pathways. The discovery of PRgenes in Eucalyptus is desirable as these genes have pre-viously been shown to afford broad spectrum resistancein other crops. We present our progress in exploitingthe Eucalyptus genomic and transcriptomic data for thediscovery of tree defense genes and explore the applica-tion thereof in determining which pathways areactivated in response to various pathogens.Materials and methods

Background The International Year of Forests 2011 [http://www. un.org/en/events/iyof2011/] will be a milestone for forest tree genomics. The draft genome sequence of Eucalyptus grandis was released in January 2011 in the USA (Phytozome [http://www.phytozome.net]) and in Belgium (BOGAS, [http://bioinformatics.psb.ugent.be/webtools/bogas/]). The genome sequencing was funded by the US Department of Energy (DOE) and performed at the DOE Joint Genome Institute (JGI) in collaboration with members of the Eucalyptus Genome Network (EUCAGEN, [http://www.eucagen.org]) who contributed genetic materials, linkage maps, EST resources and bioinformatics support. The E. grandis genome together with that of Populus trichocarpa[1]and other woody plant genomes recently completed (e.g. Vitis, Cacao, Prunus, Citrus and Malus)will provide excellent opportunities for comparative studies of the unique biology of woody plants. Eucalypts are currently the most widely grown hardwood fibre crop in the world and eucalypt breeding programs will benefit greatly from the new genomic resources. The reference genome sequence of Eucalyptus, a foundation tree genus in Australia comprising more than 70% of the native forest estate, will also offer important benefits for ecological and evolutionary biology studies. We report the sequencing, assembly and annotation of the E. grandis genome. Genome sequencing and assembly Whole-genome (8X) shotgun sequencing was performed for a partially inbred (S1), 17-year-old tree of E. grandis (est. genome size 640 Mbp, n = 11), BRASUZ1 (Suzano, Brazil). A total of 7.7 million Sanger reads (5.4 Gbp) were produced from plasmid, fosmid and BAC libraries. An inbred genotype was selected to circumvent perceived problems with the assembly of a highly heterozygous eucalypt genome. However, microsatellite genotyping showed that BRASUZ1 was much less homozygous than expected, with large parts of the genome remaining heterozygous presumably due to viability selection. This finding was confirmed during the assembly of the S1 genome approximately 25% of the assembly occurred in two haplotypes of 3-4X coverage, while the remainder of the genome assembled into a single haplotype of 6-7X coverage. Linkage maps with over 2400 DArT and microsatellite markers were subsequently used as a framework for the assembly of 11 large chromosome scaffolds. The chromosome scaffolds contained 88% (605 Mbp) of the draft assembly, with the remainder of the assembly sequence (85 Mbp) in 4941 smaller scaffolds. Based on similarity searches with 1.6 million ESTs from BRASUZ1, it was estimated that 96% of expressed gene loci were included in the 11 chromosome assemblies.

Background Eucalyptus species and their hybrids encompass approximately 40% of forestry plantation area in South Africa and contribute significantly to the paper pulp industry due to their favourable wood fibre properties. Eucalypt plantation trees are affected by numerous pathogens during their lifetime, some of which can cause severe losses such as Phytophtora spp and Chrysoporthe spp. Plant defence mechanisms against pathogens is currently better understood in the model plant Arabidopsis thaliana where it has been shown that the salicylic acid (SA) and jasmonic acid (JA) signalling pathways enhance resistance against biotrophic and necrotrophic pathogens, respectively [1] . This process involves the up-regulation of specific defence genes which are considered to be marker (diagnostic) genes for the two signalling pathways [2,3].

Background Despite its importance as a widely-planted crop tree, eucalypt species and hybrids are relatively difficult to micropropagate, culture and genetically transform in vitro. Compared to other plant species, few non-commercial laboratories are proficient at Eucalyptus tissue culture and transformation. We have undertaken to establish and transform several eucalypt clones in the laboratory. Our main aims include the identification of clones amenable to culturing and transformation, and the development of robust and transferable micropropagation, organogenesis and transformation protocols to enable routine production of transgenic eucalypts for public sector research. Efficient transformation protocols are essential to take full value of the eucalypt genome for functional genomics, ecophysiology, and biotechnology.

Background Transcription factors (TFs) play important roles in the regulation of secondary cell wall (SCW) biosynthesis in herbaceous and woody plants. In Arabidopsis, the onset of SCW deposition is initiated by a nexus of NAC, MYB, homeodomain and several other families of TFs, which function in a transcriptional network regulating SCW biosynthetic genes. NAC family members SND1/ NST1 and VND6/VND7 have been identified as functionally redundant master regulators of SCW formation in fibres and vessels, respectively [1,2]. Arabidopsis plants overexpressing SND2, an indirect target of fibre master regulator SND1, exhibited increased SCW thickness in inflorescence stem fibres, whilst dominant repression lines exhibited a decrease in fibre SCW thickness associated with a reduction in glucose and xylose cell wall sugar content [3]. The ability of SND2 to transactivate the CesA8 promoter [3] suggested that SND2 may regulate cellulose biosynthetic genes during fibre SCW formation. The evaluation of this hypothesis necessitates the identification of all downstream genes potentially regulated by SND2, and the analysis of SCW chemistry and morphology in overexpression lines. The aim of this ongoing study is to further elucidate the role of SND2 in fibre SCW formation through microarray analysis of overexpression lines and by independent confirmation of the effect of SND2 overexpression on fibre SCW chemistry and morphology in Arabidopsis plants.

Background Genetic linkage maps have been essential tools to examine the inheritance of qualitative and quantitative traits, to carry out comparative mapping and to provide markers for molecular breeding applications. Linkage maps for species of Eucalyptus have been reported for several pedigrees using different molecular marker technologies [1]. However improved marker density, throughput and transferability across species are necessary to increase resolution of current maps for a variety of genomic applications. We report the development of a high density linkage map for Eucalyptus based on microsatellites and DArT (Diversity Arrays Technology) markers generated by a standardized genotyping microarray [2]. DNA probes that constitute the DArT microarray were sequenced and positioned on the reference Eucalyptus genome providing information about their sequence content, their distribution relative to annotated genes as well as the relationship between physical and recombination distance in the Eucalyptus genome.

Background The hemicellulose xylan constitutes the major non-cellulosic component of plant secondary cell walls. It has been shown that xylan adsorbs to cellulose fibres and also covalently binds a carbon moiety of lignin [1,2]. Eucalyptus is an important hardwood tree genus used in the pulp and paper industry and has potential as biofuel feedstock. Xylan removal is expensive and uses environmentally harsh chemical treatments [3]. Previous studies have shown that endo-(1-4)-b-xylanase enzymes belonging to glycoside hydrolase (GH) family 10 internally attacks the xylan backbone resulting in shorter xylo-saccharide chains [4]. The recently sequenced Eucalyptusgrandis genome (DOE-JGI, http://www.phytozome.net) provides a unique opportunity to analyze the native endo-(1-4)-beta-xylanase proteins involved in xylan modification in eucalypt fibre cell walls. Detailed knowledge of endogenous xylanolytic enzymes from Eucalyptus could facilitate the development of strategies to enhance the processing of woody biomass for cellulose and biofuel production. The aims of this study are to identify xylem secondary cell wall-related endo-(1-4)-bxylanase genes in the E. grandis genome and to perform a comparative analysis of the Eucalyptus xylanasepeptide sequences with those of previously studied Arabidopsis orthologs to provide a framework for assigning function to the Eucalyptus enzymes. Results Analysis of the E. grandis genome sequence on Phytozome v7.0 (http://www.phytozome.net) for putative endo-(1-4)-b-xylanase genes resulted in the identification of 18 putative GH10 family members. The expression profile of each family member was assessed (via mRNA-Seq analysis, http://eucspresso.bi.up.ac.za/) to identify members with putative roles in xylem secondary cell wall metabolism. Egrandis_v1_0.001952m (designated EgrXYN1) showed the highest xylem to phloem and xylem to leaf expression ratios of the expressed E. grandis GH10 genes [5]. BLAST analysis (

Background The fast growth and good wood properties of Eucalyptus tree species and hybrids make them excellent renewable sources of fiber for pulp and paper production, and woody biomass for bioenergy production. Our research is aimed at understanding the genetic regulation of wood formation in eucalypts, with a focus on transcriptomes, regulatory sequences and gene families involved in secondary cell wall biosynthesis.

BackgroundAgrobacterium mediated gene transfer technology offersthe potential to introduce novel, high-value traits intoselected, elite tree genotypes. Significant progress hasbeen made in the regeneration and genetic transforma-tion of Eucalyptus trees, but transformation efficiency hasgenerally been low for eucalypts [1], especially for eliteclonal genotypes. As a preliminary step towards theregeneration of explants derived from clonal material andthe production of transgenic plants from commerciallyimportant Eucalyptus genotypes, there is a need to iden-tify genotypes exhibiting high regeneration capacity. Inthis ongoing study, we are comparing eightEucalyptusgenotypes for callus induction and shoot regenerationpotential. Browning of callus tissue and surrounding cul-ture media is a common obstacle limiting regeneration ofshoots in eucalypts. Hence, a second aim is to minimizethe oxidation of phenolic compounds released from thewounded tissue and improve shoot regeneration rates.MethodsShoot buds collected from potted ramets of eight Euca-lyptus clones (Sappi and Mondi, South Africa) wereestablished underin vitro conditionson MS [2] basalmedium containing BAP (Benzyl adenine purine). Theestablished clones consisted of four E. grandis (G1, G2,G3 and G4), oneE. grandis x E. nitens (GN1), two E.grandis x E. camaldulensis (GC1 and GC2) and one E.grandis x E. urophylla (GU1) genotype. Leaf explantswere excised from in vitro shoot cultures and culturedon MS basal medium containing BAP, NAA(Naphthalene acetic acid) and TDZ (Thidiazuron).Browning and necrosis of the callus as well as surround-ing culture media was observed in all cultures and PVP(polyvinylpyrrolidone) was therefore added to the mediato reduce oxidation.A standardised regeneration protocol was used for theoptimization of Agrobacteriummediated genetic trans-formation of selected Eucalyptus clones. The Agrobac-terium tumefaciens strain AGL1 containing pMDC162with the uidA gene (b-glucuronidase; GUS) andhpt(hygromycin phosphotransferase) gene was used fortransformation. To minimize extensive exudation ofphenolic compounds from the wounded explants, theleaves were subjected to different wounding methods.Intact leaves were excised fromin vitro shoot culturesand preconditioned for 24 hrs on callus induction med-ium followed by wounding with a surgical blade prior toAgrobacterium infection. The leaves were wounded intwo different ways: (a) removing the edges of the leaf,making 8-10 small wounds and cutting the leaf transver-sally into two equal halves and (b) making 5-6 smallwounds in the intact leaf without damaging the rest ofthe leaf. Alternatively, the leaves were wounded prior to24 h preconditioning using the first wounding method(a). The explants were immersed in a bacterial solution(A

Background Plants are able to incite a type of broad spectrum resistance against pathogens upon pre-treatment with biological or chemical inducers. Systemic Acquired Resistance (SAR) and Induced Systemic Resistance (ISR) are two types of induced resistance which lead to the accumulation of specific pathogenesis-related (PR) proteins. Non-pathogenic rhizobacteria are inducing agents for ISR and increased levels of ethylene (ET) and jasmonate (JA) are associated with this pathway, whereas SAR is associated with an increase in salicylic acid (SA) levels [1]. Pinus patula and P. radiata are commercially planted conifer species in South Africa, but are both highly susceptible to the causal agent of pitch canker, Fusarium circinatum. Annually, the forestry sector suffers substantial economic losses due to this disease which affects 20-30% of the planting stock. Bonello et al. (2001) showed that repeated mechanical inoculation of P. radiata with F. circinatum activated induced resistance, enhancing the protection of the tree against subsequent pathogen challenge [2]. Detailed knowledge of the molecular mechanisms underlying induced resistance may be useful to develop strategies to control diseases of pine trees. This study aimed to compare the efficiency of ten biological and chemical inducers in inciting resistance against F. circinatum. Additionally the molecular basis of this induced resistance was investigated by analyzing the response of selected putative defence response genes.

Background E. grandis is used extensively for the production of pulp and paper due to its rapid growth, good form and ease of vegetative propagation. E. urophylla exhibits tolerance to fungal diseases that limit the growth of E. grandis in tropical and subtropical regions. Interspecific hybrids of these two species are, therefore, commonly used to produce fast-growing, disease tolerant hybrids for clonal eucalypt plantations in tropical and subtropical regions (e.g., South Africa, Congo and Brazil). These hybrids often exhibit superior growth and wood quality compared to the pure species, but the underlying genetic basis of the observed hybrid superiority remains unclear. Phenotypic variation observed in interspecific mapping populations has been used to identify QTLs in several genetic linkage studies in Eucalyptus. However, QTL intervals have generally been wide (20 to 30 cM) and may include several hundred genes. To bridge the gap between fine mapping and QTL validation studies, the expression levels of genes and metabolites in individuals from the segregating population can be treated as quantitative traits and used for eQTL and mQTL mapping, respectively. Co-localization of wood property, expression and metabolite QTLs will facilitate the identification of positional candidate genes and other components of regulatory networks underlying phenotypic variation. Methods To identify genetic factors controlling growth and wood property traits in Eucalyptus, an F2 pseudo-backcross mapping family derived from a cross between an F1 hybrid (GUSAP1, E. grandisxE. urophylla, Sappi Forest Research) and an E. urophylla parent (USAP1), consisting of 555 individuals, was used for genetic linkage map construction using microsatellite and DArT markers. Phenotypic trait assessment included physical measurements of tree diameter and wood density performed on 319 three-year-old individuals. Klason (acid-soluble & -insoluble) lignin and cell wall sugar content were determined for a selection of 100 backcross progeny and used for near-infrared analysis (NIRA) calibration. NIRA predictions for glucose, xylose, arabinose, cellulose and total lignin content, as well as pulp yield were made for all 315 individuals. Total lignin and S:G ratios were also separately measured for the 315 individuals. Immature xylem tissues, collected from 192 backcross progeny, were used for metabolite profiling (ORNL, Oak Ridge, TN) and Illumina mRNA-Seq (PE50, BGI Americas) quantification of transcript levels.