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Summary Eucalyptus is among the most widely planted taxa of forest trees worldwide. However, its spread as an exotic or genetically engineered form can create ecological and social problems. To mitigate gene flow via pollen and seeds, we mutated the Eucalyptus orthologue of LEAFY (LFY) by transforming a Eucalyptus grandis × urophylla wild‐type hybrid and two Flowering Locus T (FT) overexpressing (and flowering) lines with CRISPR Cas9 targeting its LFY orthologue, ELFY. We achieved high rates of elfy biallelic knockouts, often approaching 100% of transgene insertion events. Frameshift mutations and deletions removing conserved amino acids caused strong floral alterations, including indeterminacy in floral development and an absence of male and female gametes. These mutants were otherwise visibly normal and did not differ statistically from transgenic controls in juvenile vegetative growth rate or leaf morphology in greenhouse trials. Genes upstream or near to ELFY in the floral development pathway were overexpressed, whereas floral organ identity genes downstream of ELFY were severely depressed. We conclude that disruption of ELFY function appears to be a useful tool for sexual containment, without causing statistically significant or large adverse effects on juvenile vegetative growth or leaf morphology.

Gall-inducing insects and their hosts present some of the most intricate plant-herbivore interactions. Oviposition on the host is often the first cue of future herbivory and events at this early time point can affect later life stages. Many gallers are devastating plant pests, yet little information regarding the plant-insect molecular interplay exists, particularly following egg deposition. We studied the physiological and transcriptional responses of Eucalyptus following oviposition by the gall-inducing wasp, Leptocybe invasa, to explore potential mechanisms governing defence responses and gall development. RNA sequencing and microscopy were used to explore a susceptible Eucalyptus-L. invasa interaction. Infested and control material was compared over time (1-3, 7 and 90 days post oviposition) to examine the transcriptional and morphological changes. Oviposition induces accumulation of reactive oxygen species and phenolics which is reflected in the transcriptome analysis. Gene expression supports phytohormones and 10 transcription factor subfamilies as key regulators. The egg and oviposition fluid stimulate cell division resulting in gall development. Eucalyptus responses to oviposition are apparent within 24 hr. Putative defences include the oxidative burst and barrier reinforcement. However, egg and oviposition fluid stimuli may redirect these responses towards gall development.

The galling insect, Leptocybe invasa, causes significant losses in plantations of various Eucalyptus species and hybrids, threatening its economic viability. We applied a genome-wide association study (GWAS) to identify single-nucleotide polymorphism (SNP) markers associated with resistance to L. invasa. A total of 563 insect-challenged Eucalyptus grandis trees, from 61 half-sib families, were genotyped using the EUChip60K SNP chip, and we identified 15,445 informative SNP markers in the test population. Multi-locus mixed-model (MLMM) analysis identified 35 SNP markers putatively associated with resistance to L. invasa based on four discreet classes of insect damage scores: (0) not infested, (1) infested showing evidence of oviposition but no gall development, (2) infested with galls on leaves, midribs or petioles and (3) stunting and lethal gall formation. MLMM analysis identified three associated genomic regions on chromosomes 3, 7 and 8 jointly explaining 17.6% of the total phenotypic variation. SNP analysis of a validation population of 494 E. grandis trees confirmed seven SNP markers that were also detected in the initial association analysis. Based on transcriptome profiles of resistant and susceptible genotypes from an independent experiment, we identified several putative candidate genes in associated genomic loci including Nucleotide-binding ARC- domain (NB-ARC) and toll-interleukin-1-receptor-Nucleotide binding signal- Leucine rich repeat (TIR-NBS-LRR) genes. Our results suggest that Leptocybe resistance in E. grandis may be influenced by a few large-effect loci in combination with minor effect loci segregating in our test and validation populations.

Accessible chromatin changes dynamically during development and harbours functional regulatory regions which are poorly understood in the context of wood development. We explored the importance of accessible chromatin in Eucalyptus grandis in immature xylem generally, and MYB transcription factor-mediated transcriptional programmes specifically. We identified biologically reproducible DNase I Hypersensitive Sites (DHSs) and assessed their functional significance in immature xylem through their associations with gene expression, epigenomic data and DNA sequence conservation. We identified in vitro DNA binding sites for six secondary cell wall-associated Eucalyptus MYB (EgrMYB) transcription factors using DAP-seq, reconstructed protein-DNA networks of predicted targets based on binding sites within or outside DHSs and assessed biological enrichment of these networks with published datasets. 25 319 identified immature xylem DHSs were associated with increased transcription and significantly enriched for various epigenetic signatures (H3K4me3, H3K27me3, RNA pol II), conserved noncoding sequences and depleted single nucleotide variants. Predicted networks built from EgrMYB binding sites located in accessible chromatin were significantly enriched for systems biology datasets relevant to wood formation, whereas those occurring in inaccessible chromatin were not. Our study demonstrates that DHSs in E. grandis immature xylem, most of which are intergenic, are of functional significance to gene regulation in this tissue.

We performed gene and genome targeted SNP discovery towards the development of a genome-wide, multispecies genotyping array for tropical pines. Pooled RNA-seq data from shoots of seedlings from five tropical pine species was used to identify transcript-based SNPs resulting in 1.3 million candidate Affymetrix SNP probe sets. In addition, we used a custom 40 K probe set to perform capture-seq in pooled DNA from 81 provenances representing the natural ranges of six tropical pine species in Mexico and Central America resulting in 563 K candidate SNP probe sets. Altogether, 300 K RNA-seq (72%) and 120 K capture-seq (28%) derived SNP probe sets were tiled on a 420 K screening array that was used to genotype 576 trees representing the 81 provenances and commercial breeding material. Based on the screening array results, 50 K SNPs were selected for commercial SNP array production including 20 K polymorphic SNPs for P. patula, P. tecunumanii, P. oocarpa and P. caribaea, 15 K for P. greggii and P. maximinoi, 13 K for P. elliottii and 8K for P. pseudostrobus. We included 9.7 K ancestry informative SNPs that will be valuable for species and hybrid discrimination. Of the 50 K SNP markers, 25% are polymorphic in only one species, while 75% are shared by two or more species. The Pitro50K SNP chip will be useful for population genomics and molecular breeding in this group of pine species that, together with their hybrids, represent the majority of fast-growing tropical and subtropical pine plantations globally.

As the second most consumed non-alcoholic beverage, the tea plant (Camellia sinensis) has high economic value. Tea improvement efforts that largely target economic traits such as yield have traditionally relied on conventional breeding approaches. The tea plant’s perennial nature and its long generation time make conventional approaches time-consuming and labour-intensive. Biotechnology provides a complementary tool for accelerating tea improvement programmes through marker-assisted selection (MAS). Quantitative trait loci (QTLs) identified on linkage maps are an essential prerequisite to the implementation of MAS. QTL analysis was performed on yield data over 3 years (2010–2012) across two sites (Timbilil and Kangaita, in Kenya), based on two parental framework linkage maps arising from a population of 261 F1 progeny, derived from a reciprocal cross between GW Ejulu and TRFK 303/577. The maps contain 15 linkage groups each, this corresponds to the haploid chromosome number of tea (2n=2x=30). The total length of the parental maps was 1028.1 cM for GW Ejulu and 1026.6 cM for TRFK 303/577 with an average locus spacing of 5.5 cM and 5.4 cM, respectively. A total of 13 QTLs were identified over the three measurement years. The 13 QTLs had LOD values ranging from 1.98 to 7.24 and explained 3.4% to 12% of the phenotypic variation. The two sites had seven mutually detected QTLs.

Genomic selection (GS) can substantially reduce breeding cycle times in forest trees compared to traditional breeding cycles. Practical implementation of GS in tree breeding requires an assessment of significant drivers of genetic gains over time, which may differ among species and breeding objectives. We present results of a GS study of growth and wood quality traits in an operational Eucalyptus grandis breeding program in South Africa. The training population consisted of 1575 full and half-sib individuals, genotyped with the Eucalyptus (EUChip60K) SNP chip resulting in 15,040 informative SNP markers. The accuracy of the GS models ranged from 0.47 (diameter) to 0.67 (fibre width). We compared a 4-year GS breeding cycle equivalent to half of a traditional 8-year E. grandis breeding cycle and obtained GS efficiencies ranging from 1.20 (wood density) to 1.62 (fibre length). Simulated over 17 years, the ratio of the accumulated genetic gains between three GS cycles and two traditional breeding cycles ranged from 1.53 (diameter) to 3.35 (wood density). To realise these genetic gains per unit time in E. grandis breeding, we show that significant adjustments have to be made to integrate GS into operational breeding steps.

Secondary cell wall (SCW) deposition in Arabidopsis is regulated among others by NAC transcription factors, where SND1 chiefly initiates xylem fibre differentiation while VND6 controls metaxylem vessel SCW development, especially programmed cell death and wall patterning. The translational relevance of Arabidopsis SCW regulation theory and the utility of characterized transcription factors as modular synthetic biology tools for improving commercial fibre crops is unclear. We investigated inter-lineage gene activation dynamics for potential fibre and vessel differentiation regulators from the widely grown hardwood Eucalyptus grandis (Myrtales). EgrNAC26, a VND6 homolog, and EgrNAC61, an SND1 homolog, were transiently expressed in Arabidopsis mesophyll protoplasts in parallel to determine early and late (i.e. 7 and 14 hours post-transfection) gene targets. Surprisingly, across the time series EgrNAC26 activated only a subset of SCW-related transcription factors and biosynthetic genes activated by EgrNAC61, specializing instead in targeting vessel-specific wall pit and programmed cell death markers. Promoters of EgrNAC26 and EgrNAC61 both induced reporter gene expression in vessels of young Arabidopsis plants, with EgrNAC61 also conferring xylem- and cork cambium-preferential expression in Populus. Our results demonstrate partial conservation, with notable exceptions, of SND1 and VND6 homologs in Eucalyptus and a first report of cork cambium expression for EgrNAC61.

Supplementary Table 1. Environmental and phenotype data (full population) for the three Eucalyptus grandis sites surveyed for Leptocybe invasa infestation. [Excel file]

Lignocellulosic biomass is a promising feedstock for the manufacture of biodegradable and renewable bioproducts. However, the complex lignocellulosic polymeric structure of woody tissue is difficult to access without extensive industrial pre-treatment. Enzyme processing of partly depolymerised biomass is an established technology, and there is evidence that high temperature (extremely thermophilic) lignocellulose degrading enzymes [carbohydrate active enzymes (CAZymes)] may enhance processing efficiency. However, wild-type thermophilic CAZymes will not necessarily be functionally optimal under industrial pre-treatment conditions. With recent advances in synthetic biology, it is now potentially possible to build CAZyme constructs from individual protein domains, tailored to the conditions of specific industrial processes. In this review, we identify a 'toolbox' of thermostable CAZyme domains from extremely thermophilic organisms and highlight recent advances in CAZyme engineering which will allow for the rational design of CAZymes tailored to specific aspects of lignocellulose digestion.

Conversion of lignocellulosic biomass to biofuels and biomaterials suffers from high production costs associated with biomass pretreatment and enzymatic hydrolysis. In-planta expression of lignocellulose-digesting enzymes is a promising approach to reduce these cost elements. However, this approach faces a number of challenges, including auto-hydrolysis of developing cell walls, plant growth and yield penalties, low expression levels and the limited stability of expressed enzymes at the high temperatures generally used for biomass processing to release fermentable sugars. To overcome these challenges we expressed codon-optimized recombinant hyperthermophilic endoglucanase (EG) and xylanase (Xyn) genes in A. thaliana. Transgenic Arabidopsis lines expressing EG and Xyn enzymes at high levels without any obvious plant growth or yield penalties were selected for further analysis. The highest enzyme activities were observed in the dry stems of transgenic lines, indicating that the enzymes were not degraded during stem senescence and storage. Biomass from transgenic lines exhibited improved saccharification efficiency relative to WT control plants. We conclude that the expression of hyperthermophilic enzymes in plants is a promising approach for combining pretreatment and enzymatic hydrolysis processes in lignocellulosic digestion. This study provides a valid foundation for further studies involving in planta co-expression of core and accessory lignocellulose-digesting enzymes.

Despite the considerable contribution of xylem development (xylogenesis) to plant biomass accumulation, its epigenetic regulation is poorly understood. Furthermore, the relative contributions of histone modifications to transcriptional regulation is not well studied in plants. We investigated the biological relevance of H3K4me3 and H3K27me3 in secondary xylem development using ChIP-seq and their association with transcript levels among other histone modifications in woody and herbaceous models. In developing secondary xylem of the woody model Eucalyptus grandis, H3K4me3 and H3K27me3 genomic spans were distinctly associated with xylogenesis-related processes, with (late) lignification pathways enriched for putative bivalent domains, but not early secondary cell wall polysaccharide deposition. H3K27me3-occupied genes, of which 753 (~31%) are novel targets, were enriched for transcriptional regulation and flower development and had significant preferential expression in roots. Linear regression models of the ChIP-seq profiles predicted ~50% of transcript abundance measured with strand-specific RNA-seq, confirmed in a parallel analysis in Arabidopsis where integration of seven additional histone modifications each contributed smaller proportions of unique information to the predictive models. This study uncovers the biological importance of histone modification antagonism and genomic span in xylogenesis and quantifies for the first time the relative correlations of histone modifications with transcript abundance in plants.

We used a systems genetics approach to elucidate the molecular mechanisms of the responses of maize to grey leaf spot (GLS) disease caused by Cercospora zeina, a threat to maize production globally. Expression analysis of earleaf samples in a subtropical maize recombinant inbred line population (CML444 × SC Malawi) subjected in the field to C. zeina infection allowed detection of 20 206 expression quantitative trait loci (eQTLs). Four trans-eQTL hotspots coincided with GLS disease QTLs mapped in the same field experiment. Co-expression network analysis identified three expression modules correlated with GLS disease scores. The module (GY-s) most highly correlated with susceptibility (r = 0.71; 179 genes) was enriched for the glyoxylate pathway, lipid metabolism, diterpenoid biosynthesis and responses to pathogen molecules such as chitin. The GY-s module was enriched for genes with trans-eQTLs in hotspots on chromosomes 9 and 10, which also coincided with phenotypic QTLs for susceptibility to GLS. This transcriptional network has significant overlap with the GLS susceptibility response of maize line B73, and may reflect pathogen manipulation for nutrient acquisition and/or unsuccessful defence responses, such as kauralexin production by the diterpenoid biosynthesis pathway. The co-expression module that correlated best with resistance (TQ-r; 1498 genes) was enriched for genes with trans-eQTLs in hotspots coinciding with GLS resistance QTLs on chromosome 9. Jasmonate responses were implicated in resistance to GLS through co-expression of COI1 and enrichment of genes with the Gene Ontology term 'cullin-RING ubiquitin ligase complex' in the TQ-r module. Consistent with this, JAZ repressor expression was highly correlated with the severity of GLS disease in the GY-s susceptibility network.

Human life intimately depends on plants for food, biomaterials, health, energy, and a sustainable environment. Various plants have been genetically improved mostly through breeding, along with limited modification via genetic engineering, yet they are still not able to meet the ever-increasing needs, in terms of both quantity and quality, resulting from the rapid increase in world population and expected standards of living. A step change that may address these challenges would be to expand the potential of plants using biosystems design approaches. This represents a shift in plant science research from relatively simple trial-and-error approaches to innovative strategies based on predictive models of biological systems. Plant biosystems design seeks to accelerate plant genetic improvement using genome editing and genetic circuit engineering or create novel plant systems through de novo synthesis of plant genomes. From this perspective, we present a comprehensive roadmap of plant biosystems design covering theories, principles, and technical methods, along with potential applications in basic and applied plant biology research. We highlight current challenges, future opportunities, and research priorities, along with a framework for international collaboration, towards rapid advancement of this emerging interdisciplinary area of research. Finally, we discuss the importance of social responsibility in utilizing plant biosystems design and suggest strategies for improving public perception, trust, and acceptance.

The genetic consequences of adaptation to changing environments can be deciphered using population genomics, which may help predict species' responses to global climate change. Towards this, we used genome-wide SNP marker analysis to determine population structure and patterns of genetic differentiation in terms of neutral and adaptive genetic variation in the natural range of Eucalyptus grandis, a widely cultivated subtropical and temperate species, serving as genomic reference for the genus. We analysed introgression patterns at subchromosomal resolution using a modified ancestry mapping approach and identified provenances with extensive interspecific introgression in response to increased aridity. Furthermore, we describe potentially adaptive genetic variation as explained by environment-associated SNP markers, which also led to the discovery of what is likely a large structural variant. Finally, we show that genes linked to these markers are enriched for biotic and abiotic stress responses.

Fusarium circinatum poses a serious threat to many pine species in both commercial and natural pine forests. Knowledge regarding the molecular basis of pine-F. circinatum host-pathogen interactions could assist efforts to produce more resistant planting stock. This study aimed to identify molecular responses underlying resistance against F. circinatum. A dual RNA-seq approach was used to investigate host and pathogen expression in F. circinatum challenged Pinus tecunumanii (resistant) and Pinus patula (susceptible), at three- and seven-days post inoculation. RNA-seq reads were mapped to combined host-pathogen references for both pine species to identify differentially expressed genes (DEGs). F. circinatum genes expressed during infection showed decreased ergosterol biosynthesis in P. tecunumanii relative to P. patula. For P. tecunumanii, enriched gene ontologies and DEGs indicated roles for auxin-, ethylene-, jasmonate- and salicylate-mediated phytohormone signalling. Correspondingly, key phytohormone signaling components were down-regulated in P. patula. Key F. circinatum ergosterol biosynthesis genes were expressed at lower levels during infection of the resistant relative to the susceptible host. This study further suggests that coordination of phytohormone signaling is required for F. circinatum resistance in P. tecunumanii, while a comparatively delayed response and impaired phytohormone signaling contributes to susceptibility in P. patula.

Tea is the world’s most consumed beverage, next to water. This makes it an economically important crop. However, the genetic relationship between the cultivars at the Tea Research Foundation of Central Africa (TRFCA) have not been classified. This study assessed 56 tea accessions from the TRFCA. The 56 accessions were split into five groups, namely, Cambod type, China type, TRFCA, Tea Research Institute (TRI), and unknown. A total of 16,382 genome-wide genetic markers were developed using a next-generation sequencing (NGS) platform at Diversity Array Technology (DArT). The current study also explored the usefulness of the DArT-seq markers for tea germplasm analysis. The genetic relationships amongst the cultivars were analysed using the neighbour-joining method and UPGMA; this was successful in clustering the different cultivars into groups of origin. Principal coordinate analysis (PCoA) showed that 33 TRFCA-derived cultivars were distributed in all four quadrants. Analysis of molecular variance (AMOVA) revealed that there was a higher proportion of genetic variation (94%) within the groups than there was amongst the groups (6%). Nei’s unbiased genetic distances amongst groups suggested that the 33 TRFCA-derived cultivars had a low genetic distance from the other four groups. This confirms the PCoA inference that the 33 TRFCA had a high genetic admixture. The genetic structure was utilised to assign three cultivars of unknown origin, to the Cambod-type group. Two cultivars were closely related to the China-type group. Our findings are a useful guide for future tea breeding programmes in Southern Africa.

Lignocellulosic biomass, encompassing cellulose, lignin and hemicellulose in plant secondary cell walls (SCWs), is the most abundant source of renewable materials on earth. Currently, fast-growing woody dicots such as Eucalyptus and Populus trees are major lignocellulosic (wood fiber) feedstocks for bioproducts such as pulp, paper, cellulose, textiles, bioplastics and other biomaterials. Processing wood for these products entails separating the biomass into its three main components as efficiently as possible without compromising yield. Glucuronoxylan (xylan), the main hemicellulose present in the SCWs of hardwood trees carries chemical modifications that are associated with SCW composition and ultrastructure, and affect the recalcitrance of woody biomass to industrial processing. In this review we highlight the importance of xylan properties for industrial wood fiber processing and how gaining a greater understanding of xylan biosynthesis, specifically xylan modification, could yield novel biotechnology approaches to reduce recalcitrance or introduce novel processing traits. Altering xylan modification patterns has recently become a focus of plant SCW studies due to early findings that altered modification patterns can yield beneficial biomass processing traits. Additionally, it has been noted that plants with altered xylan composition display metabolic differences linked to changes in precursor usage. We explore the possibility of using systems biology and systems genetics approaches to gain insight into the coordination of SCW formation with other interdependent biological processes. Acetyl-CoA, s-adenosylmethionine and nucleotide sugars are precursors needed for xylan modification, however, the pathways which produce metabolic pools during different stages of fiber cell wall formation still have to be identified and their co-regulation during SCW formation elucidated. The crucial dependence on precursor metabolism provides an opportunity to alter xylan modification patterns through metabolic engineering of one or more of these interdependent pathways. The complexity of xylan biosynthesis and modification is currently a stumbling point, but it may provide new avenues for woody biomass engineering that are not possible for other biopolymers.

In woody plants, xylogenesis is an exceptionally strong carbon sink requiring robust transcriptional control and dynamic coordination of cellular and metabolic processes directing carbon allocation and partitioning into secondary cell wall biosynthesis. As a biological process, wood formation is an excellent candidate for systems modeling due to the strong correlation patterns and interconnectedness observed for transcriptional and metabolic component traits contributing to complex phenotypes such as cell wall chemistry and ultrastructure. Genetic variation in undomesticated tree populations provides abundant perturbation of systems components, adding another dimension to plant systems biology (besides spatial and temporal variation). High-throughput analysis of molecular component traits in adult trees has provided the first insights into the systems genetics of wood, an important renewable feedstock for biomaterials and bioenergy.

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.

Fast-growing forest plantations are sustainable feedstocks of plant biomass that can serve as alternatives to fossil carbon resources for materials, chemicals, and energy. Their ability to efficiently harvest light energy and carbon from the atmosphere and sequester this into metabolic precursors for lignocellulosic biopolymers and a wide range of plant specialized metabolites make them excellent biochemical production platforms and living biorefineries. Their large sizes have facilitated multi-omics analyses and systems modeling of key biological processes such as lignin biosynthesis in trees. High-throughput 'omics' approaches have also been applied in segregating tree populations where genetic variation creates abundant genetic perturbations of system components allowing construction of systems genetics models linking genes and pathways to complex trait variation. With this information in hand, it is now possible to start using synthetic biology and genome editing techniques in a bioengineering approach based on a deeper understanding and rational design of biological parts, devices, and integrated systems. However, the complexity of the biology and interacting components will require investment in big data informatics, machine learning, and intuitive visualization to fully explore multi-dimensional patterns and identify emergent properties of biological systems. Predictive systems models could be tested rapidly through high-throughput synthetic biology approaches and multigene editing. Such a bioengineering paradigm, together with accelerated genomic breeding, will be crucial for the development of a new generation of woody biorefinery crops.

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Supplementary Material: Fig. S1 Phylogram representing a maximum likelihood analysis of RWA proteins in Arabidopsis, Eucalyptus and Populus coupled with relative and absolute percentile expression in xylem and leaf, and domain structure. Fig. S2 Phylogram representing a maximum likelihood analysis of GUX proteins in Arabidopsis, Eucalyptus and Populus coupled with relative and absolute percentile expression in xylem and leaf, and domain structure. Fig. S3 Phylogram representing a maximum likelihood analysis of DUF579/GXM proteins in Arabidopsis, Eucalyptus and Populus coupled with relative and absolute percentile expression in xylem and leaf, and domain structure. Fig. S4 Phylogram representing a maximum likelihood analysis of DUF231/TBL proteins from clade IV in Arabidopsis, Eucalyptus and Populus coupled with relative and absolute percentile expression in xylem and leaf, and domain structure. Fig. S5 Phylogram representing a maximum likelihood analysis of the full complement of DUF231/TBL proteins in Arabidopsis, Eucalyptus and Populus coupled with relative and absolute percentile expression in xylem and leaf, and domain structure. Fig. S6 F‐score threshold determined from the number of genes that each query gene included in the network by using a joint likelihood curve. Fig. S7 Co‐expression and clustering of 1136 genes obtained for genes which passed set criteria divided into five expression modules. Fig. S8 Seventy percent of genes co‐expressed with xylan modification were found to have at least one trans‐eQTL affecting its expression. Fig. S9 Identification of the seven highly enriched trans‐eQTL hotspot loci. Fig. S10 Principal component analysis of the five module eigengenes. Fig. S11 Model of genetic regulation that shapes the expression modules. Fig. S12 Numbers of genes affected by each of the seven hotspot loci and their expression module membership. Fig. S13 Nucleotide sugar interconversion is highly represented in EM5 and regulated primarily by HS_10.3 and HS_10.4. Fig. S14 Complete annotation of Fig. 3 containing genes which were not present in EMs or affected by hotspot loci. Fig. S15 Complete annotation of Fig. 5 containing genes which were not present in EMs or affected by hotspot loci. Methods S1 Network based co‐expression analysis and community detection clustering. Methods S2 Global eQTL mapping and hotspot locus detection. Notes S1 Protein sequences of GUX, DUF579, RWA and TBL proteins used for phylogenetic reconstruction.

Subcellular compartmentation of plant biosynthetic pathways in the mitochondria and plastids requires coordinated regulation of nuclear encoded genes, and the role of these genes has been largely ignored by wood researchers. In this study, we constructed a targeted systems genetics coexpression network of xylogenesis in Eucalyptus using plastid and mitochondrial carbon metabolic genes and compared the resulting clusters to the aspen xylem developmental series. The constructed network clusters reveal the organization of transcriptional modules regulating subcellular metabolic functions in plastids and mitochondria. Overlapping genes between the plastid and mitochondrial networks implicate the common transcriptional regulation of carbon metabolism during xylem secondary growth. We show that the central processes of organellar carbon metabolism are distinctly coordinated across the developmental stages of wood formation and are specifically associated with primary growth and secondary cell wall deposition. We also demonstrate that, during xylogenesis, plastid-targeted carbon metabolism is partially regulated by the central clock for carbon allocation towards primary and secondary xylem growth, and we discuss these networks in the context of previously established associations with wood-related complex traits. This study provides a new resolution into the integration and transcriptional regulation of plastid- and mitochondrial-localized carbon metabolism during xylogenesis.

Background Fusarium circinatum is a pressing threat to the cultivation of many economically important pine tree species. Efforts to develop effective disease management strategies can be aided by investigating the molecular mechanisms involved in the host-pathogen interaction between F. circinatum and pine species. Pinus tecunumanii and Pinus patula are two closely related tropical pine species that differ widely in their resistance to F. circinatum challenge, being resistant and susceptible respectively, providing the potential for a useful pathosystem to investigate the molecular responses underlying resistance to F. circinatum. However, no genomic resources are available for P. tecunumanii. Pathogenesis-related proteins are classes of proteins that play important roles in plant-microbe interactions, e.g. chitinases; proteins that break down the major structural component of fungal cell walls. Generating a reference sequence for P. tecunumanii and characterizing pathogenesis related gene families in these two pine species is an important step towards unravelling the pine-F. circinatum interaction. Results Eight reference based and 12 de novo assembled transcriptomes were produced, for juvenile shoot tissue from both species. EvidentialGene pipeline redundancy reduction, expression filtering, protein clustering and taxonomic filtering produced a 50 Mb shoot transcriptome consisting of 28,621 contigs for P. tecunumanii and a 72 Mb shoot transcriptome consisting of 52,735 contigs for P. patula. Predicted protein sequences encoded by the assembled transcriptomes were clustered with reference proteomes from 92 other species to identify pathogenesis related gene families in P. patula, P. tecunumanii and other pine species. Conclusions The P. tecunumanii transcriptome is the first gene catalogue for the species, representing an important resource for studying resistance to the pitch canker pathogen, F. circinatum. This study also constitutes, to our knowledge, the largest index of gymnosperm PR-genes to date. Electronic supplementary material The online version of this article (10.1186/s12864-018-5015-0) contains supplementary material, which is available to authorized users.

Background Land plant organellar genomes have significant impact on metabolism and adaptation, and as such, accurate assembly and annotation of plant organellar genomes is an important tool in understanding the evolutionary history and interactions between these genomes. Intracellular DNA transfer is ongoing between the nuclear and organellar genomes, and can lead to significant genomic variation between, and within, species that impacts downstream analysis of genomes and transcriptomes. Results In order to facilitate further studies of cytonuclear interactions in Eucalyptus, we report an updated annotation of the E. grandis plastid genome, and the second sequenced and annotated mitochondrial genome of the Myrtales, that of E. grandis. The 478,813 bp mitochondrial genome shows the conserved protein coding regions and gene order rearrangements typical of land plants. There have been widespread insertions of organellar DNA into the E. grandis nuclear genome, which span 141 annotated nuclear genes. Further, we identify predicted editing sites to allow for the discrimination of RNA-sequencing reads between nuclear and organellar gene copies, finding that nuclear copies of organellar genes are not expressed in E. grandis. Conclusions The implications of organellar DNA transfer to the nucleus are often ignored, despite the insight they can give into the ongoing evolution of plant genomes, and the problems they can cause in many applications of genomics. Future comparisons of the transcription and regulation of organellar genes between Eucalyptus genotypes may provide insight to the cytonuclear interactions that impact economically important traits in this widely grown lignocellulosic crop species. Electronic supplementary material The online version of this article (10.1186/s12864-019-5444-4) contains supplementary material, which is available to authorized users.

Horsetails represent an enigmatic clade within the land plants. Despite consisting only of one genus (Equisetum) that contains 15 species, they are thought to represent the oldest extant genus within the vascular plants dating back possibly as far as the Triassic. Horsetails have retained several ancient features and are also characterized by a particularly high chromosome count (n = 108). Whole-genome duplications (WGDs) have been uncovered in many angiosperm clades and have been associated with the success of angiosperms, both in terms of species richness and biomass dominance, but remain understudied in nonangiosperm clades. Here, we report unambiguous evidence of an ancient WGD in the fern lineage, based on sequencing and de novo assembly of an expressed gene catalog (transcriptome) from the giant horsetail (Equisetum giganteum). We demonstrate that horsetails underwent an independent paleopolyploidy during the Late Cretaceous prior to the diversification of the genus but did not experience any recent polyploidizations that could account for their high chromosome number. We also discuss the specific retention of genes following the WGD and how this may be linked to their long-term survival.

National Research Foundation (NRF) of South Africa (Grant Numbers NBIG, 86936; Thuthuka Programme, UID 76225), Mondi and SAPPI through the Forest Molecular Genetics Programme, and the Technology and Human Resources for Industry Programme (THRIP, UID 80118).

We examined genome-wide patterns of DNA sequence diversity and divergence among six species of the important tree genus Eucalyptus and investigated their relationship with genomic architecture. Using c. 90 range-wide individuals of each Eucalyptus species (E. grandis, E. urophylla, E. globulus, E. nitens, E. dunnii and E. camaldulensis), genetic diversity and divergence were estimated from 2840 polymorphic diversity arrays technology markers covering the 11 chromosomes. Species differentiating markers (SDMs) identified in each of 15 pairwise species comparisons, along with species diversity (HHW ) and divergence (FST ), were projected onto the E. grandis reference genome. Across all species comparisons, SDMs totalled 1.1-5.3% of markers and were widely distributed throughout the genome. Marker divergence (FST and SDMs) and diversity differed among and within chromosomes. Patterns of diversity and divergence were broadly conserved across species and significantly associated with genomic features, including the proximity of markers to genes, the relative number of clusters of tandem duplications, and gene density within or among chromosomes. These results suggest that genomic architecture influences patterns of species diversity and divergence in the genus. This influence is evident across the six species, encompassing diverse phylogenetic lineages, geography and ecology.

Wood formation is a complex developmental process governed by genetic and environmental stimuli. Populus and Eucalyptus are fast-growing, high-yielding tree genera that represent ecologically and economically important species suitable for generating significant lignocellulosic biomass. Comparative analysis of the developing xylem and leaf transcriptomes of Populus trichocarpa and Eucalyptus grandis together with phylogenetic analyses identified clusters of homologous genes preferentially expressed during xylem formation in both species. A conserved set of 336 single gene pairs showed highly similar xylem preferential expression patterns, as well as evidence of high functional constraint. Individual members of multi-gene orthologous clusters known to be involved in secondary cell wall biosynthesis also showed conserved xylem expression profiles. However, species-specific expression as well as opposite (xylem versus leaf) expression patterns observed for a subset of genes suggest subtle differences in the transcriptional regulation important for xylem development in each species. Using sequence similarity and gene expression status, we identified functional homologs likely to be involved in xylem developmental and biosynthetic processes in Populus and Eucalyptus. Our study suggests that, while genes involved in secondary cell wall biosynthesis show high levels of gene expression conservation, differential regulation of some xylem development genes may give rise to unique xylem properties.

A time-course transcriptome analysis of two cassava varieties that are either resistant or susceptible to cassava brown streak disease (CBSD) was conducted using RNASeq, after graft inoculation with Ugandan cassava brown streak virus (UCBSV). From approximately 1.92 billion short reads, the largest number of differentially expressed genes (DEGs) was obtained in the resistant (Namikonga) variety at 2 days after grafting (dag) (3887 DEGs) and 5 dag (4911 DEGs). At the same time points, several defense response genes (encoding LRR-containing, NBARC-containing, pathogenesis-related, late embryogenesis abundant, selected transcription factors, chaperones, and heat shock proteins) were highly expressed in Namikonga. Also, defense-related GO terms of ‘translational elongation’, ‘translation factor activity’, ‘ribosomal subunit’ and ‘phosphorelay signal transduction’, were overrepresented in Namikonga at these time points. More reads corresponding to UCBSV sequences were recovered from the susceptible variety (Albert) (733 and 1660 read counts per million (cpm)) at 45 dag and 54 dag compared to Namikonga (10 and 117 cpm respectively). These findings suggest that Namikonga’s resistance involves restriction of multiplication of UCBSV within the host. These findings can be used with other sources of evidence to identify candidate genes and biomarkers that would contribute substantially to knowledge-based resistance breeding.

Summary Eucalyptus is a pivotal genus within the rosid order Myrtales with distinct geographic history and adaptations. Comparative analysis of protein domain evolution in the newly sequenced Eucalyptus grandis genome and other rosid lineages sheds light on the adaptive mechanisms integral to the success of this genus of woody perennials. We reconstructed the ancestral domain content to elucidate the gain, loss and expansion of protein domains and domain arrangements in Eucalyptus in the context of rosid phylogeny. We used functional gene ontology (GO) annotation of genes to investigate the possible biological and evolutionary consequences of protein domain expansion. We found that protein modulation within the angiosperms occurred primarily on the level of expansion of certain domains and arrangements. Using RNA-Seq data from E. grandis, we showed that domain expansions have contributed to tissue-specific expression of tandemly duplicated genes. Our results indicate that tandem duplication of genes, a key feature of the Eucalyptus genome, has played an important role in the expansion of domains, particularly in proteins related to the specialization of reproduction and biotic and abiotic interactions affecting root and floral biology, and that tissue-specific expression of proteins with expanded domains has facilitated subfunctionalization in domain families.

Tension wood has distinct physical and chemical properties, including altered fibre properties, cell wall composition and ultrastructure. It serves as a good system for investigating the genetic regulation of secondary cell wall biosynthesis and wood formation. The reference genome sequence for Eucalyptus grandis allows investigation of the global transcriptional reprogramming that accompanies tension wood formation in this global wood fibre crop. We report the first comprehensive analysis of physicochemical wood property changes in tension wood of Eucalyptus measured in a hybrid (E. grandis × Eucalyptus urophylla) clone, as well as genome-wide gene expression changes in xylem tissues 3 wk post-induction using RNA sequencing. We found that Eucalyptus tension wood in field-grown trees is characterized by an increase in cellulose, a reduction in lignin, xylose and mannose, and a marked increase in galactose. Gene expression profiling in tension wood-forming tissue showed corresponding down-regulation of monolignol biosynthetic genes, and differential expression of several carbohydrate active enzymes. We conclude that alterations of cell wall traits induced by tension wood formation in Eucalyptus are a consequence of a combination of down-regulation of lignin biosynthesis and hemicellulose remodelling, rather than the often proposed up-regulation of the cellulose biosynthetic pathway.

As a step toward functional annotation of genes required for floral initiation and development within the Eucalyptus genome, we used short read sequencing to analyze transcriptomes of floral buds from early and late developmental stages, and compared these with transcriptomes of diverse vegetative tissues, including leaves, roots, and stems. A subset of 4807 genes (13% of protein-coding genes) were differentially expressed between floral buds of either stage and vegetative tissues. A similar proportion of genes were differentially expressed among all tissues. A total of 479 genes were differentially expressed between early and late stages of floral development. Gene function enrichment identified 158 gene ontology classes that were overrepresented in floral tissues, including 'pollen development' and 'aromatic compound biosynthetic process'. At least 40 floral-dominant genes lacked functional annotations and thus may be novel floral transcripts. We analyzed several genes and gene families in depth, including 49 putative biomarkers of floral development, the MADS-box transcription factors, 'S-domain'-receptor-like kinases, and selected gene family members with phosphatidylethanolamine-binding protein domains. Expanded MADS-box gene subfamilies in Eucalyptus grandis included SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), SEPALLATA (SEP) and SHORT VEGETATIVE PHASE (SVP) Arabidopsis thaliana homologs. These data provide a rich resource for functional and evolutionary analysis of genes controlling eucalypt floral development, and new tools for breeding and biotechnology.

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.

SECONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND1) is a master regulator of fibre secondary wall deposition in Arabidopsis thaliana (Arabidopsis), with homologs in other angiosperms and gymnosperms. However, it is poorly understood to what extent the fibre-specific regulation of the SND1 promoter, and that of its orthologs, is conserved between diverged herbaceous and woody lineages. We performed a reciprocal reporter gene analysis of orthologous SND1 promoters from Arabidopsis (AthSND1), Eucalyptus grandis (EgrNAC61) and Populus alba &times, P. grandidentata (PagWND1A) relative to secondary cell wall-specific Cellulose Synthase4 (CesA4) and CesA7 promoters, in both a non-woody (Arabidopsis) and a woody (poplar) system. &beta, glucuronidase (GUS) reporter analysis in Arabidopsis showed that the SND1 promoter was active in vascular tissues as previously reported and showed interfascicular and xylary fibre-specific expression in inflorescence stems, while reporter constructs of the woody plant-derived promoters were partial to the (pro)cambium-phloem and protoxylem. In transgenic P. tremula &times, P. alba plants, all three orthologous SND1 promoters expressed the GUS reporter similarly and preferentially in developing secondary xylem, ray parenchyma and cork cambium. Ours is the first study to reciprocally test orthologous SND1 promoter specificity in herbaceous and woody species, revealing diverged regulatory functions in the herbaceous system.

Plantation forestry based on Eucalyptus spp. has grown dramatically during the past 100 years, mainly in the tropics and southern hemisphere where these trees are non-native. Early plantations were affected by pests and pathogens, although damage was generally minimal. This is a situation that is changing dramatically with serious pest and pathogen problems now emerging regularly and often times causing very serious damage. Given the growing movement of people and products around the world, this is a trend that is certain to continue and the threat of diseases and insect damage is now seen as one of the most important constraints to the future sustainability of Eucalypt plantation forestry globally. While this is a very troubling situation, there are also reasons to be optimistic. Established technologies such as breeding and selection of disease and insect resistant species and hybrids; enhanced through vegetative propagation have already shown substantial promise to reduce damage. Likewise, biological control is being effectively used to reduce damage due to invasive alien insect pests. New technologies including DNA-based tools for identification, detection and monitoring of pest and pathogens are increasingly valuable. Similarly, the growing availability of genome sequences for Eucalypts and for their pests and pathogens will increasingly add to our capacity to reduce damage. While there are unknown dangers such as the impact of climate change on the damage caused by pests and pathogens, we believe that continual and realistic investment in technology will ensure long term sustainability of Eucalypt plantation forestry. Failure to invest wisely will result in the collapse of plantation enterprises and concomitant loss in investments.

Lignocellulosic biomass from fast-growing plantation trees is composed of carbohydrate-rich materials deposited into plant cell walls in a coordinated manner during wood formation. The diversity and evolution of the transcriptional networks regulating this process have not been studied extensively. We investigated patterns of species-level nucleotide diversity in the promoters of cellulose synthase (CesA) genes from different Eucalyptus tree species and assessed the possible roles of DNA sequence polymorphism in the gain or loss of cis-elements harboured within the promoters. Promoter regions of three primary and three secondary cell wall-associated CesA genes were isolated from 13 Eucalyptus species and were analysed for nucleotide and cis-element diversity. Species-level nucleotide diversity (π) ranged from 0.014 to 0.068, and different CesA promoters exhibited distinct patterns of sequence conservation. A set of 22 putative cis-elements were mapped to the CesA promoters using in silico methods. Forty-two percent of the mapped cis-element occurrences contained singleton polymorphisms which resulted in either gain or loss of a cis-element in a particular Eucalyptus species. The promoters of Eucalyptus CesA genes contained regions that are highly conserved at the species (Eucalyptus) and genus (with Arabidopsis and Populus) level, suggesting the presence of regulatory modules imposing functional constraint on such regions. Nucleotide polymorphisms in the CesA promoters more frequently created new cis-element occurrences than disrupted existing cis-element occurrences, a process which may be important for the maintenance and evolution of cellulose gene regulation in plants.

As a consequence of their remarkable adaptability, fast growth, and superior wood properties, eucalypt tree plantations have emerged as key renewable feedstocks (over 20 million ha globally) for the production of pulp, paper, bioenergy, and other lignocellulosic products. However, most biomass properties such as growth, wood density, and wood chemistry are complex traits that are hard to improve in long-lived perennials. Systems genetics, a process of harnessing multiple levels of component trait information (e.g., transcript, protein, and metabolite variation) in populations that vary in complex traits, has proven effective for dissecting the genetics and biology of such traits. We have applied a network-based data integration (NBDI) method for a systems-level analysis of genes, processes and pathways underlying biomass and bioenergy-related traits using a segregating Eucalyptus hybrid population. We show that the integrative approach can link biologically meaningful sets of genes to complex traits and at the same time reveal the molecular basis of trait variation. Gene sets identified for related woody biomass traits were found to share regulatory loci, cluster in network neighborhoods, and exhibit enrichment for molecular functions such as xylan metabolism and cell wall development. These findings offer a framework for identifying the molecular underpinnings of complex biomass and bioprocessing-related traits. A more thorough understanding of the molecular basis of plant biomass traits should provide additional opportunities for the establishment of a sustainable bio-based economy.

Previously, an interspecific cross between Fusarium circinatum and Fusarium subglutinans was used to generate a genetic linkage map. A ca. 55 % of genotyped markers displayed transmission ratio distortion (TRD) that demonstrated a genome-wide distribution. The working hypothesis for this study was that TRD would be non-randomly distributed throughout the genetic linkage map. This would indicate the presence of distorting loci. Using a genome-wide threshold of α = 0.01, 79 markers displaying TRD were distributed on all 12 linkage groups (LGs). Eleven putative transmission ratio distortion loci (TRDLs), spanning eight LGs, were identified in regions containing three or more adjacent markers displaying distortion. No epistatic interactions were observed between these TRDLs. Thus, it is uncertain whether the genome-wide TRD was due to linkage between markers and genomic regions causing distortion. The parental origins of markers followed a non-random distribution throughout the linkage map, with LGs containing stretches of markers originating from only one parent. Thus, due to the nature of the interspecific cross, the current hypothesis to explain these observations is that the observed genome-wide segregation was caused by the high level of genomic divergence between the parental isolates. Therefore, homologous chromosomes do not align properly during meiosis, resulting in aberrant transmission of markers. This also explains previous observations of the preferential transmission of F. subglutinans alleles to the F1 progeny.

Eucalyptus species are cultivated for forestry and are of economic importance. The fungal stem canker pathogen Chrysoporthe austroafricana causes disease of varying severity on E. grandis. The Eucalyptus grandis-Chrysoporthe austroafricana interaction has been established as a model system for studying Eucalyptus antifungal defence. Previous studies revealed that the phytohormone salicylic acid (SA) affects the levels of resistance in highly susceptible (ZG14) and moderately resistant (TAG5) clones. The aims of this study were to examine histochemical changes in response to wounding and inoculation as well as host responses at the protein level. The anatomy and histochemical changes induced by wounding and inoculation were similar between the clones, suggesting that anatomical differences do not underlie their different levels of resistance. Tyloses and gum-like substances were present after inoculation and wounding, but cell death occurred only after inoculation. Hyphae of C. austroafricana were observed inside dead and living cells, suggesting that the possibility of a hemibiotrophic interaction requires further investigation. Proteomics analysis revealed the possible involvement of proteins associated with cell death, SA signalling and systemic resistance. In combination with previous information, this study forms a basis for future functional characterisation of candidate genes involved in resistance of E. grandis to C. austroafricana.

Chrysoporthe austroafricana is a fungal pathogen that causes the development of stem cankers on susceptible Eucalyptus grandis trees. Clones of E. grandis that are partially resistant and highly susceptible have been identified based on the extent of lesion formation on the stem upon inoculation with C. austroafricana. These interactions have been used as a model pathosystem to enhance our understanding of interactions between pathogenic fungi and woody hosts, which may be different to herbaceous hosts. In previous research, transcriptomics of host responses in these two clones to C. austroafricana suggested roles for salicylic acid and gibberellic acid phytohormone signaling in defense. However, it is unclear how the pathogen infiltrates host tissue and which pathogenicity factors facilitate its spread in the two host genotypes. The aim of this study was to investigate these two aspects of the E. grandis–C. austroafricana interaction and to test the hypothesis that the pathogen possesses mechanisms to modulate the tree phytohormone-mediated defenses. Light microscopy showed that the pathogen occurred in most cell types and structures within infected E. grandis stem tissue. Notably, the fungus appeared to spread through the stem by penetrating cell wall pits. In order to understand the molecular interaction between these organisms and predict putative pathogenicity mechanisms of C. austroafricana, fungal gene expression was studied in vitro and in planta. Fungal genes associated with cell wall degradation, carbohydrate metabolism and phytohormone manipulation were expressed in planta by C. austroafricana. These genes could be involved in fungal spread by facilitating cell wall pit degradation and manipulating phytohormone mediated defense in each host environment, respectively. Specifically, the in planta expression of an ent-kaurene oxidase and salicylate hydroxylase in C. austroafricana suggests putative mechanisms by which the pathogen can modulate the phytohormone-mediated defenses of the host. These mechanisms have been reported in herbaceous plant–pathogen interactions, supporting the notion that these aspects of the interaction are similar in a woody species. This study highlights ent-kaurene oxidase and salicylate hydroxylase as candidates for further functional characterization.

Gall-inducing insects are capable of exerting a high level of control over their hosts' cellular machinery to the extent that the plant's development, metabolism, chemistry, and physiology are all altered in favour of the insect. Many gallers are devastating pests in global agriculture and the limited understanding of their relationship with their hosts prevents the development of robust management strategies. Omics technologies are proving to be important tools in elucidating the mechanisms involved in the interaction as they facilitate analysis of plant hosts and insect effectors for which little or no prior knowledge exists. In this review, we examine the mechanisms behind insect gall development using evidence from omics-level approaches. The secretion of effector proteins and induced phytohormonal imbalances are highlighted as likely mechanisms involved in gall development. However, understanding how these components function within the system is far from complete and a number of questions need to be answered before this information can be used in the development of strategies to engineer or breed plants with enhanced resistance.

The status of genomics and genetics research in the Myrtaceae, a large family of dicotyledonous woody plants, is reviewed with Eucalyptus as the focal genus. The family contains over 5,650 species in 130 to 150 genera, predominantly of neo-tropical and Southern Hemisphere distribution. Several genera are well known for their economic importance worldwide. Myrtaceae are typically diploids with small to intermediate genome size. Microsatellites have been developed for several genera while higher throughput marker systems such as diversity arrays technology and single nucleotide polymorphism are available for Eucalyptus. Molecular data have been fundamental to current perspectives on the phylogeny, phylogeography and taxonomy of the Myrtaceae, while numerous studies of genetic diversity have been carried out particularly as it relates to endangered, rare, fragmented, overharvested or economically important species. Large expressed sequence tag collections for species of Eucalyptus have recently become public to support the annotation of the Eucalyptus grandis genome. Transcriptomics in Eucalyptus has advanced by microarrays and next-generation sequencing focusing on wood development. Linkage maps for Eucalyptus display high synteny across species and have been extensively used to map quantitative trait loci for a number of traits including growth, wood quality, disease and insect resistance. Candidate gene-based association genetics have successfully found marker–trait associations for wood and fiber traits. Genomic selection experiments have demonstrated clear potential to improve the efficiency of breeding programs while freeze-tolerant transgenic Eucalyptus trials have recently been initiated. The recently released E. grandis genome, sequenced to an average coverage of 8×, will open up exceptional opportunities to advance Myrtaceae genetics and genomics research.

Fast-growing, short-rotation forest trees, such as Populus and Eucalyptus, produce large amounts of cellulose-rich biomass that could be utilized for bioenergy and biopolymer production. Major obstacles need to be overcome before the deployment of these genera as energy crops, including the effective removal of lignin and the subsequent liberation of carbohydrate constituents from wood cell walls. However, significant opportunities exist to both select for and engineer the structure and interaction of cell wall biopolymers, which could afford a means to improve processing and product development. The molecular underpinnings and regulation of cell wall carbohydrate biosynthesis are rapidly being elucidated, and are providing tools to strategically develop and guide the targeted modification required to adapt forest trees for the emerging bioeconomy. Much insight has already been gained from the perturbation of individual genes and pathways, but it is not known to what extent the natural variation in the sequence and expression of these same genes underlies the inherent variation in wood properties of field-grown trees. The integration of data from next-generation genomic technologies applied in natural and experimental populations will enable a systems genetics approach to study cell wall carbohydrate production in trees, and should advance the development of future woody bioenergy and biopolymer crops.