Project description:Terpenes are economically and ecologically important phytochemicals. Their synthesis is controlled by the terpene synthase (TPS) gene family, which is highly diversified throughout the plant kingdom. The plant family Myrtaceae are characterised by especially high terpene concentrations, and considerable variation in terpene profiles. Many Myrtaceae are grown commercially for terpene products including the eucalypts Corymbia and Eucalyptus. Eucalyptus grandis has the largest TPS gene family of plants currently sequenced, which is largely conserved in the closely related E. globulus. However, the TPS gene family has been well studied only in these two eucalypt species. The recent assembly of two Corymbia citriodora subsp. variegata genomes presents an opportunity to examine the conservation of this important gene family across more divergent eucalypt lineages. Manual annotation of the TPS gene family in C. citriodora subsp. variegata revealed a similar overall number, and relative subfamily representation, to that previously reported in E. grandis and E. globulus. Many of the TPS genes were in physical clusters that varied considerably between Eucalyptus and Corymbia, with several instances of translocation, expansion/contraction and loss. Notably, there was greater conservation in the subfamilies involved in primary metabolism than those involved in secondary metabolism, likely reflecting different selective constraints. The variation in cluster size within subfamilies and the broad conservation between the eucalypts in the face of this variation are discussed, highlighting the potential contribution of selection, concerted evolution and stochastic processes. These findings provide the foundation to better understand terpene evolution within the ecologically and economically important Myrtaceae.
Project description:The <i>Eucalyptus</i> genus is a hyper-diverse group of long-lived trees from the Myrtaceae family, consisting of more than 700 species. <i>Eucalyptus</i> are widely distributed across their native Australian landscape and are the most widely planted hardwood forest trees in the world. The ecological and economic success of <i>Eucalyptus</i> trees is due, in part, to their ability to produce a plethora of specialized metabolites, which moderate abiotic and biotic interactions. Formylated phloroglucinol compounds (FPCs) are an important class of specialized metabolites in the Myrtaceae family, particularly abundant in <i>Eucalyptus</i>. FPCs are mono- to tetra-formylated phloroglucinol based derivatives, often with an attached terpene moiety. These compounds provide chemical defense against herbivory and display various bioactivities of pharmaceutical relevance. Despite their ecological and economic importance, and continued improvements into analytical techniques, FPCs have proved challenging to study. Here we present a simple and reliable method for FPCs extraction, identification and quantification by UHPLC-DAD-ESI-Q-TOF-MS/MS. The method was applied to leaf, flower bud, and flower samples of nine different eucalypt species, using a small amount of plant material. Authentic analytical standards were used to provide high resolution mass spectra and fragmentation patterns. A robust method provides opportunities for future investigations into the identification and quantification of FPCs in complex biological samples with high confidence. Furthermore, we present for the first time the tissue-based localization of FPCs in stem, leaf, and flower bud of <i>Eucalyptus</i> species measured by mass spectrometry imaging, providing important information for biosynthetic pathway discovery studies and for understanding the role of those compounds <i>in planta</i>.
Project description:The Xklp2 (TPX2) proteins belong to the microtubule-associated (MAP) family of proteins. All members of the family contain the conserved TPX2 motif, which can interact with microtubules, regulate microtubule dynamics or assist with different microtubule functions, for example, maintenance of cell morphology or regulation of cell growth and development. However, the role of members of the TPX family have not been studied in the model tree species Eucalyptus to date. Here, we report the identification of the members of the TPX2 family in Eucalyptus grandis (Eg) and analyse the expression patterns and functions of these genes.In present study, a comprehensive analysis of the plant TPX2 family proteins was performed. Phylogenetic analyses indicated that the genes can be classified into 6 distinct subfamilies. A genome-wide survey identified 12 members of the TPX2 family in the sequenced genome of Eucalyptus grandis. The basic genetic properties of the TPX2 family in Eucalyptus were analysed. Our results suggest that the TPX2 family proteins within different sub-groups are relatively conserved but there are important differences between groups. Quantitative real-time PCR (qRT-PCR) was performed to confirm the expression levels of the genes in different tissues. The results showed that in the whole plant, the levels of EgWDL5 transcript are the highest, followed by those of EgWDL4. Compared with other tissues, the level of the EgMAP20 transcript is the highest in the root. Over-expression of EgMAP20 in Arabidopsis resulted in organ twisting. The cotyledon petioles showed left-handed twisting while the hypocotyl epidermal cells produced right-handed helical twisting. Finally, EgMAP20, EgWDL3 and EgWDL3L were all able to decorate microtubules.Plant TPX2 family proteins were systematically analysed using bioinformatics methods. There are 12 TPX2 family proteins in Eucalyptus. We have performed an initial characterization of the functions of several members of the TPX2 family. We found that the gene products are localized to the microtubule cytoskeleton. Our results lay the foundation for future efforts to reveal the biological significance of TPX2 family proteins in Eucalyptus.
Project description:BACKGROUND:F1 hybrid clones of Eucalyptus grandis and E. urophylla are widely grown for pulp and paper production in tropical and subtropical regions. Volume growth and wood quality are priority objectives in Eucalyptus tree improvement. The molecular basis of quantitative variation and trait expression in eucalypt hybrids, however, remains largely unknown. The recent availability of a draft genome sequence (http://www.phytozome.net) and genome-wide genotyping platforms, combined with high levels of genetic variation and high linkage disequilibrium in hybrid crosses, greatly facilitate the detection of quantitative trait loci (QTLs) as well as underlying candidate genes for growth and wood property traits. In this study, we used Diversity Arrays Technology markers to assess the genetic architecture of volume growth (diameter at breast height, DBH) and wood basic density in four-year-old progeny of an interspecific backcross pedigree of E. grandis and E. urophylla. In addition, we used Illumina RNA-Seq expression profiling in the E. urophylla backcross family to identify cis- and trans-acting polymorphisms (eQTLs) affecting transcript abundance of genes underlying QTLs for wood basic density. RESULTS:A total of five QTLs for DBH and 12 for wood basic density were identified in the two backcross families. Individual QTLs for DBH and wood basic density explained 3.1 to 12.2% of phenotypic variation. Candidate genes underlying QTLs for wood basic density on linkage groups 8 and 9 were found to share trans-acting eQTLs located on linkage groups 4 and 10, which in turn coincided with QTLs for wood basic density suggesting that these QTLs represent segregating components of an underlying transcriptional network. CONCLUSION:This is the first demonstration of the use of next-generation expression profiling to quantify transcript abundance in a segregating tree population and identify candidate genes potentially affecting wood property variation. The QTLs identified in this study provide a resource for identifying candidate genes and developing molecular markers for marker-assisted breeding of volume growth and wood basic density. Our results suggest that integrated analysis of transcript and trait variation in eucalypt hybrids can be used to dissect the molecular basis of quantitative variation in wood property traits.
Project description:BACKGROUND: Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing. RESULTS: We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1) digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes. CONCLUSIONS: The two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae, including genome sequencing, gene isolation, functional and comparative genomics. Because they have been constructed using the same tree (E. grandis BRASUZ1) whose full genome is being sequenced, they should prove instrumental for assembly and gap filling of the upcoming Eucalyptus reference genome sequence.
Project description:In order to pinpoint the most differentially expressed genes between Eucalyptus grandis leaf blades and vascular (xylem) tissues as well as between E. grandis and Eucalyptus globulus xylem tissues, a total number of nine 50mer-oligoprobes covering the length of each one of 21,432 unique sequences derived from the Genolyptus EST dataset were synthesized “on-chip” in duplicate, randomly distributed in two blocks of each slide. Probes were also synthesized from ten cDNA sequences encoding known human proteins as negative controls, totaling 21,442 sequences. Leaves and xylem samples were taken from two E. grandis clonal trees, i.e., both derived from the same matrix tree and harboring the same genotype. Two additional xylem samples were collected from two other E. grandis clonal trees of a different genotype, as well as from two E. globulus clonal trees. Therefore, ten cDNA samples and ten identical chips were produced at Roche NimbleGen for the microarray assays, with a total number of 385,956 features per slide. Besides the discovery of differentially expressed genes between leaf and xylem, we wanted to test the validity of the assumed “technical” and “biological duplicates” since all trees were field-grown and four years-old in age. A ten chip study using total RNA recovered from mature leaf and vascular (xylem) tissues of Eucalyptus grandis and xylem from Eucalyptus globulus trees. Two clonal trees of E. grandis (E.grandis_Clone A_Ramet 1 and E.grandis_Clone A_Ramet 2), derived from a single matrix tree and therefore genomically identical, were the source of two samples of leaf RNA and two samples of xylem RNA, individually hybridized to four chips after cDNA synthesis/Cy3 labeling. Two other clonal trees of E. grandis (E.grandis_Clone B_Ramet 1 and E.grandis_Clone B_Ramet 2), derived from a different matrix tree, were the source of two additional samples of xylem RNA individually hybridized to four chips after cDNA synthesis/Cy3 labeling. Likewise, two pairs of clonal trees of E. globulus (E.globulus_Clone A_Ramet 1 and E.globulus_Clone A_Ramet 2/ E.globulus_Clone B_Ramet 1 and E.globulus_Clone B_Ramet 2), derived from two distinct matrix trees, were the source of four additional samples of xylem RNA, individually hybridized to four chips after cDNA synthesis/Cy3 labeling. Each chip measures the expression level of 21,432 genes from Eucalyptus sp. and ten human genes (negative controls) with nine 50-mer probe pairs (PM/MM) per gene in two separate blocks per chip (technical duplicate), totalizing 18 hybridization signal values per gene per chip.