The pulse of the tree is under genetic control: eucalyptus as a case study.

The pulse of the tree (diurnal cycle of stem radius fluctuations) has been widely studied as a way of analyzing tree responses to the environment, including the phenotypic plasticity of tree-water relationships in particular. However, the genetic basis of this daily phenotype and its interplay with the environment remain largely unexplored. We characterized the genetic and environmental determinants of this response, by monitoring daily stem radius fluctuation (dSRF) on 210 trees from a Eucalyptus urophylla × E. grandis full-sib family over 2 years. The dSRF signal was broken down into hydraulic capacitance, assessed as the daily amplitude of shrinkage (DA), and net growth, estimated as the change in maximum radius between two consecutive days (ΔR). The environmental determinants of these two traits were clearly different: DA was positively correlated with atmospheric variables relating to water demand, while ΔR was associated with soil water content. The heritability for these two traits ranged from low to moderate over time, revealing a time-dependent or environment-dependent complex genetic determinism. We identified 686 and 384 daily quantitative trait loci (QTL) representing 32 and 31 QTL regions for DA and ΔR, respectively. The identification of gene networks underlying the 27 major genomics regions for both traits generated additional hypotheses concerning the biological mechanisms involved in response to water demand and supply. This study highlights that environmentally induced changes in daily stem radius fluctuation are genetically controlled in trees and suggests that these daily responses integrated over time shape the genetic architecture of mature traits.

Genome-wide variation in recombination rate in Eucalyptus.

BACKGROUND: Meiotic recombination is a fundamental evolutionary process. It not only generates diversity, but influences the efficacy of natural selection and genome evolution. There can be significant heterogeneity in recombination rates within and between species, however this variation is not well understood outside of a few model taxa, particularly in forest trees. Eucalypts are forest trees of global economic importance, and dominate many Australian ecosystems. We studied recombination rate in Eucalyptus globulus using genetic linkage maps constructed in 10 unrelated individuals, and markers anchored to the Eucalyptus reference genome. This experimental design provided the replication to study whether recombination rate varied between individuals and chromosomes, and allowed us to study the genomic attributes and population genetic parameters correlated with this variation. RESULTS: Recombination rate varied significantly between individuals (range = 2.71 to 3.51 centimorgans/megabase [cM/Mb]), but was not significantly influenced by sex or cross type (F1 vs. F2). Significant differences in recombination rate between chromosomes were also evident (range = 1.98 to 3.81 cM/Mb), beyond those which were due to variation in chromosome size. Variation in chromosomal recombination rate was significantly correlated with gene density (r = 0.94), GC content (r = 0.90), and the number of tandem duplicated genes (r = -0.72) per chromosome. Notably, chromosome level recombination rate was also negatively correlated with the average genetic diversity across six species from an independent set of samples (r = -0.75). CONCLUSIONS: The correlations with genomic attributes are consistent with findings in other taxa, however, the direction of the correlation between diversity and recombination rate is opposite to that commonly observed. We argue this is likely to reflect the interaction of selection and specific genome architecture of Eucalyptus. Interestingly, the differences amongst chromosomes in recombination rates appear stable across Eucalyptus species. Together with the strong correlations between recombination rate and features of the Eucalyptus reference genome, we maintain these findings provide further evidence for a broad conservation of genome architecture across the globally significant lineages of Eucalyptus.

Genetic architecture of carbon isotope composition and growth in Eucalyptus across multiple environments.

In the context of climate change, the water-use efficiency (WUE) of highly productive tree varieties, such as eucalypts, has become a major issue for breeding programmes. This study set out to dissect the genetic architecture of carbon isotope composition (δ(13) C), a proxy of WUE, across several environments. A family of Eucalyptus urophylla × E. grandis was planted in three trials and phenotyped for δ(13) C and growth traits. High-resolution genetic maps enabled us to target genomic regions underlying δ(13) C quantitative trait loci (QTLs) on the E. grandis genome. Of the 15 QTLs identified for δ(13) C, nine were stable across the environments and three displayed significant QTL-by-environment interaction, suggesting medium to high genetic determinism for this trait. Only one colocalization was found between growth and δ(13) C. Gene ontology (GO) term enrichment analysis suggested candidate genes related to foliar δ(13) C, including two involved in the regulation of stomatal movements. This study provides the first report of the genetic architecture of δ(13) C and its relation to growth in Eucalyptus. The low correlations found between the two traits at phenotypic and genetic levels suggest the possibility of improving the WUE of Eucalyptus varieties without having an impact on breeding for growth.

High-resolution genetic maps of Eucalyptus improve Eucalyptus grandis genome assembly.

Genetic maps are key tools in genetic research as they constitute the framework for many applications, such as quantitative trait locus analysis, and support the assembly of genome sequences. The resequencing of the two parents of a cross between Eucalyptus urophylla and Eucalyptus grandis was used to design a single nucleotide polymorphism (SNP) array of 6000 markers evenly distributed along the E. grandis genome. The genotyping of 1025 offspring enabled the construction of two high-resolution genetic maps containing 1832 and 1773 markers with an average marker interval of 0.45 and 0.5 cM for E. grandis and E. urophylla, respectively. The comparison between genetic maps and the reference genome highlighted 85% of collinear regions. A total of 43 noncollinear regions and 13 nonsynthetic regions were detected and corrected in the new genome assembly. This improved version contains 4943 scaffolds totalling 691.3 Mb of which 88.6% were captured by the 11 chromosomes. The mapping data were also used to investigate the effect of population size and number of markers on linkage mapping accuracy. This study provides the most reliable linkage maps for Eucalyptus and version 2.0 of the E. grandis genome.

High-density linkage mapping in a pine tree reveals a genomic region associated with inbreeding depression and provides clues to the extent and distribution of meiotic recombination.

BACKGROUND: The availability of a large expressed sequence tags (EST) resource and recent advances in high-throughput genotyping technology have made it possible to develop highly multiplexed SNP arrays for multi-objective genetic applications, including the construction of meiotic maps. Such approaches are particularly useful in species with a large genome size, precluding the use of whole-genome shotgun assembly with current technologies. RESULTS: In this study, a 12 k-SNP genotyping array was developed for maritime pine from an extensive EST resource assembled into a unigene set. The offspring of three-generation outbred and inbred mapping pedigrees were then genotyped. The inbred pedigree consisted of a classical F2 population resulting from the selfing of a single inter-provenance (Landes x Corsica) hybrid tree, whereas the outbred pedigree (G2) resulted from a controlled cross of two intra-provenance (Landes x Landes) hybrid trees. This resulted in the generation of three linkage maps based on SNP markers: one from the parental genotype of the F2 population (1,131 markers in 1,708 centimorgan (cM)), and one for each parent of the G2 population (1,015 and 1,110 markers in 1,447 and 1,425 cM for the female and male parents, respectively). A comparison of segregation patterns in the progeny obtained from the two types of mating (inbreeding and outbreeding) led to the identification of a chromosomal region carrying an embryo viability locus with a semi-lethal allele. Following selfing and segregation, zygote mortality resulted in a deficit of Corsican homozygous genotypes in the F2 population. This dataset was also used to study the extent and distribution of meiotic recombination along the length of the chromosomes and the effect of sex and/or genetic background on recombination. The genetic background of trees in which meiotic recombination occurred was found to have a significant effect on the frequency of recombination. Furthermore, only a small proportion of the recombination hot- and cold-spots were common to all three genotypes, suggesting that the spatial pattern of recombination was genetically variable. CONCLUSION: This study led to the development of classical genomic tools for this ecologically and economically important species. It also identified a chromosomal region bearing a semi-lethal recessive allele and demonstrated the genetic variability of recombination rate over the genome.

Plasticity of primary and secondary growth dynamics in Eucalyptus hybrids: a quantitative genetics and QTL mapping perspective.

BACKGROUND: The genetic basis of growth traits has been widely studied in forest trees. Quantitative trait locus (QTL) studies have highlighted the presence of both stable and unstable genomic regions accounting for biomass production with respect to tree age and genetic background, but results remain scarce regarding the interplay between QTLs and the environment. In this study, our main objective was to dissect the genetic architecture of the growth trajectory with emphasis on genotype x environment interaction by measuring primary and secondary growth covering intervals connected with environmental variations. RESULTS: Three different trials with the same family of Eucalyptus urophylla x E. grandis hybrids (with different genotypes) were planted in the Republic of Congo, corresponding to two QTL mapping experiments and one clonal test. Height and radial growths were monitored at regular intervals from the seedling stage to five years old. The correlation between growth increments and an aridity index revealed that growth before two years old (r = 0.5; 0.69) was more responsive to changes in water availability than late growth (r = 0.39; 0.42) for both height and circumference. We found a regular increase in heritability with time for cumulative growth for both height [0.06 - 0.33] and circumference [0.06 - 0.38]. Heritabilities for incremental growth were more heterogeneous over time even if ranges of variation were similar (height [0-0.31]; circumference [0.19 to 0.48]). Within the trials, QTL analysis revealed collocations between primary and secondary growth QTLs as well as between early growth increments and final growth QTLs. Between trials, few common QTLs were detected highlighting a strong environmental effect on the genetic architecture of growth, validated by significant QTL x E interactions. CONCLUSION: These results suggest that early growth responses to water availability determine the genetic architecture of total growth at the mature stage and highlight the importance of considering growth as a composite trait (such as yields for annual plants) for a better understanding of its genetic bases.

Soil water stress affects both cuticular wax content and cuticle-related gene expression in young saplings of maritime pine (Pinus pinaster Ait).

BACKGROUND: The cuticle is a hydrophobic barrier located at the aerial surface of all terrestrial plants. Recent studies performed on model plants, such as Arabidopsis thaliana, have suggested that the cuticle may be involved in drought stress adaptation, preventing non-stomatal water loss. Although forest trees will face more intense drought stresses (in duration and intensity) with global warming, very few studies on the role of the cuticle in drought stress adaptation in these long-lived organisms have been so far reported. RESULTS: This aspect was investigated in a conifer, maritime pine (Pinus pinaster Ait.), in a factorial design with two genetic units (two half-sib families with different growth rates) and two treatments (irrigated vs non-irrigated), in field conditions. Saplings were grown in an open-sided greenhouse and half were irrigated three times per week for two growing seasons. Needles were sampled three times per year for cuticular wax (composition and content) and transcriptome (of 11 genes involved in cuticle biosynthesis) analysis. Non-irrigated saplings (i) had a higher cuticular wax content than irrigated saplings and (ii) overexpressed most of the genes studied. Both these trends were more marked in the faster growing family. CONCLUSIONS: The higher cuticular wax content observed in the non-irrigated treatment associated with strong modifications in products from the decarbonylation pathway suggest that cuticular wax may be involved in drought stress adaptation in maritime pine. This study provides also a set of promising candidate genes for future forward genetic studies in conifers.

Proteomic plasticity of two Eucalyptus genotypes under contrasted water regimes in the field.

Water deficit affects tree growth and limits wood production. In an attempt to identify the molecular triggers of adaptation mechanisms to water deficit in Eucalyptus, we investigated protein expression patterns of two ecophysiologically contrasted Eucalyptus genotypes. They were grown in the field in either natural conditions or irrigated for 7 weeks during the dry season in the Republic of Congo. At the phenotypic level, genotype (G), treatment (T) and/or G × T interaction effects were observed for above- and below-ground biomass-related traits. At the molecular level, changes in protein abundance were recorded in leaves (acidic pH 4-7, and basic pH 7-11, proteomes) and stems (acidic proteome) using two-dimensional gel electrophoresis (2-DE). One third of the detected protein spots displayed significant G, T and/or G × T effects, and 158 of them were identified by tandem mass spectrometry (LC-MS/MS) analysis. Thus, several proteins whose molecular plasticity was genetically controlled (i.e. G × T effect) were revealed, highlighting adaptive mechanisms to water deficit specific to each genotype, namely cell wall modification, cell detoxification and osmoregulation. Transcript abundances corresponding to G × T proteins were also investigated by quantitative RT-PCR. These proteins represent relevant targets to improve drought resistance in this ecologically and economically important forest tree genus.

RNA-Seq reveals genotype-specific molecular responses to water deficit in eucalyptus.

BACKGROUND: In a context of climate change, phenotypic plasticity provides long-lived species, such as trees, with the means to adapt to environmental variations occurring within a single generation. In eucalyptus plantations, water availability is a key factor limiting productivity. However, the molecular mechanisms underlying the adaptation of eucalyptus to water shortage remain unclear. In this study, we compared the molecular responses of two commercial eucalyptus hybrids during the dry season. Both hybrids differ in productivity when grown under water deficit. RESULTS: Pyrosequencing of RNA extracted from shoot apices provided extensive transcriptome coverage - a catalog of 129,993 unigenes (49,748 contigs and 80,245 singletons) was generated from 398 million base pairs, or 1.14 million reads. The pyrosequencing data enriched considerably existing Eucalyptus EST collections, adding 36,985 unigenes not previously represented. Digital analysis of read abundance in 14,460 contigs identified 1,280 that were differentially expressed between the two genotypes, 155 contigs showing differential expression between treatments (irrigated vs. non irrigated conditions during the dry season), and 274 contigs with significant genotype-by-treatment interaction. The more productive genotype displayed a larger set of genes responding to water stress. Moreover, stress signal transduction seemed to involve different pathways in the two genotypes, suggesting that water shortage induces distinct cellular stress cascades. Similarly, the response of functional proteins also varied widely between genotypes: the most productive genotype decreased expression of genes related to photosystem, transport and secondary metabolism, whereas genes related to primary metabolism and cell organisation were over-expressed. CONCLUSIONS: For the most productive genotype, the ability to express a broader set of genes in response to water availability appears to be a key characteristic in the maintenance of biomass growth during the dry season. Its strategy may involve a decrease of photosynthetic activity during the dry season associated with resources reallocation through major changes in the expression of primary metabolism associated genes. Further efforts will be needed to assess the adaptive nature of the genes highlighted in this study.

Comprehensive genetic dissection of wood properties in a widely-grown tropical tree: Eucalyptus.

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

Overexpression of EgROP1, a Eucalyptus vascular-expressed Rac-like small GTPase, affects secondary xylem formation in Arabidopsis thaliana.

To better understand the genetic control of secondary xylem formation in trees we analysed genes expressed during Eucalyptus xylem development. Using eucalyptus xylem cDNA libraries, we identified EgROP1, a member of the plant ROP family of Rho-like GTPases. These signalling proteins are central regulators of many important processes in plants, but information on their role in xylogenesis is scarce. Quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) confirmed that EgROP1 was preferentially expressed in the cambial zone and differentiating xylem in eucalyptus. Genetic mapping performed in a eucalyptus breeding population established a link between EgROP1 sequence polymorphisms and quantitative trait loci (QTLs) related to lignin profiles and fibre morphology. Overexpression of various forms of EgROP1 in Arabidopsis thaliana altered anisotropic cell growth in transgenic leaves, but most importantly affected vessel element and fibre growth in secondary xylem. Patches of fibre-like cells in the secondary xylem of transgenic plants showed changes in secondary cell wall thickness, lignin and xylan composition. These results suggest a role for EgROP1 in fibre cell morphology and secondary cell wall formation making it a good candidate gene for marker-based selection of eucalyptus trees.

Evidence of intense chromosomal shuffling during conifer evolution.

While recent advances have been gained on genome evolution in angiosperm lineages, virtually nothing is known about karyotype evolution in the other group of seed plants, the gymnosperms. Here we used high density gene-based linkage mapping to compare the karyotype structure of two families of conifers (the most abundant group of gymnosperms) separated around 290 million years ago: Pinaceae and Cupressaceae. We propose for the first time a model based on the fusion of 20 ancestral chromosomal blocks that may have shaped the modern karyotpes of Pinaceae (with n=12) and Cupressaceae (with n=11). The considerable difference in modern genome organization between these two lineages contrasts strongly with the remarkable level of synteny already reported within the Pinaceae. It also suggests a convergent evolutionary mechanism of chromosomal block shuffling that has shaped the genomes of the spermatophytes.

Interaction between environmental factors affects the accumulation of root proteins in hydroponically grown Eucalyptus globulus (Labill.).

Eucalyptus globulus (Labill.) is used for pulp and paper production worldwide. In this report we studied changes in protein expression in one osmotically stressed elite clone widely used in industrial plantations in Spain. High molecular weight polyethylene glycol (PEG) was used as an osmoticum in the growing medium. Roots of rooted cuttings were sampled after 3 and 36 h of treatment. Water potential and abscissic acid content were measured in shoot and root apices to characterize the physiological states of the plants. Total soluble proteins from roots were extracted and separated using two-dimensional gel electrophoresis (2-DE). Gels were stained with Coomassie brillant blue for quantitative analysis of protein accumulation. From a total of 406 reproducible spots, 34 were found to be differentially expressed depending on treatment (osmotic versus control condition) and/or stress duration (3 h versus 36 h), and were further characterized by tandem mass spectrometry. Several proteins were reliably identified including adenosine kinase, actin, stress-related proteins as well as proteins associated to cellular processes, among which some residents of the endoplasmic reticulum. This study constitutes the first investigation of the root proteome in this important forest tree genus.

Proteomics research on forest trees, the most recalcitrant and orphan plant species.

The contribution of proteomics to the knowledge of forest tree (the most recalcitrant and almost forgotten plant species) biology is being reviewed and discussed, based on the author's own research work and papers published up to November 2010. This review is organized in four introductory sections starting with the definition of forest trees (1), the description of the environmental and economic importance (2) and its derived current priorities and research lines for breeding and conservation (3) including forest tree genomics (4). These precede the main body of this review: a general overview to proteomics (5) for introducing the forest tree proteomics section (6). Proteomics, defined as scientific discipline or experimental approach, it will be discussed both from a conceptual and methodological point of view, commenting on realities, challenges and limitations. Proteomics research in woody plants is limited to a reduced number of genera, including Pinus, Picea, Populus, Eucalyptus, and Fagus, mainly using first-generation approaches, e.g., those based on two-dimensional electrophoresis coupled to mass spectrometry. This area joins the own limitations of the technique and the difficulty and recalcitrance of the plant species as an experimental system. Furthermore, it contributes to a deeper knowledge of some biological processes, namely growth, development, organogenesis, and responses to stresses, as it is also used in the characterization and cataloguing of natural populations and biodiversity (proteotyping) and in assisting breeding programmes.

Proteomic analysis of differentially expressed proteins in hepatocellular carcinoma developed in patients with chronic viral hepatitis C.

Hepatocellular carcinoma (HCC) is a major complication of chronic viral hepatitis C. Therapy for HCC is still disappointing. It is thus of great importance to identify novel HCC markers for early detection of the disease, and tumor-specific proteins as potential therapeutic targets. We have used a proteomic approach to identify new proteins involved in HCC development. Four cases of HCC developing from chronic viral hepatitis C were analyzed by two-dimensional electrophoresis (2-DE), and results were compared to those of paired adjacent non-tumorous liver tissues. For MS fingerprinting, protein spots with differential intensity between HCC and non-tumorous liver were directly cut out of gels and processed for MALDI-MS and nano-LC-ESI-MS/MS analysis. Approximately 850 spots were visualized in each gel. The comparative analysis of paired samples indicated that 345 protein spots showed significant differences in expression level between non-tumor and tumor tissue. Among the 345 protein spots analyzed, 238 spots corresponding to 155 different proteins were identified; 49 proteins were up-regulated, whereas 106 proteins were down-regulated. Among these 155 proteins, 91 proteins were regulated in at least three cases. Although 52 out of these 91 proteins have been already described by previous proteomic or transcriptomic studies, or are already known to be involved in hepatocarcinogenesis, this experiment revealed 39 new proteins differentially expressed in HCC developing from viral hepatitis C. Variations in protein accumulation were confirmed for two selected proteins (apolipoprotein E, chloride intracellular channel 1) by Western blotting in ten additional cases of HCC developing in patients with viral hepatitis C.

The proteome of maritime pine wood forming tissue.

Wood is one of our most important natural resources. Surprisingly, we know hardly anything about the details of the process of wood formation. The aim of this work was to describe the main proteins expressed in wood forming tissue of a conifer species (Pinus pinaster Ait.). Using high resolution 2-DE with linear pH gradient ranging from 4 to 7, a total of 1039 spots were detected. Out of the 240 spots analyzed by MS/MS, 67.9% were identified, 16.7% presented no homology in the databases, and 15.4% corresponded to protein mixtures. Out of the 57 spots analyzed by MALDI-MS, only 15.8% were identified. Most of the 175 identified proteins play a role in either defense (19.4%), carbohydrates (16.6%) and amino acid (14.9%) metabolisms, genes and proteins expression (13.1%), cytoskeleton (8%), cell wall biosynthesis (5.7%), secondary (5.1%) and primary (4%) metabolisms. A summary of the identified proteins, their putative functions, and behavior in different types of wood are presented. This information was introduced into the PROTICdb database and is accessible at http://cbib1.cbib.u-bordeaux2.fr/Protic/Protic/home/index.php. Finally, the average protein amount was compared with their respective transcript abundance as quantified through EST counting in a cDNA-library constructed with mRNA extracted from wood forming tissue.