Globulus Clones Research Articles

Relationships between diameter growth and functional wood anatomy in Eucalyptus globulus clones

Aim of study: We aimed to 1) analyze the variability of wood hydraulic anatomical traits in 10 clones of E. globulus Labill. with different growth rates, and 2) determine whether the magnitude of diameter growth affects the relationships between anatomical variables and diameter at breast height (DBH). Area of study: 25-year-old common garden trial in Balcarce, Buenos Aires, Argentina. Material and methods: We measured vessel diameter and number per unit area in transverse histological sections of stem wood, and calculated the proportion of vessel lumens, vessel composition (S), and theoretical specific hydraulic conductivity (Ks) of 10 E. globulus clones of high (HG) and low (LG) mean growth rates (measured as DBH) under field conditions. Main results: There was a difference in the range of variability in hydraulic anatomy between HG and LG clones, with LG clones showing a wider range. HG clones had wood with larger and fewer vessels and higher S compared to LG clones, with similar Ks between both growth groups. No clear or strong trends were observed between wood anatomy and DBH within the HG and LG groups, but across all clones a high correlation (Spearman coefficient r; p<0.001) was observed between vessel number ‒ DBH (r= -0.68), and S ‒ DBH (r= 0.74). These correlations were driven by contrasting mean values of both growth groups. Research highlights: Commercial E. globulus clones present a relatively large variation in anatomical and hydraulic strategies. However, in contrast to what is postulated for various woody species, there was no clear relationship between theoretical hydraulic efficiency and individual diameter growth rate in the genotypes studied.

Early testing for improving growth under water shortage in Eucalyptus globulus Labill.

Aim of study: We aimed at identifying differences in the response to water shortage between Eucalyptus globulus clones. Area of study: Field trials were established in SW Spain. Material and methods: Potted plants from six clones were grown in a greenhouse for 35 days under two watering regimes. Two clones were F0 genotypes and the other four were F1 (hybrid) genotypes, including one inbred clone. Differences in stomatal conductance, hydraulic traits, growth and specific leaf area (SLA) were analyzed. Main results: Water shortage decreased SLA, growth in height and leaf area and leaf-specific hydraulic conductivity (KLmax). We measured the highest growth in F1 genotypes and the lowest in the clone in which SLA was lowest. The inbred clone showed the highest growth reduction under water shortage. There was substantial hysteresis between leaf water potential (Y) and native embolism, most probably a result of combined cavitation and refilling. High losses of hydraulic conductance were compatible with high stomatal conductances. Maximum values of stomatal conductance decreased with the soil water content estimated from predawn Y and were lowest in the inbred clone, showing less plasticity and a diminished ability to cope with high temperatures, which could explain its poor development under field conditions. Research highlights: Soil water content and predawn Y appeared as critical factors controlling stomata closure, while stomatal conductance and SLA could be useful to predict differences in growth and survival from early trials.

Effect of different cytokinin sources on in vitro/ex vitro multiplication and rooting rate in Eucalyptus spp.

Eucalyptus globulus Labill. is of great interest to the pulp industry. However, the species is highly susceptible to leaf spot caused by Teratosphaeria nubilosa and has a low rooting capacity. In order to overcome these limitations, INIA has been implementing a genetic improvement program for this species and the generation of interspecific hybrids. The strategy is based on the selection of E. globulus clones with high precocity or foliage turnover, since the adult foliage is more tolerant to T. nubilosa. In addition, controlled crosses of E. globulus and E. maidenii with E. grandis are carried out to incorporate disease resistance and ease of rooting. The multiplication of the selected materials is carried out by micropropagation, but the low rooting capacity is a trait that needs improvement. The cytokines necessary for in vitro proliferation can have a negative effect on the rooting process. Benzyladenine is the most commonly used cytokine because of its low cost and easy availability, with some negative effects on rooting, while meta-topolin is a cytokine of lesser impact. The objective of this study was to evaluate the effect of benzyladenine and meta-topolin on the multiplication rate, rooting percentage and plantlets survival of E. globulus, E. grandis × E. globulus, and E. grandis × E. maidenii. In multiplication media, benzyladenine was supplied at 0.4 µM, while two concentrations of meta-topolin (0.4 and 2 µM) were used. Preliminary results indicated a higher multiplication rate with the inclusion of meta-topolin in both concentrations. The in vitro rooting percentage of explants treated with meta-topolin was 51% and 44%, at 0.4 and 2 µM, respectively, while with benzyladenine was lesser (37%). After 28 days post-transplanting, survival was higher in meta-topolin-derived plants. Meta-topolin improved multiplication, in vitro rooting and acclimation of plants. The use of this hormone could improve micropropagation protocols, facilitating rooting and multiplication of valuable genotypes.

Exploring reproductive biology in Eucalyptus globulus clones under distinct climatic conditions

AbstractDespite the great economic benefits, one of the major threats of introducing species into exotic ranges is their ability to regenerate and spread to novel environments. For example, Eucalyptus globulus plantations have been established in many parts of the world, using seedlings or clonal material, the latter often from advanced generation tree improvement programs. However, major concerns exist about this species’ ability to invade non-native areas. Therefore, it is necessary to study whether breeding programs affect this species’ reproductive biology to assess the risk of invasiveness of these improved trees. In this sense, this work compared the E. globulus phenological behaviour and reproductive structures production of three clonal and one seedling-based stand across two distinct climatic zones (mesic and xeric) in Portugal. Through binocular observation, four different stands (seedlings descending from open-pollinated crosses and three clonal-stands) were monitored every three weeks for different reproductive phenological stages, namely the presence of flowers and capsules. Clonal stands presented a shorter flowering season, less abundant reproductive structures, and higher phenological variability across sites than seedling-based stands. Phenological patterns and fructification abundance also varied between the three clonal stands. These results suggest that clones with decreased reproductive ability can be chosen during the breeding process, lowering the dispersal risk while enhancing productivity. Thus, understanding the phenological behaviour of E. globulus is critical to more sustainable agroforestry by reducing the spread risks.

Contrasting ethylene-mediated responses to waterlogging in four Eucalyptus globulus Labill. clones

We investigated the effects of overabundant watering in Eucalyptus globulus, a widely used species for pulp production with a low to moderate tolerance to waterlogging. We hypothesized that (1) overabundant watering would result stressful and trigger some of the well-known physiological responses to waterlogging and (2) different clones may show a different response to overabundant watering. Twelve potted plants from four E. globulus clones were watered with nozzles for twenty minutes, twice a day along 21 days. Other eight plants per clone were kept as controls and watered every two to four days. Overabundant watering caused a decrease in total leaf area after 21 days of treatment. Chlorophyll content and net photosynthetic rate were lower in plants under overabundant watering with no significant decrease in the rate of transpiration and no photoinhibition. Root pressure-generated xylem sap exudation was 3–17 times higher in plants under overabundant watering. We found significant differences between clones in ethylene production, growth, chlorophyll fluorescence parameters and root thickening. The highest increase in ethylene production was measured in clone c14, in which we measured the highest decrease in growth, chlorophyll content and the electron transport rate under overabundant watering. In conclusion, overabundant watering imposed a moderate stress, which allowed the plants to respond at the root and leaf level trying to drain the excess of soil water through transpiration. Our results support the viability of early testing for waterlogging resistance in Eucalyptus globulus clones, in order to select the best attributes to identify waterlogging-resistant genotypes.

Gene expression analysis in Eucalyptus globulus exposed to drought stress in a controlled and a field environment indicates different strategies for short- and longer-term acclimation.

Previous knowledge suggested the involvement of specific pathways/proteins that could be identified as potential molecular indicators linked to enhanced drought tolerance in Eucalyptus globulus. Here, we looked for specific variations in key transcripts of two Eucalyptus globulus clones (AL-18 and AL-13) exposed to water deficit and rehydration with two main goals: (i) to check if and how transcripts potentially associated with stress response and protection are modulated in a controlled experiment; and (ii) to verify if the transcript response is robust in a field case study. Our results showed that the controlled experiment induced a severe acute stress that resulted in a strong realignment of gene expression resulting from an overwhelming of physiological adjustments to water limitation. A number of transcripts exhibited altered abundance after the acute water stress: reduction of RuBisCO activase and mitochondrial glycine cleavage system H protein, and increase of isoflavone reductase. Malate dehydrogenase, catalase, dehydration response element B1A and potassium channel GORK showed a different abundance pattern in each clone. The stress in the field was more moderate and chronic and the plants were able to deal with the stress primarily through physiological adjustments resulting in much smaller changes in gene expression. The transcripts of clone AL-18 showed few alterations between irrigated and non-irrigated plants throughout the experiment, while the transcript changes found in clone AL-13 highlighted the impact of early rewatering rather than growing under extended drought typical of a Mediterranean summer. Although a few concurrent responses were found, the results obtained in the field study draw a very distinct picture when compared with the controlled experiment.

Combined Drought and Heat Activates Protective Responses in Eucalyptus globulus That Are Not Activated When Subjected to Drought or Heat Stress Alone.

Aiming to mimic a more realistic field condition and to determine convergent and divergent responses of individual stresses in relation to their combination, we explored physiological, biochemical, and metabolomic alterations after drought and heat stress imposition (alone and combined) and recovery, using a drought-tolerant Eucalyptus globulus clone. When plants were exposed to drought alone, the main responses included reduced pre-dawn water potential (Ψpd) and gas exchange. This was accompanied by increases in malondialdehyde (MDA) and total glutathione, indicative of oxidative stress. Abscisic acid (ABA) levels increased while the content of jasmonic acid (JA) fell. Metabolic alterations included reductions in the levels of sugar phosphates accompanied by increases in starch and non-structural carbohydrates. Levels of α-glycerophosphate and shikimate were also reduced while free amino acids increased. On the other hand, heat alone triggered an increase in relative water content (RWC) and Ψpd. Photosynthetic rate and pigments were reduced accompanied by a reduction in water use efficiency. Heat-induced a reduction of salicylic acid (SA) and JA content. Sugar alcohols and several amino acids were enhanced by the heat treatment while starch, fructose-6-phosphate, glucose-6-phosphate, and α-glycerophosphate were reduced. Contrary to what was observed under drought, heat stress activated the shikimic acid pathway. Drought-stressed plants subject to a heat shock exhibited a sharp decrease in gas exchange, Ψpd and JA, no alterations in electrolyte leakage, MDA, starch, and pigments and increased glutathione pool in relation to control. Comparing this with drought stress alone, subjecting drought stressed plants to an additional heat stress alleviated Ψpd and MDA, maintained an increased glutathione pool and reduced starch content and non-structural carbohydrates. A novel response triggered by the combined stress was the accumulation of cinnamate. Regarding recovery, most of the parameters affected by each stress condition reversed after re-establishment of control growing conditions. These results highlight that the combination of drought and heat provides significant protection from more detrimental effects of drought-stressed eucalypts, confirming that combined stress alter plant metabolism in a novel manner that cannot be extrapolated by the sum of the different stresses applied individually.

IBA AND MICROCUTTING COLLECTIONS IN THE MICROPROPAGATION OF Eucalyptus spp HYBRID CLONES.1

ABSTRACT This study aims to evaluate the effect of IBA concentrations and microcuttings successive collections in the micropropagation of Eucalyptus grandis x E. urophylla and Eucalyptus urophylla x E. globulus clones. Clumps containing six to eight buds of clones established in vitro were transferred to a 250 mL glass flask in JADS semisolid medium. Successive collections were performed every 20 days for Eucalyptus grandis x E. urophylla clone and every 30 days for Eucalyptus urophylla x E. globulus clone. The following variables were evaluated under in vitro conditions: number of shoots > 0.5 cm, number of microcuttings > 2 cm, length of the longest microcutting, and shoots vigor. Under ex vitro conditions, in the greenhouse and shade house, the following variables were evaluated: seedling height, percentage of survival, stem diameter, percentage of root observed at the lower end of the tube, and seedling vigor. In full sun (ex vitro), the following variables were analyzed: seedling height, stem diameter, survival, number of roots, root volume, seedling vigor, and shoot and root dry matter. Good in vitro microcuttings productivity was observed over the successive collections. IBA levels were adjusted for each clone, ranging from 0.25 to 0.50 mg L-1 for Eucalyptus grandis x E. urophylla clone, and from 0.75 to 1.0 mg L-1 for Eucalyptus urophylla x E. globulus clone. IBA concentrations led to residual effects under ex vitro conditions, providing good rooting and survival for Eucalyptus grandis x E. urophylla and Eucalyptus urophylla x E. globulus clones at IBA concentrations between 0.25 and 0.50 mg L-1 and between 0.50 and 1.0 mg L-1, respectively.

Efficiency of EUChip60K pipeline in fingerprinting clonal population of Eucalyptus globulus

EUChip60K is a good option to evaluate clonal identity in a breeding population of E. globulus. Breeding populations normally are composed of numerous progenies which become the base for the development of new genetic material for propagation and to advance generations of breeding. Errors in clonal identity could have a great impact on expected gain from the breeding programs. In most breeding programs, a moderate percentage of genetic material is often mislabeled. Molecular markers, such as microsatellites, are being currently used to detect such errors. These markers have been demonstrated to be efficient and low cost for massive analysis of samples in a forest breeding program. However, they have a very low transferability across laboratories and the analysis can be time consuming. In this study, we show the high reproducibility level for genotyping Eucalyptus globulus clones and demonstrate the high ability to identify errors between clones using the EUChip60K, a SNP panel with around 60K SNP markers. Even though EUChip60K is a multi-species array for Eucalyptus, it could be easily applied to an E. globulus clonal program. This is the first case of study to assess the EUChip60K performance to evaluate identity errors in a clonal population of E. globulus.

ASSESSMENT OF GENETIC STABILITY OF MICROPROPAGATED Eucalyptus globulus Labill HYBRID CLONES BY MEANS OF FLOW CYTOMETRY AND MICROSATELLITES MARKERS

ABSTRACT Flow cytometry and microsatellite markers were used to determine a genetic fidelity of micropropagated plants from the two Eucalyptus urophylla x E. globulus clones and a Eucalyptus grandis x E. globulus clone derived from adult material. Clones were repeatedly subcultured for 25 subcultures on MS medium supplemented with BA (2.22 µM) and ANA (0.05 µM) for in vitro shoot multiplication. The elongation was performed in MS culture medium supplemented with AIB (2.46 µM) and BA(0.22 µM). The ex vitro rooting and acclimatization phases were lead at the same time. The micropropagated clones showed genetic stability by flow cytometry and microsatellite markers. The results proved that micropropagation, for purposes of rejuvenation, can be a viable technique to generate genetically stable or identical E. globulus hybrid clones.

Integrated proteomics and metabolomics to unlock global and clonal responses of Eucalyptus globulus recovery from water deficit

Water availability is well known for impacting productivity of Eucalyptus but comprehensive knowledge on cellular pathways involved in recovery and tolerance is scarce. In this context, we aimed to unveil putative mechanisms that account for drought recovery of E. globulus, and to identify specific strategies that make a clone more adapted to water deficit. We resorted to comparative proteome (using difference gel electrophoresis) and metabolome [using Gas chromatography–mass spectrometry (GC–MS)] analyses in two E. globulus clones that exhibit physiological differences in their capacity to tolerate water shortage and restoration; also, interpretable networks were constructed coupled with previously assessed physiological matrices in order to interrogate the large datasets generated and develop a clear and integrative analysis. Our study enabled the separation and isolation of 2031 peptide spots, 217 of which were identified. GC–MS yielded the detection of 121 polar metabolites. Water shortage negatively affected photosynthesis, gene regulation, cell growth and secondary metabolites; enhanced photo protection, osmoprotection, and other defence-related pathways; and caused a shift from chloroplastic to mitochondrial energy generation. Recovery was characterised by upregulation of all previously described pathways. The analysis of the resilient clone AL-18, which presented a network very distinct from the responsive clone AL-10, reinforced the role of specific photosynthetic and defence-related proteins as key players in mediating drought tolerance and revealed new players: glutamine synthetase, malate dehydrogenase and isoflavone reductase-like protein. This study provides a set of novel proteins and pathways involved in drought stress that represent potential drought tolerance markers for early selection of Eucalyptus.

Early growth of invasive acacias as a potential biomass-for-energy source under Mediterranean conditions

Most of the short rotation energy coppices used in Northern and Central Europe may be of limited value under Mediterranean climates. Abandoned acacia stands with an invasive behaviour, of environmental concern in many Mediterranean countries, may perform better as biomass producers under summer drought than those coppices. Seedlings of invasive Acacia dealbata, A. melanoxylon and A. pycnantha were planted to test for early growth performance under rain-fed and irrigated conditions. Rooted cuttings of an E. globulus clone were also established as a reference for biomass production. Plants were harvested after one year, sorted by biomass components and oven-dried. Above-ground dry weight was calculated on an area basis, accounting for survival. A. dealbata and A. melanoxylon showed low survival and biomass production, even irrigated, whereas A. pycnantha had better survival and growth performance than eucalypt, even in rain-fed plots, suggesting potential for the harvest of this drought-adapted species as a biomass source.

Physiological and biochemical responses to severe drought stress of nine Eucalyptus globulus clones: a multivariate approach.

Seasonal drought, typical of temperate and Mediterranean environments, creates problems in establishing plantations and affects development and yield, and it has been widely studied in numerous species. Forestry fast-growing species such as Eucalyptus spp. are an important resource in such environments, selected clones being generally used for production purposes in plantations in these areas. However, use of mono-specific plantations increases risk of plant loss due to abiotic stresses, making it essential to understand differences in an individual clone's physiological responses to drought stress. In order to study clonal differences in drought responses, nine Eucalyptus globulus (Labill.) clones (C14, C46, C97, C120, C222, C371, C405, C491 and C601) were gradually subjected to severe drought stress (<14% of field capacity). A total of 31 parameters, physiological (e.g., photosynthesis, gas exchange), biochemical (e.g., chlorophyll content) and hormonal (abscisic acid [ABA] content), were analysed by classic and multivariate techniques. Relationships between parameters were established, allowing related measurements to be grouped into functional units (pigment, growth, water and ABA). Differences in these units showed that there were two distinct groups of E. globulus clones on the basis of their different strategies when faced with drought stress. The C14 group (C14, C120, C405, C491 and C601) clones behave as water savers, maintaining high water content and showing high stomatal adjustment, and reducing their aerial growth to a great extent. The C46 group (C46, C97, C222 and C371) clones behave as water spenders, reducing their water content drastically and presenting osmotic adjustment. The latter maintains the highest growth rate under the conditions tested. The method presented here can be used to identify appropriate E. globulus clones for drought environments, facilitating the selection of material for production and repopulation environments.

Pre and post-severance effects of light quality on carbohydrate dynamics and microcutting adventitious rooting of two Eucalyptus species of contrasting recalcitrance

Adventitious rooting is a complex developmental response affected by genetic and environmental factors. Radiation quality effects on adventitious rooting depend on characteristics such as species, growth stage, irradiance, spectral quality, and time of exposure. Eucalyptus is an essential genus for the paper industry, and high yield plantations depend on adventitious rooting of selected genotypes. This work addressed two hypotheses: (1) radiation quality equally affects adventitious rooting in Eucalyptus species of different recalcitrance; (2) adventitious rooting outcome depends on both donor plant and cutting radiation quality treatments. To that end, the easy-to-root Eucalyptus grandis and the recalcitrant Eucalyptus globulus were evaluated. The effect of white, blue, red and far-red radiation enrichment on microcuttings and donor plants of both species was evaluated in relation to rooting. There was no effect of radiation quality on adventitious rooting of E. grandis or when radiation treatments were applied to E. globulus microcuttings. In contrast, donor plants of E. globulus, grown in medium devoid of sucrose and exposed to far-red radiation, yielded microcuttings showing higher rooting percentage, even in the absence of exogenous auxin in the rooting medium. Sucrose in donor plant medium abolished the positive effect of far-red radiation. An increase in endogenous soluble sugars and starch contents in basal microcuttings was associated with far-red radiation treatment of donor plants. These results underline the importance of appropriate carbohydrate partitioning in donor plants for adventitious rooting of cuttings and provide a basis for understanding and overcoming rooting recalcitrance in E. globulus clones.

Estabelecimento &lt;i&gt;in vitro&lt;/i&gt; de clones híbridos de &lt;i&gt;Eucalyptus globulus&lt;/i&gt;

http://dx.doi.org/10.5902/198050986626O presente estudo teve como objetivos avaliar o comportamento de clones de Eucalyptus grandis W.Hill ex Maiden x Eucalyptus globulus Labill e Eucalyptus urophylla S.T.Blake x Eucalyptus globulus Labill e o efeito de diferentes introduções in vitro (aos 30, 90 e 150 dias após a poda do ápice das minicepas) na fase de estabelecimento in vitro. As minicepas, fornecedoras dos explantes para a introdução in vitro, foram conduzidas em minijardim clonal semi-hidropônico. Segmentos nodais de 21 clones de Eucalyptus urophylla x Eucalyptus globulus e 8 clones de Eucalyptus grandis x Eucalyptus globulus foram coletados, desinfestados e inoculados em meio de cultura MS, suplementado com 0,5 mg L-1 de BAP e 0,1 mg L-1 de ANA. Após 30 dias em cultura, foram feitas as avaliações e concluiu-se que houve variação entre clones, bem como entre introduções in vitro, para as variáveis estudadas, sendo possível o estabelecimento in vitro da maioria dos clones.

Drought effects on water use efficiency, freezing tolerance and survival of Eucalyptus globulus and Eucalyptus globulus × nitens cuttings

Genetic improvement of Eucalyptus genotypes for drought and frost resistance is essential for successful intensive management of commercial plantations. Understanding the physiological mechanisms that relate water use and frost resistance for highly deployed genotypes may allow for better prediction of their future performance, genetic selection and seedling management for site specific purposes. We studied whether instantaneous water use efficiency (WUE i ) may serve as drought, freezing and photoinhibition tolerance predictor by studying its response on six E. globulus clones (Eg1–Eg6) and four E. globulus × E. nitens hybrid seedlings (Egn1–Egn4) under drought and irrigated (control) treatments. Net photosynthesis (A) and transpiration (E) were studied using a gas exchange system in order to calculate WUE i (A/E). Simultaneous chlorophyll a fluorescence measurements were performed to assess the non photochemical quenching components. Frost tolerance of plants under control and drought treatments were evaluated by measuring temperatures that exert 50% photoinactivation of photosystem II. Finally, drought tolerance was evaluated by plant survival within each genotype after rehydration. Our results showed significant genotype variability in the rate of soil and xylem water potential decrease during drought. While most of the genotypes reached −4.0 MPa in about 35 days of drought, genotypes Eg6 and Egn4 required 56 days of drought to reach this xylem water potential. WUE i exhibited significant differences among genotypes and irrigation treatments. Genotypes Eg5 and Egn4 increased their WUE i between 70 and 80% after drought. This was associated with a more conservative control of water loss at the stomatal level combined with maintenance of relatively higher rates of net photosynthesis than the other genotypes under drought conditions. Plants exposed to drought were more freezing tolerant than control plants, having in average 3°C lower LT50 than well irrigated ones. There was no a clear correlation between WUE i and drought tolerance or drought-induced photoinhibition, however WUE i was inversely correlated with LT50. Our results suggest that WUE i is not suitable by itself to select drought tolerant genotypes, but may provide evidence for discarding drought sensitive genotypes. In addition, it could provide valuable information to select for freezing tolerance.

Genomic of Eucalyptus globulusfor pulp and biofuels

Eucalyptus is a species of great interest for the pulp and paper industry. Worldwide there are 20 million hectares planted and within Chile a total of 700.000 ha, being the main species grown for short-fibre pulp Eucalyptus globulus, which is extensively used for pulping due to its fast growth, high pulp yield, and good fiber properties. Genetic programs of this specie have been oriented in improving commercial traits as volume, growth and form and recently traits as pulp ability have been included. Understanding the genomics of wood formation and identifying genes that are responsible for the traits of interest is a major challenge. An increase in 2% of pulp yield in trees can translate in a large economical gain, but most importantly there will be less pressure for land. There are two ways of reaching this goal, one through the use of genetic engineering and the second by genetic improvement, in both cases it is important to understand and identify the main genes involved in conferring the traits of interest. To identify these genes, different studies have been carried out, mainly by using EST libraries, finding a large number of sequences. Although there is large information regarding the enzymes and genes involved in the lignin pathway, little information is available regarding other metabolic pathways as cellulose and hemicellulose, as well as genes responsible for traits as density, cell wall, among others. In this study a detailed phenotypic characterization of 100 different genotypes was made, determining pulp content, lignin, cellulose, hemicellulose and Syringyl/Guaiacyl (S/G) ratio, among others. By employing NIR models a larger number of E. globulus clones were characterized for the same properties. From the 300 genotypes studied, “contrasting genotypes”, with high (good) and with low (bad) density and pulp yield were selected. It was found that clones with high density and pulp yield had high glucan content, lignin rich in S units, high β-O-4 linkages and low lignin, and xylan content. Out of these contrasting genotypes, one of each, low and high pulp yield and density, were further studied to find candidate genes involved in pulp ability. An EST library was made for each genotype, which was sequenced by the 454 platform, giving a total of 21,000 sequences, out of which 250 were differentially expressed. Due to the low amount of Eucalyptus sequences available, 28% of these sequences blasted to ESTs from wine grape, 22% to poplar and less than 5% to eucalypts. From these sequences, genes involved in lignin pathway, cellulose biosynthesis as well a transcription factors were identified. F5H was one of the main genes studied, which was characterized at a biochemical and expression level.

Eucalyptusresearch in the post-genome era

With the efforts of the Joint Genome Institute (JGI) through the EUCAGEN Eucalyptus Genome Network reaching the final release of a Eucalyptus grandis reference genome (BRASUZ1), it is anticipated that this accomplishment will profoundly shape the future research of this global tree genus [1]. One of the first steps toward this end has been the refinement of the genome annotation. Robust models have been generated for protein-coding genes. Here we report the annotation of other pivotal genome constituents, transposable elements (TE) and micro RNA genes. TEs are not only the most dominant elements but are also major drivers of genome plasticity. Several bioinformatic strategies were employed to perform “de novo” and homology based prediction of repetitive elements [2]in the current genome release (version 1.0). A total of 53 distinct TE families could be identified, with the retrotransposon super-family being widely over-represented, as observed for the majority of plant genomes sequenced. Micro RNAs (miRNA), key players in post-transcriptional gene regulation, were annotated using a combination of massively parallel sequencing of small RNA libraries and a genome-wide computational screening to ascertain a compatible secondary structure of the precursor. Both experimental and “in silico” evidences enabled the annotation of 206 distinct miRNA loci comprising 36 different mir gene families including several miRNA isoforms. The blueprint provided by a high-quality reference genome, both in terms of sequence completeness and annotation, will leverage efforts to better characterize intra- and inter-specific sequence variation underlying the marked phenotypic differences among the hundreds of species comprising this genus. Recent advances of DNA sequencing technologies permit a comprehensive interrogation of several other individuals at a fraction of the cost. In this context, we have used Illumina (2x75bp) short read sequencing data of E. globulus clone X46 generated by JGI and made available through the EUCAGEN network to carry out two comparative genomics experiments. From 40X raw sequence data provided it was possible to use an equivalent of 20X coverage (~12Gbp). We first attempted to perform a de novo assembly using VELVET [3]. A total of 161,000 contigs were obtained the largest one sizing at ~3,5kb. In spite of the easy access and low cost of next generation sequencing technologies, these results suggest that even for relatively small forest tree genomes, current technical and computational limitations preclude comprehensive assembly, likely due to the ubiquitous occurrence of repetitive elements in such genomes. Nevertheless when we mapped the E. globulus sequencing data against the BRASUZ1 reference genome, 55% of the reads could be mapped with high confidence. From these, approximately 800,000 high quality single nucleotide polymorphisms (SNPs) could be identified clearly showing the key role that the reference genome will have for future genomic undertakings. The sheer number of molecular markers discovered in this experiment not only fosters more powerful studies on the evolutionary history and population genomics of eucalypts, but also inaugurates a new era in molecular breeding of species of this genus, providing genome-wide coverage for genomic selection and association studies.

Enraizamento de miniestacas de clones híbridos de Eucalyptus globulus

Este estudo teve como objetivo avaliar o efeito do regulador de crescimento ácido indolbutírico (AIB) no enraizamento de miniestacas apicais e intermediárias de clones de Eucalyptus urophylla x E. globulus e Eucalyptus grandis x E. globulus. O enraizamento das miniestacas foi conduzido durante 30 dias em casa de vegetação climatizada e, em seguida, as mudas foram transferidas para casa de sombra, permanecendo por 10 dias. O delineamento experimental utilizado foi em blocos ao acaso, em arranjo fatorial triplo, constituído de dois tipos de miniestacas (apicais e intermediárias), três concentrações de AIB (0, 2000 e 4000 mg L-1) e 11 clones, em três repetições, compostas de parcelas com 12 miniestacas por repetição. Não houve efeito do AIB sobre as características avaliadas. A sobrevivência e o enraizamento das miniestacas apicais foram significativamente superiores. Houve diferença significativa entre clones quanto às características avaliadas, exceto a sobrevivência após 30 dias na casa de vegetação.

Lignin composition and structure in young versus adult Eucalyptus globulus plants.

Lignin changes during plant growth were investigated in a selected Eucalyptus globulus clone. The lignin composition and structure were studied in situ by a new procedure enabling the acquisition of two-dimensional nuclear magnetic resonance (2D-NMR) spectra on wood gels formed in the NMR tube as well as by analytical pyrolysis-gas chromatography-mass spectrometry. In addition, milled-wood lignins were isolated and analyzed by 2D-NMR, pyrolysis-gas chromatography-mass spectrometry, and thioacidolysis. The data indicated that p-hydroxyphenyl and guaiacyl units are deposited at the earlier stages, whereas the woods are enriched in syringyl (S) lignin during late lignification. Wood 2D-NMR showed that β-O-4' and resinol linkages were predominant in the eucalypt lignin, whereas other substructures were present in much lower amounts. Interestingly, open β-1' structures could be detected in the isolated lignins. Phenylcoumarans and cinnamyl end groups were depleted with age, spirodienone abundance increased, and the main substructures (β-O-4' and resinols) were scarcely modified. Thioacidolysis revealed a higher predominance of S units in the ether-linked lignin than in the total lignin and, in agreement with NMR, also indicated that resinols are the most important nonether linkages. Dimer analysis showed that most of the resinol-type structures comprised two S units (syringaresinol), the crossed guaiacyl-S resinol appearing as a minor substructure and pinoresinol being totally absent. Changes in hemicelluloses were also shown by the 2D-NMR spectra of the wood gels without polysaccharide isolation. These include decreases of methyl galacturonosyl, arabinosyl, and galactosyl (anomeric) signals, assigned to pectin and related neutral polysaccharides, and increases of xylosyl (which are approximately 50% acetylated) and 4-O-methylglucuronosyl signals.