Microsatellite markers in candidate genes for wood properties and its application in functional diversity assessment in eucalyptus globulus

Background: Functional genetic markers have important implications for genetic analysis by providing direct estimation of functional diversity. Although high throughput sequencing techniques for functional diversity analysis are being developed nowadays, the use of already well established variable markers present in candidate genes is still an interesting alternative for mapping purposes and functional diversity studies. SSR markers are routinely used in most plant and animal breeding programs for many species including Eucalyptus. SSR markers derived from candidate genes (SSR-CG) can be used effectively in co-segregation studies and marker-assisted diversity management. Results: In the present study, eight new non reported SSRs were identified in seven candidate genes for wood properties in Eucalyptus globulus: cinnamoyl CoA reductase (CCR), homocysteine S-methyltransferase (HMT), shikimate kinase (SK), xyloglucan endotransglycosylase 2 (XTH2), cellulose synthase 3 (CesA3), glutathione S-transferase (GST) and the transcription factor LIM1. Microsatellites were located in promoters, introns and exons, being most of them CT dinucleotide repeats. Genetic diversity of these eight CG-derived SSR-markers was explored in 54 unrelated genotypes. Except for XTH2, high levels of polymorphism were detected: 93 alleles (mean of 13.1 sd 1.6 alleles per locus), a mean effective number of alleles (Ne) of 5.4 (sd 1.6), polymorphic information content values (PIC) from 0.617 to 0.855 and probability of Identity (PI) ranging from 0.030 to 0.151. Conclusions: This is the first report on the identification, characterization and diversity analysis of microsatellite markers located inside wood quality candidate genes (CG) from Eucalyptus globulus...

Utilization of rocks and ectomycorrhizal fungi to promote growth of eucalypt

The utilization of rocks as fertilizers is limited by their low solubility. However, solubilization may be achieved by some micro-organisms, such as ectomycorrhizal fungi (ECMf). The aim of this study was to evaluate the potential of seven isolates of ECMf to solubilize two rocks, alkaline breccia and granite, and to liberate potassium and phosphorus for Eucalyptus dunnii seedlings under greenhouse conditions. Fungal inoculants were produced in a peat-vermiculite-liquid medium mixture and added to the planting substrate at 10 percent. Rocks were ground up and added at 0.500 mg and 16.0 mg per plant, as a source of phosphorus and potassium, respectively. Other nutrients were added and E. dunnii seeds were sown. Control plants, non-inoculated, were fertilized with the same amount of phosphorus and potassium using soluble forms. After 90 days, the plant height, shoot dry weight, root length, phosphorus and potassium contents, and mycorrhizal colonization were evaluated. Alkaline breccia was more efficient than granite as a source of phosphorus and potassium for the plants, and may be an alternative to conventional fertilizers. Isolates UFSC-Pt22 (Pisolithus sp.) and UFSC-Pt186 (Pisolithus microcarpus) were the most efficient in promoting plant growth, mainly when combined with alkaline breccia to replace potassium and phosphorus fertilizers, respectively.

Efeito da estrutura da paisagem e do genótipo de Eucalyptus na abundância e controle biológico de Glycaspis brimblecombei Moore (Hemiptera: Psyllidae) / Effects of landscape structure and Eucalyptus genotype on the abundance and biological control of Glycaspis brimblecombei Moore (Hemiptera: Psyllidae)

Glycaspis brimblecombei Moore is an Australian native pest of Eucalyptus detected in Brazil in 2003. Since then, it has spread fast and colonized plantations in several states of the country. This study aimed to investigate the influence of cerrado remnants on the abundance and biological control of G. brimblecombei. We placed yellow sticky card traps to capture insects in four plantations of hybrid clones of Eucalyptus urophylla x Eucalyptus grandis ("Urograndis") and four plantations of E. urophylla x Eucalyptus camaldulensis ("Urocam"). Traps were placed in three areas of these plantations: center, border with cerrado and border without cerrado. We also collected leaves from the same clones to estimate psyllid egg and lerp abundance. The abundance of G. brimblecombei was lower in the plantation-cerrado border, and the inverse pattern was observed for microhymenopterans. The leaf abaxial surface showed a higher abundance of eggs and nymphs, probably as a consequence of a lower parasitism rate and mechanical removal by wind and rain. Egg number was higher on Urograndis than in Urocam clones, but the number of psyllid lerps was higher in the latter. Thus, the establishment of first instars is probably a critical event to psyllid infestation, and these differences may be caused by morphological, anatomical and biochemical leaf features of distinct clones. Our results suggest that the maintenance of native vegetation around plantations is a promising management practice to promote the natural biological control of G.brimblecombei, a strategy that would also enhance the preservation of cerrado remnants.

Genetic control of Eucalyptus urophylla and E. grandis resistance to canker caused by Chrysoporthe cubensis

Chrysophorte cubensis induced canker occurs in nearly all tropical and subtropical regions where eucalypts are planted, causing losses in both wood quality and volume productivity, especially so in the warmer and more humid regions of Brazil. The wide inter and intra-specific genetic variability of resistance to canker among Eucalyptus species facilitates the selection of resistant plants. In this study, we evaluated resistance to this pathogen in five Eucalyptus grandis (G) and 15 E. urophylla (U) trees, as well as in 495 individuals from 27 progenies derived from crosses between the trees. In the field, six-months-old test seedlings were inoculated with C. cubensis. Lesion length in the xylem and bark was measured eight months later. The results demonstrated that xylem lesions could preferentially be used for the selection of resistant clones. Eight trees (7 U and 1 G) were susceptible, and the remainder (8 U and 4 G) resistant. Individual narrow and broad sense heritability estimates were 17 and 81 percent, respectively, thereby suggesting that canker resistance is quantitative and highly dependent on dominance and epistasis.

Leaf anatomy and morphometry in three eucalypt clones treated with glyphosate / Anatomia e morfometria foliar em clones de eucalipto tratados com glyphosate

This work aimed to evaluate the effects of simulated drift of glyphosate on the morphoanatomy of three eucalypt clones and to correlate the intoxication symptoms on a microscopic scale with those observed in this visual analysis. The effects of glyphosate drift were proportional to the five doses tested, with Eucalyptus urophylla being more tolerant to the herbicide than E. grandis and urograndis hybrid. The symptoms of intoxication which were similar for the different clones at 7 and 15 days after application were characterized by leaf wilting, chlorosis and curling and, at the highest rates, by necrosis, leaf senescence and death. Anatomically glyphosate doses higher than 86.4 g.ha-1 caused cellular plasmolysis, hypertrophy and hyperplasia, formation of the cicatrization tissue and dead cells on the adaxial epidermis. The spongy parenchyma had a decrease, and the palisade parenchyma and leaf blade thickness had an increase. The increased thickness in leaf blade and palisade parenchyma may be related to the plant response to glyphosate action, as a form of recovering the photosynthetically active area reduced by necroses and leaf senescence caused by the herbicide.

Este trabalho teve como objetivo avaliar os efeitos da deriva simulada de glyphosate na morfoanatomia de três clones de eucalipto e correlacionar os sintomas de intoxicação em escala microscópica com aqueles observados à vista desarmada. Os efeitos da deriva do glyphosate foram proporcionais às doses testadas, sendo Eucalyptus urophylla mais tolerante ao herbicida que E. grandis e o híbrido urograndis. Os sintomas de intoxicação foram semelhantes para os diferentes clones testados, tanto aos 7 quanto aos 15 dias após a aplicação, sendo caracterizados, morfologicamente, por murcha, clorose e enrolamento foliar e, no caso das maiores doses, por necrose, senescência foliar e morte das plantas de eucalipto. Anatomicamente, doses de glyphosate superiores a 86,4 g.ha-1 provocaram plasmólise, hipertrofia e hiperplasia celular, formação de tecido de cicatrização e morte das células da face adaxial da epiderme. Observou-se diminuição na espessura do parênquima lacunoso e aumento na espessura do parênquima paliçádico e da lâmina foliar. O aumento na espessura da folha e do parênquima paliçádico podem estar relacionados à resposta das plantas ao glyphosate, como forma de compensar a área fotossinteticamente reduzida pelas necroses e senescência causadas pelo herbicida.

Eucalyptus microsatellites mined in silico: survey and evaluation.

Eucalyptus is an important short rotation pulpy woody plant, grown widely in the tropics. Recently, many genomic programmes are underway leading to the accumulation of voluminous genomic and expressed sequence tag sequences in public databases. These sequences can be utilized for analysis of simple sequence repeats (SSRs) and single nucleotide polymorphism (SNPs) available in the transcribed genes. In this study, in silico analysis of 15,285 sequences representing partial and full-length mRNA from Eucalyptus species for their use in developing SSRs or microsatellites were carried out. A total of 875 EST-SSRs were identified from 772 SSR containing ESTs. Motif size of 6 for dinucleotide and 5 for trinucleotide, tetranucleotide, and pentanucleotides were considered in locating the microsatellites. The average frequency of identified SSRs was 12.9%. The dinucleotide repeats were the most abundant among the dinucleotide, trinucleotide and tetranucleotide motifs and accounted for 50.9% of the Eucalyptus genome. Primer designing analysis showed that 571 sequences with SSRs had sufficient flanking regions for polymerase chain reaction (PCR) primer synthesis. Evaluation of the usefulness of the SSRs showed that EST-derived SSRs can generate polymorphic markers as all the primers showed allelic diversity among the 16 provenances of E. tereticornis.

Plant microsatellite genotyping with 4-color fluorescent detection using multiple-tailed primers

We extended the concept of fluorescent microsatellite genotyping with a single-universal tailed primer to the simultaneous use of three different tailed primers to allow multiplexed 4-color detection for medium throughput genotyping of plant species. The method was tested on Eucalyptus DNA samples using three forward primer sequences of human microsatellite markers labeled with different fluorescent dyes. The robustness of the method was tested for the simultaneous detection and genetic analysis of microsatellites in a genetic mapping experiment. This method allows reliable and cost-effective genotyping with the same level of multiplexing attained in regular microsatellite fluorescent detection assays. Besides the enhanced quality of the genotypic data provided by the fluorescent detection method when compared to colorimetric ones, the economy brought about by this method becomes greater with an increasing number of microsatellite markers. This method has been particularly useful for genotyping populations of several tropical tree species addressing community-wide population genetics and conservation questions.

In silico evaluation of the Eucalyptus transcriptome

The expressed sequence tags (ESTs) produced in the Forests project provide an invaluable opportunity to assess the Eucalyptus transcriptome. Besides providing information on the different proteins produced by this plant, it is possible to infer gene expression profiles because non-normalized cDNA libraries were used. The EST frequency from any gene is correlated to the transcript levels in the tissues from which the cDNA libraries were constructed. The goal of this work was to identify Eucalyptus genes that showed either differential expression pattern or were ubiquitously expressed in the tissues sampled in the Forests project. Six robust statistical tests and very restrictive rules were applied to gain confidence in the in silico data aiming to avoid false positives. Several genes with interesting expression profiles were identified and some of them were validated by RT-PCR

Eucalyptus ESTs involved in the production of 9-cis epoxycarotenoid dioxygenase, a regulatory enzyme of abscisic acid production

Abscisic acid (ABA) regulates stress responses in plants, and genomic tools can help us to understand the mechanisms involved in that process. FAPESP, a Brazilian research foundation, in association with four private forestry companies, has established the FORESTs database (https://forests.esalq.usp.br). A search was carried out in the Eucalyptus expressed sequence tag database to find ESTs involved with 9-cis epoxycarotenoid dioxygenase (NCED), the regulatory enzyme for ABA biosynthesis, using the basic local BLAST alignment tool. We found four clusters (EGEZLV2206B11.g, EGJMWD2252H08.g, EGBFRT3107F10.g, and EGEQFB1200H10.g), which represent similar sequences of the gene that produces NCED. Data showed that the EGBFRT3107F10.g cluster was similar to the maize (Zea mays) NCED enzyme, while EGEZLV2206B11.g and EGJMWD2252H08.g clusters were similar to the avocado (Persea americana) NCED enzyme. All Eucalyptus clusters were expressed in several tissues, especially in flower buds, where ABA has a special participation during the floral development process

Identification and frequency of transposable elements in Eucalyptus

Transposable elements (TE) are major components of eukaryotic genomes and involved in cell regulation and organism evolution. We have analyzed 123,889 expressed sequence tags of the Eucalyptus Genome Project database and found 124 sequences representing 76 TE in 9 groups, of which copia, MuDR and FAR1 groups were the most abundant. The low amount of sequences of TE may reflect the high efficiency of repression of these elements, a process that is called TE silencing. Frequency of groups of TE in Eucalyptus libraries which were prepared with different tissues or physiologic conditions from seedlings or adult plants indicated that developing plants experience the expression of a much wider spectrum of TE groups than that seen in adult plants. These are preliminary results that identify the most relevant TE groups involved with Eucalyptus development, which is important for industrial wood production

Using the FORESTS and KEGG databases to investigate the metabolic network of Eucalyptus

In this work we apply a bioinformatics approach to determine the most important enzymes of the metabolic network of Eucalyptus to determine the coverage of the genome in the FORESTS library. We conclude that the library does not cover completely the metabolism of the organism. However, some important pathways could be analyzed, especially the lignin synthesis. We found that four of the most important enzymes predicted are involved in this pathway

Boron transport in Eucalyptus. 2. Identification in silico of a putative boron transporter for xylem loading in eucalypt

Boron (B) is a low mobility plant micronutrient whose molecular mechanisms of absorption and translocation are still controversial. Many factors are involved in tolerance to Boron excess or deficiency. Recently, the first protein linked to boron transport in biological systems, BOR1, was characterized in Arabidopsis thaliana. This protein is involved in boron xylem loading and is similar to bicarbonate transporters found in animals. There are indications that BOR1 is a member of a conserved protein family in plants. In this work, FORESTS database was used to identify sequences similar to this protein family, looking for a probable BOR1 homolog in eucalypt. We found five consensus sequences similar to BOR1; three of them were then used in multiple alignment analysis. Based on amino acid similarity and in silico expression patterns, a consensus sequence was identified as a candidate BOR1 homolog, helping deeper experimental assays that could identify the function of this protein family in Eucalyptus

Genes encoding enzymes of the lignin biosynthesis pathway in Eucalyptus

Eucalyptus ESTs libraries were screened for genes involved in lignin biosynthesis. This search was performed under the perspective of recent revisions on the monolignols biosynthetic pathway. Eucalyptus orthologues of all genes of the phenylpropanoid pathway leading to lignin biosynthesis reported in other plant species were identified. A library made with mRNAs extracted from wood was enriched for genes involved in lignin biosynthesis and allowed to infer the isoforms of each gene family that play a major role in wood lignin formation. Analysis of the wood library suggests that, besides the enzymes of the phenylpropanoids pathway, chitinases, laccases, and dirigent proteins are also important for lignification. Colocalization of several enzymes on the endoplasmic reticulum membrane, as predicted by amino acid sequence analysis, supports the existence of metabolic channeling in the phenylpropanoid pathway. This study establishes a framework for future investigations on gene expression level, protein expression and enzymatic assays, sequence polymorphisms, and genetic engineering

Satellyptus: analysis and database of microsatellites from ESTs of Eucalyptus

The main goal of our research was to search for SSRs in the Eucalyptus EST FORESTs database (using a software for mining SSR-motifs). With this objective, we created a database for cataloging Eucalyptus EST-derived SSRs, and developed a bioinformatics tool, named Satellyptus, for finding and analyzing microsatellites in the Eucalyptus EST database. The search for microsatellites in the FORESTs database containing 71,115 Eucalyptus EST sequences (52.09 Mb) revealed 20,530 SSRs in 15,621 ESTs. The SSR abundance detected on the Eucalyptus ESTs database (29% or one microsatellite every four sequences) is considered very high for plants. Amongst the categories of SSR motifs, the dimeric (37%) and trimeric ones (33%) predominated. The AG/CT motif was the most frequent (35.15%) followed by the trimeric CCG/CGG (12.81%). From a random sample of 1,217 sequences, 343 microsatellites in 265 SSR-containing sequences were identified. Approximately 48% of these ESTs containing microsatellites were homologous to proteins with known biological function. Most of the microsatellites detected in Eucalyptus ESTs were positioned at either the 5 or 3 end. Our next priority involves the design of flanking primers for codominant SSR loci, which could lead to the development of a set of microsatellite-based markers suitable for marker-assisted Eucalyptus breeding programs

In silico characterization of microsatellites in Eucalyptus spp: abundance, length variation and transposon associations

This study assessed the abundance of microsatellites, or simple sequence repeats (SSR), in 19 Eucalyptus EST libraries from FORESTs, containing cDNA sequences from five species: E. grandis, E. globulus, E. saligna, E. urophylla and E. camaldulensis. Overall, a total of 11,534 SSRs and 8,447 SSR-containing sequences (25.5% of total ESTs) were identified, with an average of 1 SSR/2.5 kb when considering all motifs and 1 SSR/3.1 kb when mononucleotides were not included. Dimeric repeats were the most abundant (41.03%), followed by trimerics (36.11%) and monomerics (19.59%). The most frequent motifs were A/T (87.24%) for monomerics, AG/CT (94.44%) for dimerics, CCG/CGG (37.87%) for trimerics, AAGG/CCTT (18.75%) for tetramerics, AGAGG/CCTCT (14.04%) for pentamerics and ACGGCG/CGCCGT (6.30%) for hexamerics. According to sequence length, Class II or potentially variable markers were the most commonly found, followed by Class III. Two sequences presented high similarity to previously published Eucalyptus sequences from the NCBI database, EMBRA_72 and EMBRA_122. Local blastn search for transposons did not reveal the presence of any transposable elements with a cut-off value of 10-50. The large number of microsatellites identified will contribute to the refinement of marker-assisted mapping and to the discovery of novel markers for virtually all genes of economic interest

Eucalyptus ESTs associated with resistance to herbicide inhibitors of aromatic and branched-chain amino acid synthesis

Herbicides inhibit enzymatic systems of plants. Acetolactate synthase (ALS, EC = 4.1.3.18) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) are key enzymes for herbicide action. Hundreds of compounds inhibit ALS. This enzyme is highly variable, enabling the selective control of weeds in a number of crops. Glyphosate, the only commercial herbicide inhibiting EPSPS is widely used for non-selective control of weeds in many crops. Recently, transgenic crops resistant to glyphosate were developed and have been used by farmers. The aim of this study was the data mining of eucalypt expressed sequence tags (ESTs) in the FORESTs Genome Project database (https://forests.esalq.usp.br) related to these enzymes. Representative amino acid sequences from the NCBI database associated with ALS and EPSPS were blasted with ESTs from the FORESTs database using the tBLASTx option of the blast tool. The best blasting reads and clusters from FORESTs, represented as nucleotide sequences, were blasted back with the NCBI database to evaluate the level of similarity with available sequences from different species. One and seven clusters were identified as showing high similarity with EPSPS and ALS sequences from the literature, respectively. The alignment of EPSPS sequences allowed the identification of conserved regions that can be used to design specific primers for additional sequencings

In silico survey of resistance (R) genes in Eucalyptus transcriptome

A major goal of plant genome research is to recognize genes responsible for important traits. Resistance genes are among the most important gene classes for plant breeding purposes being responsible for the specific immune response including pathogen recognition, and activation of plant defence mechanisms. These genes are quite abundant in higher plants, with 210 clusters found in Eucalyptus FOREST database presenting significant homology to known R-genes. All five gene classes of R-genes with their respective conserved domains are present and expressed in Eucalyptus. Most clusters identified (93) belong to the LRR-NBS-TIR (genes with three domains: Leucine-rich-repeat, Nucleotide-binding-site and Toll interleucine 1-receptor), followed by the serine-threonine-kinase class (49 clusters). Some new combinations of domains and motifs of R-genes may be present in Eucalyptus and could represent novel gene structures. Most alignments occurred with dicots (94.3%), with emphasis on Arabidopsis thaliana (Brassicaceae) sequences. All best alignments with monocots (5.2%) occurred with rice (Oryza sativa) sequences and a single cluster aligned with the gymnosperm Pinus sylvestris (0.5%). The results are discussed and compared with available data from other crops and may bring useful evidences for the understanding of defense mechanisms in Eucalyptus and other crop species

Eucalyptus ESTs corresponding to the enzyme glutamine synthetase and the protein D1, sites of action of herbicides that cause oxidative stress

This work was aimed at locating Eucalyptus ESTs corresponding to the GS enzyme (Glutamine Synthetase, EC = 6.3.1.2) and to the D1 protein, which are directly related to resistance to herbicides that promote oxidative stress. Glutamine Synthetase corresponds to the site of action of the herbicide glufosinate. Herbicides that belong to groups such as ureas, uracils, triazines and triazinones act on the D1-Qb complex (receptor of electrons from the Photosystem II) by inactivating it. The clusters EGEQRT3302E01.g, EGEQRT3001F12.b; EGEZLV1203B04.g; EGBGFB1211H06.g and EGEZLV1205F09.g enclosed complete sequences (with 356 amino acids) of the Glutamine Synthetase enzyme. The cluster EGEQSL1054G06.g is a consensus of four reads and enclosed a complete sequence of D1 Protein (with 353 amino acids). The comparison of the sequences of Protein D1 from different species showed that the substitutions of serine (S) by glycine (G) or serine (S) by threonine (T) at the position 264 could produce plants resistant to herbicides that act on electron flow on Photosystem II. The sequence of amino acids corresponding to the cluster EGEQSL1054G06.g had a serine in position 264 indicating sensitivity of the Eucalyptus plants to herbicides that act on this site

Eucalyptus ESTs corresponding to the protoporphyrinogen IX oxidase enzyme related to the synthesis of heme, chlorophyll, and to the action of herbicides

This work was aimed at locating Eucalyptus ESTs corresponding to the PROTOX or PPO enzyme (Protoporphyrinogen IX oxidase, E.C. 1.3.3.4) directly related to resistance to herbicides that promote oxidative stress, changing the functionality of this enzyme. PROTOX, which is the site of action of diphenyl-ether (oxyfluorfen, lactofen, fomesafen), oxadiazole (oxadiazon and oxadiargyl), and aryl triazolinone (sulfentrazone and carfentrazone) herbicides, acts on the synthesis route of porphyrins which is associated with the production of chlorophyll a, catalases, and peroxidases. One cluster and one single read were located, with e-values better than e-70, associated to PROTOX. The alignment results between amino acid sequences indicated that this enzyme is adequately represented in the ESTs database of the FORESTs project

In silico analysis of Eucalyptus thioredoxins

The Eucalyptus Genome Sequencing Project (FORESTs), an initiative from the Brazilian ONSA consortium (Organization for Nucleotide Sequencing and Analysis), has achieved the sequencing of 123.889 EST clones from 18 different cDNA libraries. We have investigated the FORESTs data set to identify EST clusters potentially encoding thioredoxins (TRX). Two types of thioredoxin families described in plants, chloroplastic (TRXm/f/x/y) and cytosolic (TRXh), have been found in the transcriptome. Putative typical TRXs have been identified in fifteen clusters, four m-type, seven h-type, two f-type, one cluster for each x/y-types and one putative homologue of the TDX gene from Arabidopsis thaliana. One cluster presents an atypical active site WCMPS, different from the conserved WCGPC present in the other 15 clusters, and corresponds to a subgroup of cytosolic thioredoxins. Except in specific libraries from callus, roots, seedlings and wood tissues, thioredoxin deduced ESTs are found in all remaining libraries. According to the calculated frequencies of ESTs, chloroplastic thioredoxins are preferentially present in green tissues such as leaves whilst cytoplasmic thioredoxins are more general but demonstrate elevated frequencies in seedlings and flower tissues. TRX frequency patterns in the Eucalyptus transcriptome seem to indicate a good coherence with data from Arabidopsis thaliana gene expression