Sérgio Hermínio Brommonschenkel

The genome of Eucalyptus grandis

Eucalypts are the world’s most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries

BackgroundEucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing.ResultsWe describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1) digested with Hind III and BstY I, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes.ConclusionsThe two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae, including genome sequencing, gene isolation, functional and comparative genomics. Because they have been constructed using the same tree (E. grandis BRASUZ1) whose full genome is being sequenced, they should prove instrumental for assembly and gap filling of the upcoming Eucalyptus reference genome sequence.

Genome-wide analysis of differentially expressed genes during the early stages of tomato infection by a potyvirus.

Plant responses against pathogens cause up- and downward shifts in gene expression. To identify differentially expressed genes in a plant-virus interaction, susceptible tomato plants were inoculated with the potyvirus Pepper yellow mosaic virus (PepYMV) and a subtractive library was constructed from inoculated leaves at 72 h after inoculation. Several genes were identified as upregulated, including genes involved in plant defense responses (e.g., pathogenesis-related protein 5), regulation of the cell cycle (e.g., cytokinin-repressed proteins), signal transduction (e.g., CAX-interacting protein 4, SNF1 kinase), transcriptional regulators (e.g., WRKY and SCARECROW transcription factors), stress response proteins (e.g., Hsp90, DNA-J, 20S proteasome alpha subunit B, translationally controlled tumor protein), ubiquitins (e.g., polyubiquitin, ubiquitin activating enzyme 2), among others. Downregulated genes were also identified, which likewise display identity with genes involved in several metabolic pathways. Differential expression of selected genes was validated by macroarray analysis and quantitative real-time polymerase chain reaction. The possible roles played by some of these genes in the viral infection cycle are discussed.

Genetic mapping provides evidence for the role of additive and non-additive QTLs in the response of inter-specific hybrids of Eucalyptus to Puccinia psidii rust infection

Eucalypts are susceptible to a wide range of diseases. One of the most important diseases that affect Eucalyptus plantations worldwide is caused by the rust fungus Puccinia psidii. Here, we provide evidence on the complex genetic control of rust resistance in Eucalyptus inter-specific hybrids, by analyzing a number of full-sib families that display different patterns of segregation for rust resistance. These families are totally unrelated to those previously used in other inheritance studies of rust resistance. By using a full genome scan with 114 genetic markers (microsatellites and expressed sequence tag derived microsatellites) we also corroborated the existence and segregation of a resistance locus, explaining 11.5% of the phenotypic variation, on linkage group 3, corresponding to Ppr1. This find represents an additional validation of this locus in totally unrelated pedigree. We have also detected significant additive × additive digenic interactions with LOD >10.0 on several linkage groups. The additive and epistatic QTLs identified explain between 29.8 and 44.8% of the phenotypic variability for rust resistance. The recognition that both additive and non-additive genetic variation (epistasis) are important contributors to rust resistance in eucalypts reveals the complexity of this host-pathogen interaction and helps explain the success that breeding has achieved by selecting rust-resistant clones, where all the additive and non-additive effects are readily captured. The positioning of epistatic QTLs also provides starting points to look for the underlying genes or genomic regions controlling this phenotype on the upcoming E. grandis genome sequence.

Genetic diversity of populations of Hemileia vastatrix from organic and conventional coffee plantations in Brazil

Despite the importance of coffee leaf rust (Hemileia vastatrix), population studies of the pathogen are mostly focussed on identification of physiological races and there is no information on the genetic diversity and structure of populations of H. vastatrix in Brazil. In this study, 120 isolates of H. vastatrix were collected from organic and conventional coffee production systems from two coffee-producing regions in the state of Minas Gerais, Brazil. To test the hypothesis that H. vastatrix populations differ between conventional and organic plantations, random amplified polymorphicDNAmarkers were used to determine the genetic diversity and structure of the populations of H. vastatrix sampled from 12 fields within the two regions. Across all loci, high values of genetic diversity (h=0.28) and 92 haplotypes were detected. Based on the cluster analysis and q test, there was no association between origin of the isolate with either cropping system or region. In addition, considering the AMOVA analysis, most of the genetic variance occurred within populations (≈80%). There is evidence of random mating in the population of H. vastatrix in Brazil, which can partially explain the high genetic variability of the pathogen. Therefore, we conclude that genetic diversity in H. vastatrix populations in Minas Gerais was high and no clear clustering of isolates was detected that allowed establishment of correlation of pathogen diversity with geographical origin or coffee cropping system.

Componentes de resistência em cebola a Colletotrichum gloeosporioides

Despite the importance of onion (Allium cepa) leaf anthracnose caused by Colletotrichum gloeosporioides in Latin America, Africa, and Asia, few studies have focused on host resistance to the pathogen. Therefore, in this study, resistance components of two cultivars and eight accessions of onion to four isolates of C. gloeosporioides were evaluated under greenhouse conditions. Inoculations were performed either by spraying inoculum suspension or by placing a mycelial disc on the leaf. The cultivars and accessions differed significantly regarding initial infection frequency and monocyclic progress rate (rg) with the spray-inoculation, and regarding incubation period and lesion area with the mycelial-disc inoculation. Correlation coefficient (r) values were estimated between the components with the mycelial disk inoculations. Values of r were 0.98 between disease severity visually assessed nine days after inoculation (SEV9) and area under the disease progress curve (AUDPC), 0.80 between SEV9 and disease severity assessed with the leaf area meter (SEV), 0.72 between SEV9 and rg, 0.64 between SEV9 and infection frequency nine days after inoculation, 0.81 between SEV and AUDPC, and 0.64 between SEV and rg. Considering both the significant r values associated with SEV9 and that to estimate SEV9 there is no need of rating diagrams, this component is potentially useful to evaluate onion germplasm against C. gloeosporiodes. The spray inoculation procedure was faster, simpler, and provided higher infection efficiency and lower variability than the mycelial disk inoculation technique. Therefore, this should be the preferred inoculation procedure when assessing onion germplasm.

PCR amplification and sequence analyses of reverse transcriptase-like genes in Crinipellis perniciosa isolates

A analise de sequencias de transcriptase reversa (RT) e uma etapa importante para descobrir a presenca de elementos transponiveis e investigar o seu papel na geracao de variabilidade genetica em C. perniciosa. Sequencias putativas de TR foram analisadas no genoma do fitopatogeno C. perniciosa, o agente causal da doenca vassoura-de-bruxa no cacau. Um fragmento de 394 pb foi amplificado a partir do DNA genomico de diferentes isolados de C. perniciosa, pertencentes aos biotipos C, L e S e a distintas areas geograficas. A clivagem dos produtos de PCR com diferentes enzimas de restricao e sequenciamento de varios fragmentos de TR indicou a presenca de diferentes sequencias mostrando eventos de transicao G:C para A:T. A analise por hibridizacao revelou alto numero de sinais sugerindo a presenca de copias de TR com diferentes perfis entre os isolados dos biotipos C, S e L. As comparacoes de sequencias dos peptideos preditos indicam uma relacao proxima com a proteina TR de retrotransposons-LTR da familia gypsy.

The Hemileia vastatrix effector HvEC‐016 suppresses bacterial blight symptoms in coffee genotypes with the SH1 rust resistance gene

Summary A number of genes that confer resistance to coffee leaf rust (SH1–SH9) have been identified within the genus Coffea, but despite many years of research on this pathosystem, the complementary avirulence genes of Hemileia vastatrix have not been reported. After identification of H. vastatrix effector candidate genes (HvECs) expressed at different stages of its lifecycle, we established an assay to characterize HvEC proteins by delivering them into coffee cells via the type‐three secretion system (T3SS) of Pseudomonas syringae pv. garcae (Psgc). Employing a calmodulin‐dependent adenylate cyclase assay, we demonstrate that Psgc recognizes a heterologous P. syringae T3SS secretion signal which enables us to translocate HvECs into the cytoplasm of coffee cells. Using this Psgc‐adapted effector detector vector (EDV) system, we found that HvEC‐016 suppresses the growth of Psgc on coffee genotypes with the SH1 resistance gene. Suppression of bacterial blight symptoms in SH1 plants was associated with reduced bacterial multiplication. By contrast, HvEC‐016 enhanced bacterial multiplication in SH1‐lacking plants. Our findings suggest that HvEC‐016 may be recognized by the plant immune system in a SH1‐dependent manner. Thus, our experimental approach is an effective tool for the characterization of effector/avirulence proteins of this important pathogen.

Influência do tratamento com etileno sobre o teor de sólidos solúveis e a cor de pimentas

The goal of the present work was evaluate the genetic variability regarding the accumulation of total soluble solids, degradation of chlorophyll, synthesis of carotenoids and number of days to full ripening of Capsicum fruits treated with ethephon. The treatment with ethephon altered the content of soluble solids in the fruits of accessions BGH 4366 (C. baccatum) and BGH 4708 (C. frutescens), while the remaining accessions did not show any changes when treated with ethephon. The ripening was characterized by the degradation of chlorophyll and increase in the synthesis of carotenoids in all fruit, but there was no variability among the accessions when treated with ethephon. The ethephon was efficient in inducing the ripening of BGH 4179 (C. frutescens), BGH 6029 (C. baccatum) and Ca 6 (C. annuum) accessions.

Integração de pAN7-1 no genoma de Magnaporthe grisea mediada por enzima de restrição

To investigate insertional mutagenesis in Magnaporthe grisea, we tested the transformation of protoplasts produced after protocol optimization, and analyzed the integration efficiency of pAN7-1 into the M. grisea genome mediated by the restriction endonuclease Hind III. The I-22 protoplasts were readily transformed for hygromycin resistance. When pAN7-1 was linearized with Hind III and used to transform fungal protoplasts in the presence of the corresponding enzyme, the transformation efficiency was increased 1.1 to 8.1-fold. The optimal Hind III concentration for enhanced transformation corresponded to 5 unit/transformation mix. This concentration led to average frequency of 332 transformants/µg de pAN7-1/107 protoplasts. The presence of selection gene hph in the 18 transformant genome was confirmed by PCR.