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Quantifying species trophic interaction strengths is crucial for understanding community dynamics and has significant implications for pest management and species conservation. DNA-based methods to identify species interactions have revolutionized these efforts, but a significant limitation is the poor ability to quantify the strength of trophic interactions, that is the biomass or number of prey consumed. We present an improved pipeline, called Lazaro, to map unassembled shotgun reads to a comprehensive arthropod mitogenome database and show that the number of prey reads detected is quantitatively predicted from the prey biomass consumed, even for indirect predation. Two feeding bioassays were performed: starved coccinellid larvae consuming different numbers of aphids (Prey Quantity bioassay), and starved coccinellid larvae consuming a chrysopid larvae that had consumed aphids (Direct and Indirect Predation bioassay). Prey taxonomic assignment against a mitochondrial genome database had high accuracy (99.8% positive predictive value) and the number of prey reads was directly related to the number of prey consumed and inversely related to the elapsed time since consumption with high significance (r2 = .932, p = 4.92E-6). Aphids were detected up to 6 h after direct predation plus 3 h after indirect predation (9 h in total) and detection was related to the predator-specific decay rates. Lazaro enabled quantitative predictions of prey consumption across multiple trophic levels with high taxonomic resolution while eliminating all false positives, except for a few confirmed contaminants, and may be valuable for characterizing prey consumed by field-sampled predators. Moreover, Lazaro is readily applicable for species diversity determination from any degraded environmental DNA.
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Afídeos , Besouros , Animais , Cadeia Alimentar , Besouros/genética , Comportamento Predatório , Afídeos/genética , DNA/genéticaRESUMO
The root-knot nematode (RKN), Meloidogyne incognita, is a devastating soybean pathogen worldwide. The use of resistant cultivars is the most effective method to prevent economic losses caused by RKNs. To elucidate the mechanisms involved in resistance to RKN, we determined the proteome and transcriptome profiles from roots of susceptible (BRS133) and highly tolerant (PI 595099) Glycine max genotypes 4, 12, and 30 days after RKN infestation. After in silico analysis, we described major defense molecules and mechanisms considered constitutive responses to nematode infestation, such as mTOR, PI3K-Akt, relaxin, and thermogenesis. The integrated data allowed us to identify protein families and metabolic pathways exclusively regulated in tolerant soybean genotypes. Among them, we highlighted the phenylpropanoid pathway as an early, robust, and systemic defense process capable of controlling M. incognita reproduction. Associated with this metabolic pathway, 29 differentially expressed genes encoding 11 different enzymes were identified, mainly from the flavonoid and derivative pathways. Based on differential expression in transcriptomic and proteomic data, as well as in the expression profile by RT-qPCR, and previous studies, we selected and overexpressed the GmPR10 gene in transgenic tobacco to assess its protective effect against M. incognita. Transgenic plants of the T2 generation showed up to 58% reduction in the M. incognita reproduction factor. Finally, data suggest that GmPR10 overexpression can be effective against the plant parasitic nematode M. incognita, but its mechanism of action remains unclear. These findings will help develop new engineered soybean genotypes with higher performance in response to RKN infections.
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Maternal nutrition is critical in mammalian development, influencing the epigenetic reprogramming of gametes, embryos, and fetal programming. We evaluated the effects of different levels of sulfur (S) and cobalt (Co) in the maternal diet throughout the pre- and periconceptional periods on the biochemical and reproductive parameters of the donors and the DNA methylome of the progeny in Bos indicus cattle. The low-S/Co group differed from the control with respect to homocysteine, folic acid, B12, insulin growth factor 1, and glucose. The oocyte yield was lower in heifers from the low S/Co group than that in the control heifers. Embryos from the low-S/Co group exhibited 2320 differentially methylated regions (DMRs) across the genome compared with the control embryos. We also characterized candidate DMRs linked to the DNMT1 and DNMT3B genes in the blood and sperm cells of the adult progeny. A DMR located in DNMT1 that was identified in embryos remained differentially methylated in the sperm of the progeny from the low-S/Co group. Therefore, we associated changes in specific compounds in the maternal diet with DNA methylation modifications in the progeny. Our results help to elucidate the impact of maternal nutrition on epigenetic reprogramming in livestock, opening new avenues of research to study the effect of disturbed epigenetic patterns in early life on health and fertility in adulthood. Considering that cattle are physiologically similar to humans with respect to gestational length, our study may serve as a model for studies related to the developmental origin of health and disease in humans.
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Cobalto , Epigenoma , Animais , Bovinos , Cobalto/metabolismo , Metilação de DNA , Feminino , Mamíferos , Oócitos/metabolismo , Enxofre/metabolismoRESUMO
Nematodes and drought are major constraints in tropical agriculture and often occur simultaneously. Plant responses to these stresses are complex and require crosstalk between biotic and abiotic signaling pathways. In this study, we explored the transcriptome data of wild Arachis species subjected to drought (A-metaDEG) and the root-knot nematode Meloidogyne arenaria (B-metaDEG) via meta-analysis, to identify core-stress responsive genes to each individual and concurrent stresses in these species. Transcriptome analysis of a nematode/drought bioassay (cross-stress) showed that the set of stress responsive DEGs to concurrent stress is distinct from those resulting from overlapping A- and B-metaDEGs, indicating a specialized and unique response to combined stresses in wild Arachis. Whilst individual biotic and abiotic stresses elicit hormone-responsive genes, most notably in the jasmonic and abscisic acid pathways, combined stresses seem to trigger mainly the ethylene hormone pathway. The overexpression of a cross-stress tolerance candidate gene identified here, an endochitinase-encoding gene (AsECHI) from Arachis stenosperma, reduced up to 30% of M. incognita infection and increased post-drought recovery in Arabidopsis plants submitted to both stresses. The elucidation of the network of cross-stress responsive genes in Arachis contributes to better understanding the complex regulation of biotic and abiotic responses in plants facilitating more adequate crop breeding for combined stress tolerance.
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Arachis/genética , Arachis/parasitologia , Secas , Estresse Fisiológico/fisiologia , Tylenchoidea , Animais , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/genética , TranscriptomaRESUMO
The sugarcane giant borer (SGB), Telchin licus licus, is a pest that has strong economic relevance for sugarcane producers. Due to the endophytic behavior of the larva, current methods of management are inefficient. A promising biotechnological management option has been proposed based on RNA interference (RNAi), a process that uses molecules of double-stranded RNA (dsRNA) to specifically knock down essential genes and reduce insect survival. The selection of suitable target genes is often supported by omic sciences. Studies have shown that genes related to feeding adaptation processes are good candidates to be targeted by RNAi for pest management. Among those genes, esterases are highlighted because of their impact on insect development. In this study, the objective was to evaluate the transcriptome responses of the SGB's gut in order to provide curated data of genes that could be used for pest management by RNAi in future studies. Further, we validated the function of an esterase-coding gene and its potential as a target for RNAi-based control. We sequenced the gut transcriptome of SGB larvae by Illumina HiSeq and evaluated its gene expression profiles in response to different diets (sugarcane stalk and artificial diet). We obtained differentially expressed genes (DEGs) involved in detoxification, digestion, and transport, which suggest a generalist mechanism of adaptation in SGB larvae. Among the DEGs, was identified and characterized a candidate juvenile hormone esterase gene (Tljhe). We knocked down the Tljhe gene by oral delivery of dsRNA molecules and evaluated gene expression in the gut. The survival and nutritional parameters of the larvae were measured along the developmental cycle of treated insects. We found that the gene Tljhe acts as a regulator of feeding behavior. The knockdown of Tljhe triggered a forced starvation state in late larval instars that significantly reduced the fitness of the larvae. However, the mechanism of action of this gene remains unclear, and the correlation between the expression of Tljhe and the levels of juvenile hormone (JH) metabolites in the hemolymph of the SGB must be assessed in future research.
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MAIN CONCLUSION: In Brachiaria brizantha BbrizSERK1, BbrizSERK2 and BbrizSERK3 were identified. SERK expression marks somatic embryogenesis, sexual MMC, and sexual and apomictic PMC. BbrizSERK3 might have a regulatory role in reproductive development. Somatic embryogenesis receptor-like kinase (SERK) consists of plasma membrane receptor genes that have been characterized in various species, associated with several aspects of plant development, including reproduction. SERK genes are involved in anther development and in early embryo development in sexual and asexual seed formation. To comprehend the complexity of the SERK genes and their function in Brachiaria reproduction, we performed a homology-based search in a genomic database of a sexual B. brizantha and identified sequences of three SERK genes, BbrizSERK1, BbrizSERK2, and BbrizSERK3. RNASeq data showed equivalent abundance of BbrizSERK1 and BbrizSERK2 transcripts in ovaries at early megasporogenesis of sexuals and apomicts, while BbrizSERK3 transcripts were more abundant in ovaries of sexuals than in apomicts. BbrizSERK3 results in three coding sequences due to alternative splicing, among them Variant 1 results in a protein with all the predicted domains of a SERK. BbrizSERK transcripts were detected in male reproductive tissues of both sexual and apomictic plants, suggesting a role in controlling anther development. BbrizSERK transcripts were detected early in ovule development, in the integuments, and in the megaspore mother cell of the sexual plant, but not in the cells that give rise to apomictic embryo sacs, suggesting a role in female reproductive development of sexuals. This paper provides evidences that SERK genes plays a role in the onset and establishment of somatic embryogenesis and in the reproductive development of B. brizantha and suggests a distinct role of BbrizSERK in apomixis initiation.
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Brachiaria/crescimento & desenvolvimento , Brachiaria/genética , Regulação da Expressão Gênica de Plantas , Desenvolvimento Vegetal/genética , Reprodução/genética , Sementes/crescimento & desenvolvimento , Sementes/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes de Plantas , Técnicas de Embriogênese Somática de PlantasRESUMO
The sugarcane borer (Diatraea saccharalis, Fabricius, 1794) is a devastating pest that causes millions of dollars of losses each year to sugarcane producers by reducing sugar and ethanol yields. The control of this pest is difficult due to its endophytic behavior and rapid development. Pest management through biotechnological approaches has emerged in recent years as an alternative to currently applied methods. Genetic information about the target pests is often required to perform biotechnology-based management. The genomic and transcriptomic data for D. saccharalis are very limited. Herein, we report a tissue-specific transcriptome of D. saccharalis larvae and a differential expression analysis highlighting the physiological characteristics of this pest in response to two different diets: sugarcane and an artificial diet. Sequencing was performed on the Illumina HiSeq 2000 platform, and a de novo assembly was generated. A total of 27,626 protein-coding unigenes were identified, among which 1,934 sequences were differentially expressed between treatments. Processes such as defence, digestion, detoxification, signaling, and transport were highly represented among the differentially expressed genes (DEGs). Furthermore, seven aminopeptidase genes were identified as candidates to encode receptors of Cry proteins, which are toxins of Bacillus thuringiensis used to control lepidopteran pests. Since plant-insect interactions have produced a considerable number of adaptive responses in hosts and herbivorous insects, the success of phytophagous insects relies on their ability to overcome challenges such as the response to plant defences and the intake of nutrients. In this study, we identified metabolic pathways and specific genes involved in these processes. Thus, our data strongly contribute to the knowledge advancement of insect transcripts, which can be a source of target genes for pest management.
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Dieta , Mucosa Intestinal/metabolismo , Lepidópteros/genética , Transcriptoma , Aminopeptidases/genética , Aminopeptidases/metabolismo , Animais , Herbivoria/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Lepidópteros/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
We report the complete genomic sequences of seven viral isolates from the soybean looper (Chrysodeixis includens) from midwestern and southeastern Brazil. The genomes range from 138,760 to 139,637 bp in length with a G+C content of 39.2% and 140 open reading frames (ORFs).
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Coffee production is a global industry valued at approximately 173 billion US dollars. One of the main challenges facing coffee production is the management of the coffee berry borer (CBB), Hypothenemus hampei, which is considered the primary arthropod pest of coffee worldwide. Current control strategies are inefficient for CBB management. Although biotechnological alternatives, including RNA interference (RNAi), have been proposed in recent years to control insect pests, characterizing the genetics of the target pest is essential for the successful application of these emerging technologies. In this study, we employed RNA-seq to obtain the transcriptome of three developmental stages of the CBB (larva, female and male) to increase our understanding of the CBB life cycle in relation to molecular features. The CBB transcriptome was sequenced using Illumina Hiseq and assembled de novo. Differential gene expression analysis was performed across the developmental stages. The final assembly produced 29,434 unigenes, of which 4,664 transcripts were differentially expressed. Genes linked to crucial physiological functions, such as digestion and detoxification, were determined to be tightly regulated between the reproductive and nonreproductive stages of CBB. The data obtained in this study help to elucidate the critical roles that several genes play as regulatory elements in CBB development.
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Coffea/parasitologia , Genes de Insetos , Gorgulhos/crescimento & desenvolvimento , Gorgulhos/genética , Animais , Feminino , Perfilação da Expressão Gênica , Larva/genética , Larva/crescimento & desenvolvimento , Masculino , RNA-Seq , TranscriptomaRESUMO
The baculovirus, Chrysodeixis (formerly Pseudoplusia) includens nucleopolyhedrovirus (ChinNPV), is a new Alphabaculovirus pathogenic to Chrysodeixis includens Here, we report the complete genome sequences of six ChinNPV isolates. The availability of these genome sequences will provide information on ChinNPV molecular genetics, promoting understanding of its pathogenicity, diversity, and evolution.
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Wild peanut relatives (Arachis spp.) are genetically diverse and were adapted to a range of environments during the evolution course, constituting an important source of allele diversity for resistance to biotic and abiotic stresses. The wild diploid A. stenosperma harbors high levels of resistance to a variety of pathogens, including the root-knot nematode (RKN) Meloidogyne arenaria, through the onset of the Hypersensitive Response (HR). In order to identify genes and regulators triggering this defense response, a comprehensive root transcriptome analysis during the first stages of this incompatible interaction was conducted using Illumina Hi-Seq. Overall, eight cDNA libraries were produced generating 28.2 GB, which were de novo assembled into 44,132 contigs and 37,882 loci. Differentially expressed genes (DEGs) were identified and clustered according to their expression profile, with the majority being downregulated at 6 DAI, which coincides with the onset of the HR. Amongst these DEGs, 27 were selected for further qRT-PCR validation allowing the identification of nematode-responsive candidate genes that are putatively related to the resistance response. Those candidates are engaged in the salycilic (NBS-LRR, lipocalins, resveratrol synthase) and jasmonic (patatin, allene oxidase cyclase) acids pathways, and also related to hormonal balance (auxin responsive protein, GH3) and cellular plasticity and signaling (tetraspanin, integrin, expansin), with some of them showing contrasting expression behavior between Arachis RKN-resistant and susceptible genotypes. As these candidate genes activate different defensive signaling systems, the genetic (HR) and the induced resistance (IR), their pyramidding in one genotype via molecular breeding or transgenic strategy might contribute to a more durable resistance, thus improving the long-term control of RKN in peanut.
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Arachis/genética , Resistência à Doença/fisiologia , Doenças das Plantas/imunologia , Doenças das Plantas/parasitologia , Tylenchoidea/imunologia , Animais , Ciclopentanos/metabolismo , Perfilação da Expressão Gênica , Genes de Plantas , Lipocalinas/metabolismo , Oxilipinas/metabolismo , Raízes de Plantas/genética , Resveratrol , Estilbenos/metabolismoRESUMO
Olfaction plays a fundamental role in insect survival through resource location and intra and interspecific communications. We used RNA-Seq to analyze transcriptomes for odorant-binding proteins (OBPs) from major stink bug pest species in Brazil, Euschistus heros, Chinavia ubica, and Dichelops melacanthus, and from their egg parasitoid, Telenomus podisi. We identified 23 OBPs in E. heros, 25 OBPs in C. ubica, 9 OBPs in D. melacanthus, and 7 OBPs in T. podisi. The deduced amino acid sequences of the full-length OBPs had low intraspecific similarity, but very high similarity between two pairs of OBPs from E. heros and C. ubica (76.4 and 84.0%) and between two pairs of OBPs from the parasitoid and its preferred host E. heros (82.4 and 88.5%), confirmed by a high similarity of their predicted tertiary structures. The similar pairs of OBPs from E. heros and C. ubica may suggest that they have derived from a common ancestor, and retain the same biological function to bind a ligand perceived or produced in both species. The T. podisi OBPs similar to E. heros were not orthologous to any known hymenopteran OBPs, and may have evolved independently and converged to the host OBPs, providing a possible basis for the host location of T. podisi using E. heros semiochemical cues.
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Heterópteros/genética , Óvulo/parasitologia , Parasitos/genética , Receptores Odorantes/metabolismo , Transcriptoma/genética , Clima Tropical , Sequência de Aminoácidos , Animais , Simulação por Computador , Biblioteca Gênica , Ontologia Genética , Interações Hospedeiro-Parasita/genética , Modelos Moleculares , Anotação de Sequência Molecular , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Receptores Odorantes/química , Receptores Odorantes/genética , Alinhamento de Sequência , Análise de Sequência de RNA , Especificidade da EspécieRESUMO
BACKGROUND: Pseudoplusia includens single nucleopolyhedrovirus (PsinSNPV-IE) is a baculovirus recently identified in our laboratory, with high pathogenicity to the soybean looper, Chrysodeixis includens (Lepidoptera: Noctuidae) (Walker, 1858). In Brazil, the C. includens caterpillar is an emerging pest and has caused significant losses in soybean and cotton crops. The PsinSNPV genome was determined and the phylogeny of the p26 gene within the family Baculoviridae was investigated. RESULTS: The complete genome of PsinSNPV was sequenced (Roche 454 GS FLX - Titanium platform), annotated and compared with other Alphabaculoviruses, displaying a genome apparently different from other baculoviruses so far sequenced. The circular double-stranded DNA genome is 139,132 bp in length, with a GC content of 39.3 % and contains 141 open reading frames (ORFs). PsinSNPV possesses the 37 conserved baculovirus core genes, 102 genes found in other baculoviruses and 2 unique ORFs. Two baculovirus repeat ORFs (bro) homologs, bro-a (Psin33) and bro-b (Psin69), were identified and compared with Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) and Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) bro genes and showed high similarity, suggesting that these genes may be derived from an ancestor common to these viruses. The homologous repeats (hrs) are absent from the PsinSNPV genome, which is also the case in ChchNPV and TnSNPV. Two p26 gene homologs (p26a and p26b) were found in the PsinSNPV genome. P26 is thought to be required for optimal virion occlusion in the occlusion bodies (OBs), but its function is not well characterized. The P26 phylogenetic tree suggests that this gene was obtained from three independent acquisition events within the Baculoviridae family. The presence of a signal peptide only in the PsinSNPV p26a/ORF-20 homolog indicates distinct function between the two P26 proteins. CONCLUSIONS: PsinSNPV has a genomic sequence apparently different from other baculoviruses sequenced so far. The complete genome sequence of PsinSNPV will provide a valuable resource, contributing to studies on its molecular biology and functional genomics, and will promote the development of this virus as an effective bioinsecticide.
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Evolução Molecular , Produtos do Gene gag/genética , Lepidópteros/genética , Nucleopoliedrovírus/genética , Animais , Lepidópteros/virologiaRESUMO
Peanut (Arachis hypogaea L.) is an important legume cultivated mostly in drought-prone areas where its productivity can be limited by water scarcity. The development of more drought-tolerant varieties is, therefore, a priority for peanut breeding programs worldwide. In contrast to cultivated peanut, wild relatives have a broader genetic diversity and constitute a rich source of resistance/tolerance alleles to biotic and abiotic stresses. The present study takes advantage of this diversity to identify drought-responsive genes by analyzing the expression profile of two wild species, Arachis duranensis and Arachis magna (AA and BB genomes, respectively), in response to progressive water deficit in soil. Data analysis from leaves and roots of A. duranensis (454 sequencing) and A. magna (suppression subtractive hybridization (SSH)) stressed and control complementary DNA (cDNA) libraries revealed several differentially expressed genes in silico, and 44 of them were selected for further validation by quantitative RT-PCR (qRT-PCR). This allowed the identification of drought-responsive candidate genes, such as Expansin, Nitrilase, NAC, and bZIP transcription factors, displaying significant levels of differential expression during stress imposition in both species. This is the first report on identification of differentially expressed genes under drought stress and recovery in wild Arachis species. The generated transcriptome data, besides being a valuable resource for gene discovery, will allow the characterization of new alleles and development of molecular markers associated with drought responses in peanut. These together constitute important tools for the peanut breeding program and also contribute to a better comprehension of gene modulation in response to water deficit and rehydration.
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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.
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Eucalyptus/genética , Genoma de Planta , Eucalyptus/classificação , Evolução Molecular , Variação Genética , Endogamia , FilogeniaRESUMO
BACKGROUND: Although banana (Musa sp.) is an important edible crop, contributing towards poverty alleviation and food security, limited transcriptome datasets are available for use in accelerated molecular-based breeding in this genus. 454 GS-FLX Titanium technology was employed to determine the sequence of gene transcripts in genotypes of Musa acuminata ssp. burmannicoides Calcutta 4 and M. acuminata subgroup Cavendish cv. Grande Naine, contrasting in resistance to the fungal pathogen Mycosphaerella musicola, causal organism of Sigatoka leaf spot disease. To enrich for transcripts under biotic stress responses, full length-enriched cDNA libraries were prepared from whole plant leaf materials, both uninfected and artificially challenged with pathogen conidiospores. RESULTS: The study generated 846,762 high quality sequence reads, with an average length of 334 bp and totalling 283 Mbp. De novo assembly generated 36,384 and 35,269 unigene sequences for M. acuminata Calcutta 4 and Cavendish Grande Naine, respectively. A total of 64.4% of the unigenes were annotated through Basic Local Alignment Search Tool (BLAST) similarity analyses against public databases.Assembled sequences were functionally mapped to Gene Ontology (GO) terms, with unigene functions covering a diverse range of molecular functions, biological processes and cellular components. Genes from a number of defense-related pathways were observed in transcripts from each cDNA library. Over 99% of contig unigenes mapped to exon regions in the reference M. acuminata DH Pahang whole genome sequence. A total of 4068 genic-SSR loci were identified in Calcutta 4 and 4095 in Cavendish Grande Naine. A subset of 95 potential defense-related gene-derived simple sequence repeat (SSR) loci were validated for specific amplification and polymorphism across M. acuminata accessions. Fourteen loci were polymorphic, with alleles per polymorphic locus ranging from 3 to 8 and polymorphism information content ranging from 0.34 to 0.82. CONCLUSIONS: A large set of unigenes were characterized in this study for both M. acuminata Calcutta 4 and Cavendish Grande Naine, increasing the number of public domain Musa ESTs. This transcriptome is an invaluable resource for furthering our understanding of biological processes elicited during biotic stresses in Musa. Gene-based markers will facilitate molecular breeding strategies, forming the basis of genetic linkage mapping and analysis of quantitative trait loci.
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Musa/genética , Folhas de Planta/genética , Ascomicetos/genética , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Repetições de Microssatélites , Anotação de Sequência Molecular , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , TranscriptomaRESUMO
The Brazilian Cerrado is the second largest biome in Brazil and is considered a biodiversity hotspot. In this work, we compared the bacterial communities in Cerrado soil associated with four types of native vegetation (Cerrado Denso, Cerrado sensu stricto, Campo Sujo, and Mata de Galeria) by ribosomal RNA intergenic spacer analysis, terminal fragment restriction length polymorphism and pyrosequencing. The fingerprinting results were very similar. The bacterial communities of Cerrado Denso and Cerrado sensu stricto grouped together and were distinct from those in Campo Sujo and Mata de Galeria. Pyrosequencing generated approximately 40,000 16S rRNA gene sequences per sample and allowed the identification of 17 phyla in soil samples under Cerrado vegetation. Acidobacteria were dominant in all areas studied with a relative frequency of 40-47 %, followed closely by Proteobacteria accounting for 34-40 % of the sequences. Results from all molecular techniques used suggested that the bacterial communities of Cerrado sensu stricto and Cerrado Denso are very similar to each other, while Campo Sujo forms a separate group, and Mata de Galeria is the most distinct with higher species richness. This is the first extensive study of native Cerrado soil microbiota, an important but endangered biome.
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Acidobacteria/genética , Bactérias/genética , Ecossistema , Microbiologia do Solo , Acidobacteria/classificação , Acidobacteria/isolamento & purificação , Bactérias/classificação , Bactérias/isolamento & purificação , Brasil , DNA Bacteriano/análise , DNA Bacteriano/genética , DNA Espaçador Ribossômico/análise , DNA Espaçador Ribossômico/genética , Dados de Sequência Molecular , Poaceae , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Proteobactérias/classificação , Proteobactérias/genética , Proteobactérias/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Solo/análise , ÁrvoresRESUMO
A structural rationale for recent emergence of azole (imidazole and triazole) resistance associated with CYP51 mutations in the wheat pathogen Mycosphaerella graminicola is presented, attained by homology modelling of the wild type protein and 13 variant proteins. The novel molecular models of M. graminicola CYP51 are based on multiple homologues, individually identified for each variant, rather than using a single structural scaffold, providing a robust structure-function rationale for the binding of azoles, including important fungal specific regions for which no structural information is available. The wild type binding pocket reveals specific residues in close proximity to the bound azole molecules that are subject to alteration in the variants. This implicates azole ligands as important agents exerting selection on specific regions bordering the pocket, that become the focus of genetic mutation events, leading to reduced sensitivity to that group of related compounds. Collectively, the models account for several observed functional effects of specific alterations, including loss of triadimenol sensitivity in the Y137F variant, lower sensitivity to tebuconazole of I381V variants and increased resistance to prochloraz of V136A variants. Deletion of Y459 and G460, which brings about removal of that entire section of beta turn from the vicinity of the binding pocket, confers resistance to tebuconazole and epoxiconazole, but sensitivity to prochloraz in variants carrying a combination of A379G I381V ΔY459/G460. Measurements of binding pocket volume proved useful in assessment of scope for general resistance to azoles by virtue of their accommodation without bonding interaction, particularly when combined with analysis of change in positions of key amino acids. It is possible to predict the likely binding orientation of an azole molecule in any of the variant CYPs, providing potential for an in silico screening system and reliable predictive approach to assess the probability of particular variants exhibiting resistance to particular azole fungicides.
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Inibidores de 14-alfa Desmetilase/farmacologia , Ascomicetos/efeitos dos fármacos , Azóis/farmacologia , Ascomicetos/genética , Ascomicetos/metabolismo , Farmacorresistência Fúngica/genética , Compostos de Epóxi/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Imidazóis/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Esterol 14-Desmetilase/química , Esterol 14-Desmetilase/genética , Esterol 14-Desmetilase/metabolismo , Triazóis/farmacologiaRESUMO
BACKGROUND: Enzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. In this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the Interface Forming Residues (IFR). We were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table (matrix) of all amino acid positions at the interface as well as their respective occupancies. Our goal is to establish a platform for analysis of the relationship between IFR characteristics and binding properties/specificity for bi-molecular complexes. RESULTS: We propose a novel method for describing binding properties and delineating serine proteases specificity by compiling an exhaustive table of interface forming residues (IFR) for serine proteases and their inhibitors. Currently, the Protein Data Bank (PDB) does not contain all the data that our analysis would require. Therefore, an in silico approach was designed for building corresponding complexes. The IFRs are obtained by "rigid body docking" among 70 structurally aligned, sequence wise non-redundant, serine protease structures with 3 inhibitors: bovine pancreatic trypsin inhibitor (BPTI), ecotine and ovomucoid third domain inhibitor. The table (matrix) of all amino acid positions at the interface and their respective occupancy is created. We also developed a new computational protocol for predicting IFRs for those complexes which were not deciphered experimentally so far, achieving accuracy of at least 0.97. CONCLUSIONS: The serine proteases interfaces prefer polar (including glycine) residues (with some exceptions). Charged residues were found to be uniquely prevalent at the interfaces between the "miscellaneous-virus" subfamily and the three inhibitors. This prompts speculation about how important this difference in IFR characteristics is for maintaining virulence of those organisms.Our work here provides a unique tool for both structure/function relationship analysis as well as a compilation of indicators detailing how the specificity of various serine proteases may have been achieved and/or could be altered. It also indicates that the interface forming residues which also determine specificity of serine protease subfamily can not be presented in a canonical way but rather as a matrix of alternative populations of amino acids occupying variety of IFR positions.
Assuntos
Motivos de Aminoácidos/genética , Modelos Moleculares , Ligação Proteica , Serina Proteases/química , Inibidores de Serina Proteinase/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
BACKGROUND: Transcriptome sequences provide a complement to structural genomic information and provide snapshots of an organism's transcriptional profile. Such sequences also represent an alternative method for characterizing neglected species that are not expected to undergo whole-genome sequencing. One difficulty for transcriptome sequencing of these organisms is the low quality of reads and incomplete coverage of transcripts, both of which compromise further bioinformatics analyses. Another complicating factor is the lack of known protein homologs, which frustrates searches against established protein databases. This lack of homologs may be caused by divergence from well-characterized and over-represented model organisms. Another explanation is that non-coding RNAs (ncRNAs) may be caught during sequencing. NcRNAs are RNA sequences that, unlike messenger RNAs, do not code for protein products and instead perform unique functions by folding into higher order structural conformations. There is ncRNA screening software available that is specific for transcriptome sequences, but their analyses are optimized for those transcriptomes that are well represented in protein databases, and also assume that input ESTs are full-length and high quality. RESULTS: We propose an algorithm called PORTRAIT, which is suitable for ncRNA analysis of transcriptomes from poorly characterized species. Sequences are translated by software that is resistant to sequencing errors, and the predicted putative proteins, along with their source transcripts, are evaluated for coding potential by a support vector machine (SVM). Either of two SVM models may be employed: if a putative protein is found, a protein-dependent SVM model is used; if it is not found, a protein-independent SVM model is used instead. Only ab initio features are extracted, so that no homology information is needed. We illustrate the use of PORTRAIT by predicting ncRNAs from the transcriptome of the pathogenic fungus Paracoccidoides brasiliensis and five other related fungi. CONCLUSION: PORTRAIT can be integrated into pipelines, and provides a low computational cost solution for ncRNA detection in transcriptome sequencing projects.