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1.
Sensors (Basel) ; 23(5)2023 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-36905013

RESUMO

This paper reports on a compact and lightweight sensor for analysis of gases/vapors by means of a MEMS-based pre-concentrator coupled to a miniaturized infrared absorption spectroscopy (IRAS) module. The pre-concentrator was utilized to sample and trap vapors in a MEMS cartridge filled with sorbent material and to release them once concentrated by fast thermal desorption. It was also equipped with a photoionization detector for in-line detection and monitoring of the sampled concentration. The vapors released by the MEMS pre-concentrator are injected into a hollow fiber, which acts as the analysis cell of the IRAS module. The miniaturized internal volume of the hollow fiber of about 20 microliters keeps the vapors concentrated for analysis, thus allowing measurement of their infrared absorption spectrum with a signal to noise ratio high enough to identify the molecule, despite the short optical path, starting from sampled concentration in air down to parts per million. Results obtained for ammonia, sulfur hexafluoride, ethanol and isopropanol are reported to illustrate the sensor detection and identification capability. A limit of identification (LoI) of about 10 parts per million was validated in the lab for ammonia. The lightweight and low power consumption design of the sensor allowed operation onboard unmanned aerial vehicles (UAVs). The first prototype was developed within the EU Horizon 2020 project ROCSAFE for the remote assessment and forensic examination of a scene in the aftermath of industrial or terroristic accidents.

2.
Sensors (Basel) ; 22(19)2022 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-36236300

RESUMO

Starting from Quartz-Enhanced Photo-Acoustic Spectroscopy (QEPAS), we have explored the potential of a tightly linked method of gas/vapor sensing, from now on referred to as Tuning-Fork-Enhanced Photo-Acoustic Spectroscopy (TFEPAS). TFEPAS utilizes a non-piezoelectric metal or dielectric tuning fork to transduce the photoacoustic excitation and an optical interferometric readout to measure the amplitude of the tuning fork vibration. In particular, we have devised a solution based on Additive Manufacturing (AM) for the Absorption Detection Module (ADM). The novelty of our solution is that the ADM is entirely built monolithically by Micro-Metal Laser Sintering (MMLS) or other AM techniques to achieve easier and more cost-effective customization, extreme miniaturization of internal volumes, automatic alignment of the tuning fork with the acoustic micro-resonators, and operation at high temperature. This paper reports on preliminary experimental results achieved with ammonia at parts-per-million concentration in nitrogen to demonstrate the feasibility of the proposed solution. Prospectively, the proposed TFEPAS solution appears particularly suited for hyphenation to micro-Gas Chromatography and for the analysis of complex solid and liquid traces samples, including compounds with low volatility such as illicit drugs, explosives, and persistent chemical warfare agents.


Assuntos
Substâncias para a Guerra Química , Drogas Ilícitas , Acústica , Amônia , Nitrogênio , Quartzo/química , Análise Espectral/métodos
3.
Sensors (Basel) ; 23(1)2022 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-36616868

RESUMO

This paper reports on a compact, portable, and selective chemical sensor for hazardous vapors at trace levels, which is under development and validation within the EU project H2020 "RISEN". Starting from the prototype developed for a previous EU project, here, we implemented an updated two-stage purge and trap vapor pre-concentration system, a more compact MEMS- based fast gas-chromatographic separation module (Compact-GC), a new miniaturized quartz-enhanced photoacoustic spectroscopy (QEPAS) detector, and a new compact laser source. The system provides two-dimensional selectivity combining GC retention time and QEPAS spectral information and was specifically designed to be rugged, portable, suitable for on-site analysis of a crime scene, with accurate response in few minutes and in the presence of strong chemical background. The main upgrades of the sensor components and functional modules will be presented in detail, and test results with VOCs, simulants of hazardous chemical agents, and drug precursors will be reported and discussed.


Assuntos
Gases , Quartzo , Quartzo/química , Gases/análise , Análise Espectral/métodos
4.
Sensors (Basel) ; 20(1)2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31878138

RESUMO

This paper reports on a portable selective chemical sensor for hazardous vapors at trace levels, which combines a two-stage purge and trap vapor pre-concentration system, a Micro-Electro-Mechanical-System (MEMS) based fast gas-chromatographic (FAST-GC) separation column and a miniaturized quartz-enhanced photoacoustic spectroscopy (QEPAS) detector. The integrated sensing system provides two-dimensional selectivity combining GC retention time and QEPAS spectral information, and was specifically designed to be rugged and suitable to be deployed on unmanned robotic ground vehicles. This is the first demonstration of a miniaturized QEPAS device used as spectroscopic detector downstream of a FAST-GC separation column, enabling real-world analyses in dirty environments with response time of a few minutes. The main modules of the GC/QEPAS sensor device will be described in detail together with the system integration, and successful test results will be reported and discussed.

5.
Yeast ; 33(7): 277-87, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27168222

RESUMO

Nowadays, the presence of Saccharomyces cerevisiae has been assessed in both wild and human-related environments. Social wasps have been shown to maintain and vector S. cerevisiae among different environments. The availability of strains isolated from wasp intestines represents a striking opportunity to assess whether the strains found in wasp intestines are characterized by peculiar traits. We analysed strains isolated from the intestines of social wasps and compared them with strains isolated from other sources, all collected in a restricted geographic area. We evaluated the production of volatile metabolites during grape must fermentation, the resistance to different stresses and the ability to exploit various carbon sources. Wasp strains, in addition to representing a wide range of S. cerevisiae genotypes, also represent large part of the phenotypes characterizing the sympatric set of yeast strains; their higher production of acetic acid and ethyl acetate could reflect improved ability to attract insects. Our findings suggest that the relationship between yeasts and wasps should be preserved, to safeguard not only the natural variance of this microorganism but also the interests of wine-makers, who could take advantage from the exploitation of their phenotypic variability. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Saccharomyces cerevisiae/genética , Vespas/microbiologia , Animais , Variação Genética , Genótipo , Intestinos/microbiologia , Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico
6.
BMC Genomics ; 16: 306, 2015 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-25887666

RESUMO

BACKGROUND: Plastid genomes, also known as plastomes, are shaped by the selective forces acting on the fundamental cellular functions they code for and thus they are expected to preserve signatures of the adaptive path undertaken by different plant species during evolution. To identify molecular signatures of positive selection associated to adaptation to contrasting ecological niches, we sequenced with Solexa technology the plastomes of two congeneric Brassicaceae species with different habitat preference, Cardamine resedifolia and Cardamine impatiens. RESULTS: Following in-depth characterization of plastome organization, repeat patterns and gene space, the comparison of the newly sequenced plastomes between each other and with 15 fully sequenced Brassicaceae plastomes publically available in GenBank uncovered dynamic variation of the IR boundaries in the Cardamine lineage. We further detected signatures of positive selection in ten of the 75 protein-coding genes of the examined plastomes, identifying a range of chloroplast functions putatively involved in adaptive processes within the family. For instance, the three residues found to be under positive selection in RUBISCO could possibly be involved in the modulation of RUBISCO aggregation/activation and enzymatic specificty in Brassicaceae. In addition, our results points to differential evolutionary rates in Cardamine plastomes. CONCLUSIONS: Overall our results support the existence of wider signatures of positive selection in the plastome of C. resedifolia, possibly as a consequence of adaptation to high altitude environments. We further provide a first characterization of the selective patterns shaping the Brassicaceae plastomes, which could help elucidate the driving forces underlying adaptation and evolution in this important plant family.


Assuntos
Cardamine/fisiologia , Cloroplastos/genética , Genoma de Cloroplastos , Análise de Sequência de DNA/métodos , Adaptação Biológica , Cardamine/classificação , Cardamine/citologia , Cardamine/genética , Cloroplastos/metabolismo , Evolução Molecular , Dados de Sequência Molecular , Filogenia , Seleção Genética , Especificidade da Espécie
7.
Food Microbiol ; 48: 123-32, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25791000

RESUMO

The Traditional Mountain Malga (TMM) cheese is made from raw cow's milk by spontaneously fermentation in small farms called "Malga" located in Trentino region. This study was designed to characterize the lactic acid bacteria (LAB) growing on MRS medium, of TMM-cheese at the end of the ripening. Ninety-five LAB were isolated and genotypically characterized by Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) with two primers, species-specific PCR and partial sequencing of 16S rRNA gene. The 95 LAB clustered in 70 biotypes. Pediococcus pentosaceus and Lactobacillus paracasei were the dominant species. Isolates were tested for their growth properties, carbohydrate metabolism, acidifying ability, proteolytic and lipolytic activities, acetoin production, amino-peptidase (AP) activity, biogenic amines production, bile salts hydrolysis, conjugated linoleic acid and γ-aminobutyric acid production. Lb. paracasei isolates resulted to be well adapted to Malga environment and to show the best AP activity and acetoin production. TMM-cheese related LAB showed also interesting health promoting properties and produced bioactive substances. In particular, one Lb. brevis biotype produced a GABA mean value of 129 mg/L that is considered a high concentration. The results confirmed that TMM-cheese resident LAB could be exploited for dairy production.


Assuntos
Queijo/microbiologia , Lactobacillus/isolamento & purificação , Leite/microbiologia , Pediococcus/isolamento & purificação , Animais , Bovinos , Fermentação , Lactobacillus/classificação , Lactobacillus/genética , Lactobacillus/metabolismo , Dados de Sequência Molecular , Pediococcus/classificação , Pediococcus/genética , Pediococcus/metabolismo , Filogenia , Técnica de Amplificação ao Acaso de DNA Polimórfico
8.
Proc Natl Acad Sci U S A ; 109(33): 13398-403, 2012 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-22847440

RESUMO

Saccharomyces cerevisiae is one of the most important model organisms and has been a valuable asset to human civilization. However, despite its extensive use in the last 9,000 y, the existence of a seasonal cycle outside human-made environments has not yet been described. We demonstrate the role of social wasps as vector and natural reservoir of S. cerevisiae during all seasons. We provide experimental evidence that queens of social wasps overwintering as adults (Vespa crabro and Polistes spp.) can harbor yeast cells from autumn to spring and transmit them to their progeny. This result is mirrored by field surveys of the genetic variability of natural strains of yeast. Microsatellites and sequences of a selected set of loci able to recapitulate the yeast strain's evolutionary history were used to compare 17 environmental wasp isolates with a collection of strains from grapes from the same region and more than 230 strains representing worldwide yeast variation. The wasp isolates fall into subclusters representing the overall ecological and industrial yeast diversity of their geographic origin. Our findings indicate that wasps are a key environmental niche for the evolution of natural S. cerevisiae populations, the dispersion of yeast cells in the environment, and the maintenance of their diversity. The close relatedness of several wasp isolates with grape and wine isolates reflects the crucial role of human activities on yeast population structure, through clonal expansion and selection of specific strains during the biotransformation of fermented foods, followed by dispersal mediated by insects and other animals.


Assuntos
Evolução Biológica , Fenômenos Ecológicos e Ambientais , Saccharomyces cerevisiae/genética , Comportamento Social , Vespas/microbiologia , Animais , Sistema Digestório/microbiologia , Genoma Fúngico/genética , Humanos , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Saccharomyces cerevisiae/isolamento & purificação , Estações do Ano , Vespas/genética
9.
Plant J ; 71(4): 615-26, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22469001

RESUMO

Mitochondrial genomes of spermatophytes are the largest of all organellar genomes. Their large size has been attributed to various factors; however, the relative contribution of these factors to mitochondrial DNA (mtDNA) expansion remains undetermined. We estimated their relative contribution in Malus domestica (apple). The mitochondrial genome of apple has a size of 396 947 bp and a one to nine ratio of coding to non-coding DNA, close to the corresponding average values for angiosperms. We determined that 71.5% of the apple mtDNA sequence was highly similar to sequences of its nuclear DNA. Using nuclear gene exons, nuclear transposable elements and chloroplast DNA as markers of promiscuous DNA content in mtDNA, we estimated that approximately 20% of the apple mtDNA consisted of DNA sequences imported from other cell compartments, mostly from the nucleus. Similar marker-based estimates of promiscuous DNA content in the mitochondrial genomes of other species ranged between 21.2 and 25.3% of the total mtDNA length for grape, between 23.1 and 38.6% for rice, and between 47.1 and 78.4% for maize. All these estimates are conservative, because they underestimate the import of non-functional DNA. We propose that the import of promiscuous DNA is a core mechanism for mtDNA size expansion in seed plants. In apple, maize and grape this mechanism contributed far more to genome expansion than did homologous recombination. In rice the estimated contribution of both mechanisms was found to be similar.


Assuntos
Genoma Mitocondrial , Genoma de Planta , Malus/genética , Núcleo Celular/genética , DNA de Cloroplastos , DNA Mitocondrial , Evolução Molecular , Éxons , Magnoliopsida/genética , Dados de Sequência Molecular , Vírus de Plantas/genética , Biossíntese de Proteínas , Sequências Repetitivas de Ácido Nucleico , Sementes/genética
10.
BMC Genomics ; 14: 670, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24083400

RESUMO

BACKGROUND: Second generation sequencing has permitted detailed sequence characterisation at the whole genome level of a growing number of non-model organisms, but the data produced have short read-lengths and biased genome coverage leading to fragmented genome assemblies. The PacBio RS long-read sequencing platform offers the promise of increased read length and unbiased genome coverage and thus the potential to produce genome sequence data of a finished quality containing fewer gaps and longer contigs. However, these advantages come at a much greater cost per nucleotide and with a perceived increase in error-rate. In this investigation, we evaluated the performance of the PacBio RS sequencing platform through the sequencing and de novo assembly of the Potentilla micrantha chloroplast genome. RESULTS: Following error-correction, a total of 28,638 PacBio RS reads were recovered with a mean read length of 1,902 bp totalling 54,492,250 nucleotides and representing an average depth of coverage of 320× the chloroplast genome. The dataset covered the entire 154,959 bp of the chloroplast genome in a single contig (100% coverage) compared to seven contigs (90.59% coverage) recovered from an Illumina data, and revealed no bias in coverage of GC rich regions. Post-assembly the data were largely concordant with the Illumina data generated and allowed 187 ambiguities in the Illumina data to be resolved. The additional read length also permitted small differences in the two inverted repeat regions to be assigned unambiguously. CONCLUSIONS: This is the first report to our knowledge of a chloroplast genome assembled de novo using PacBio sequence data. The PacBio RS data generated here were assembled into a single large contig spanning the P. micrantha chloroplast genome, with a higher degree of accuracy than an Illumina dataset generated at a much greater depth of coverage, due to longer read lengths and lower GC bias in the data. The results we present suggest PacBio data will be of immense utility for the development of genome sequence assemblies containing fewer unresolved gaps and ambiguities and a significantly smaller number of contigs than could be produced using short-read sequence data alone.


Assuntos
Genoma de Cloroplastos/genética , Potentilla/genética , Análise de Sequência de DNA/métodos , Software , Composição de Bases/genética , Sequência de Bases , Bases de Dados Genéticas
11.
BMC Genomics ; 14: 2, 2013 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-23324311

RESUMO

BACKGROUND: Rapid development of highly saturated genetic maps aids molecular breeding, which can accelerate gain per breeding cycle in woody perennial plants such as Rubus idaeus (red raspberry). Recently, robust genotyping methods based on high-throughput sequencing were developed, which provide high marker density, but result in some genotype errors and a large number of missing genotype values. Imputation can reduce the number of missing values and can correct genotyping errors, but current methods of imputation require a reference genome and thus are not an option for most species. RESULTS: Genotyping by Sequencing (GBS) was used to produce highly saturated maps for a R. idaeus pseudo-testcross progeny. While low coverage and high variance in sequencing resulted in a large number of missing values for some individuals, a novel method of imputation based on maximum likelihood marker ordering from initial marker segregation overcame the challenge of missing values, and made map construction computationally tractable. The two resulting parental maps contained 4521 and 2391 molecular markers spanning 462.7 and 376.6 cM respectively over seven linkage groups. Detection of precise genomic regions with segregation distortion was possible because of map saturation. Microsatellites (SSRs) linked these results to published maps for cross-validation and map comparison. CONCLUSIONS: GBS together with genome-independent imputation provides a rapid method for genetic map construction in any pseudo-testcross progeny. Our method of imputation estimates the correct genotype call of missing values and corrects genotyping errors that lead to inflated map size and reduced precision in marker placement. Comparison of SSRs to published R. idaeus maps showed that the linkage maps constructed with GBS and our method of imputation were robust, and marker positioning reliable. The high marker density allowed identification of genomic regions with segregation distortion in R. idaeus, which may help to identify deleterious alleles that are the basis of inbreeding depression in the species.


Assuntos
Mapeamento Cromossômico/métodos , Genoma de Planta/genética , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Rosaceae/genética , Marcadores Genéticos/genética , Repetições de Microssatélites/genética , Polimorfismo de Nucleotídeo Único/genética
12.
New Phytol ; 198(2): 579-592, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23379348

RESUMO

Aquilegia is a well-known model system in the field of evolutionary biology, but obtaining a resolved and well-supported phylogenetic reconstruction for the genus has been hindered by its recent and rapid diversification. Here, we applied 454 next-generation sequencing to PCR amplicons of 21 of the most rapidly evolving regions of the plastome to generate c. 24 kb of sequences from each of 84 individuals from throughout the genus. The resulting phylogeny has well-supported resolution of the main lineages of the genus, although recent diversification such as in the European taxa remains unresolved. By producing a chronogram of the whole Ranunculaceae family based on published data, we inferred calibration points for dating the Aquilegia radiation. The genus originated in the upper Miocene c. 6.9 million yr ago (Ma) in Eastern Asia, and diversification occurred c. 4.8 Ma with the split of two main clades, one colonizing North America, and the other Western Eurasia through the mountains of Central Asia. This was followed by a back-to-Asia migration, originating from the European stock using a North Asian route. These results provide the first backbone phylogeny and spatiotemporal reconstruction of the Aquilegia radiation, and constitute a robust framework to address the adaptative nature of speciation within the group.


Assuntos
Aquilegia/genética , DNA de Cloroplastos/genética , Evolução Molecular , Análise de Sequência de DNA/métodos , Sequência de Bases , Análise por Conglomerados , Filogenia , Análise de Componente Principal , Fatores de Tempo
13.
BMC Genomics ; 13: 203, 2012 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-22631220

RESUMO

BACKGROUND: A whole-genome genotyping array has previously been developed for Malus using SNP data from 28 Malus genotypes. This array offers the prospect of high throughput genotyping and linkage map development for any given Malus progeny. To test the applicability of the array for mapping in diverse Malus genotypes, we applied the array to the construction of a SNP-based linkage map of an apple rootstock progeny. RESULTS: Of the 7,867 Malus SNP markers on the array, 1,823 (23.2%) were heterozygous in one of the two parents of the progeny, 1,007 (12.8%) were heterozygous in both parental genotypes, whilst just 2.8% of the 921 Pyrus SNPs were heterozygous. A linkage map spanning 1,282.2 cM was produced comprising 2,272 SNP markers, 306 SSR markers and the S-locus. The length of the M432 linkage map was increased by 52.7 cM with the addition of the SNP markers, whilst marker density increased from 3.8 cM/marker to 0.5 cM/marker. Just three regions in excess of 10 cM remain where no markers were mapped. We compared the positions of the mapped SNP markers on the M432 map with their predicted positions on the 'Golden Delicious' genome sequence. A total of 311 markers (13.7% of all mapped markers) mapped to positions that conflicted with their predicted positions on the 'Golden Delicious' pseudo-chromosomes, indicating the presence of paralogous genomic regions or mis-assignments of genome sequence contigs during the assembly and anchoring of the genome sequence. CONCLUSIONS: We incorporated data for the 2,272 SNP markers onto the map of the M432 progeny and have presented the most complete and saturated map of the full 17 linkage groups of M. pumila to date. The data were generated rapidly in a high-throughput semi-automated pipeline, permitting significant savings in time and cost over linkage map construction using microsatellites. The application of the array will permit linkage maps to be developed for QTL analyses in a cost-effective manner, and the identification of SNPs that have been assigned erroneous positions on the 'Golden Delicious' reference sequence will assist in the continued improvement of the genome sequence assembly for that variety.


Assuntos
Mapeamento Cromossômico , Genoma de Planta , Malus/genética , Polimorfismo de Nucleotídeo Único , Análise por Conglomerados , Ligação Genética , Genótipo , Heterozigoto , Repetições de Microssatélites , Análise de Sequência com Séries de Oligonucleotídeos , Locos de Características Quantitativas
14.
J Exp Bot ; 63(3): 1107-21, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22121200

RESUMO

Fruit ripening is a complex physiological process in plants whereby cell wall programmed changes occur mainly to promote seed dispersal. Cell wall modification also directly regulates the textural properties, a fundamental aspect of fruit quality. In this study, two full-sib populations of apple, with 'Fuji' as the common maternal parent, crossed with 'Delearly' and 'Pink Lady', were used to understand the control of fruit texture by QTL mapping and in silico gene mining. Texture was dissected with a novel high resolution phenomics strategy, simultaneously profiling both mechanical and acoustic fruit texture components. In 'Fuji × Delearly' nine linkage groups were associated with QTLs accounting from 15.6% to 49% of the total variance, and a highly significant QTL cluster for both textural components was mapped on chromosome 10 and co-located with Md-PG1, a polygalacturonase gene that, in apple, is known to be involved in cell wall metabolism processes. In addition, other candidate genes related to Md-NOR and Md-RIN transcription factors, Md-Pel (pectate lyase), and Md-ACS1 were mapped within statistical intervals. In 'Fuji × Pink Lady', a smaller set of linkage groups associated with the QTLs identified for fruit texture (15.9-34.6% variance) was observed. The analysis of the phenotypic variance over a two-dimensional PCA plot highlighted a transgressive segregation for this progeny, revealing two QTL sets distinctively related to both mechanical and acoustic texture components. The mining of the apple genome allowed the discovery of the gene inventory underlying each QTL, and functional profile assessment unravelled specific gene expression patterns of these candidate genes.


Assuntos
Frutas/genética , Frutas/metabolismo , Malus/genética , Malus/metabolismo , Locos de Características Quantitativas/genética , Parede Celular/genética , Parede Celular/metabolismo , Proteínas de Plantas/genética
15.
Analyst ; 137(16): 3725-31, 2012 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-22741156

RESUMO

In recent times we have seen the development of many "-omics" technologies. One of the youngest is undoubtedly metabolomics, which aims to define the whole chemical fingerprint unique to each specific organism. The development and optimisation of an untargeted high-throughput method capable of investigating the volatile fraction of a biological system represents a crucial step for the success of such holistic approaches, and specific optimisation criteria must be developed in connection with suitable experimental designs. In this paper experimental designs (D-optimal) were applied for the first time as an automatic optimisation tool to an untargeted HS-SPME-GC-TOF method. In this case, optimal conditions correspond to a maximal number of detected features, in order to provide a fingerprint that is as complete as possible. The system under study is the grape berry. Four variables were considered: the type of fibre, extraction time, equilibration time and temperature. The results show that the D-optimal design methodology provides an easily interpretable assessment of experimental settings. This and other specific properties of the D-optimal design, such as the possibility to explicitly exclude certain experimental conditions, make it an extremely suitable strategy for method optimisation in untargeted metabolomics.


Assuntos
Cromatografia Gasosa/métodos , Metabolômica/métodos , Microextração em Fase Sólida/métodos , Vitis/metabolismo
16.
Mol Biol Evol ; 26(1): 99-110, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18922764

RESUMO

The mitochondrial genome of grape (Vitis vinifera), the largest organelle genome sequenced so far, is presented. The genome is 773,279 nt long and has the highest coding capacity among known angiosperm mitochondrial DNAs (mtDNAs). The proportion of promiscuous DNA of plastid origin in the genome is also the largest ever reported for an angiosperm mtDNA, both in absolute and relative terms. In all, 42.4% of chloroplast genome of Vitis has been incorporated into its mitochondrial genome. In order to test if horizontal gene transfer (HGT) has also contributed to the gene content of the grape mtDNA, we built phylogenetic trees with the coding sequences of mitochondrial genes of grape and their homologs from plant mitochondrial genomes. Many incongruent gene tree topologies were obtained. However, the extent of incongruence between these gene trees is not significantly greater than that observed among optimal trees for chloroplast genes, the common ancestry of which has never been in doubt. In both cases, we attribute this incongruence to artifacts of tree reconstruction, insufficient numbers of characters, and gene paralogy. This finding leads us to question the recent phylogenetic interpretation of Bergthorsson et al. (2003, 2004) and Richardson and Palmer (2007) that rampant HGT into the mtDNA of Amborella best explains phylogenetic incongruence between mitochondrial gene trees for angiosperms. The only evidence for HGT into the Vitis mtDNA found involves fragments of two coding sequences stemming from two closteroviruses that cause the leaf roll disease of this plant. We also report that analysis of sequences shared by both chloroplast and mitochondrial genomes provides evidence for a previously unknown gene transfer route from the mitochondrion to the chloroplast.


Assuntos
DNA Mitocondrial/genética , Evolução Molecular , Vitis/genética , Cloroplastos/genética , DNA de Cloroplastos/genética , Transferência Genética Horizontal , Mitocôndrias/genética , Mutagênese Insercional , Biossíntese de Proteínas , Vitis/citologia
17.
BMC Genomics ; 10: 363, 2009 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-19660119

RESUMO

BACKGROUND: In response to pathogen attack, grapevine synthesizes phytoalexins belonging to the family of stilbenes. Grapevine cell cultures represent a good model system for studying the basic mechanisms of plant response to biotic and abiotic elicitors. Among these, modified beta-cyclodextrins seem to act as true elicitors inducing strong production of the stilbene resveratrol. RESULTS: The transcriptome changes of Vitis riparia x Vitis berlandieri grapevine cells in response to the modified beta-cyclodextrin, DIMEB, were analyzed 2 and 6 h after treatment using a suppression subtractive hybridization experiment and a microarray analysis respectively. At both time points, we identified a specific set of induced genes belonging to the general phenylpropanoid metabolism, including stilbenes and hydroxycinnamates, and to defence proteins such as PR proteins and chitinases. At 6 h we also observed a down-regulation of the genes involved in cell division and cell-wall loosening. CONCLUSIONS: We report the first large-scale study of the molecular effects of DIMEB, a resveratrol inducer, on grapevine cell cultures. This molecule seems to mimic a defence elicitor which enhances the physical barriers of the cell, stops cell division and induces phytoalexin synthesis.


Assuntos
Estilbenos/metabolismo , Vitis/metabolismo , beta-Ciclodextrinas/farmacologia , Divisão Celular , Parede Celular/metabolismo , DNA Complementar/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Análise de Sequência com Séries de Oligonucleotídeos , RNA de Plantas/genética , Resveratrol , Vitis/genética
18.
J Mol Evol ; 68(3): 197-204, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19247564

RESUMO

It is widely appreciated that noisy, highly variable data can impede phylogeney reconstruction. Researchers have for a long time omitted problematic data from phylogenetic analyses, such as the third-codon positions and variable regions. In the analyses of the phylogenetic relations of the angiosperms; however, inclusion of complete gene sequences into genomic-scale alignments has become a common practice. Here we demonstrate that this practice can be misleading. We show that support of the basal-most position of Amborella trichopoda among the angiosperms in the chloroplast genomic data is based only on a tiny subset (< 1% of the total alignment length) of the most variable positions in alignment, exhibiting mean maximum likelihood (ML) distance among the angiosperm operational taxonomic units (OTUs) approximately 36 substitutions/site. Exclusion of these positions leads to disappearance of the basal Amborella branch. Likewise, the recently reported sister-group relationship of Ceratophyllum to the eudicots is based on the presence of 2% of the most variable positions in the genomic alignment, exhibiting, on average, 20 substitutions/site in comparison among the angiosperm OTUs. These observations highlight a need for excluding a certain proportion of saturated positions in alignment from phylogenomic analyses.


Assuntos
Cloroplastos/genética , Genomas de Plastídeos , Magnoliopsida/genética , Filogenia , Interpretação Estatística de Dados , Bases de Dados Genéticas , Evolução Molecular , Genes de RNAr , Modelos Genéticos , Proteínas de Plantas/genética , RNA de Plantas/genética , RNA de Transferência/genética , Alinhamento de Sequência
20.
BMC Plant Biol ; 8: 128, 2008 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-19087337

RESUMO

BACKGROUND: Efforts to sequence the genomes of different organisms continue to increase. The DNA sequence is usually decoded for one individual and its application is for the whole species. The recent sequencing of the highly heterozygous Vitis vinifera L. cultivar Pinot Noir (clone ENTAV 115) genome gave rise to several thousand polymorphisms and offers a good model to study the transferability of its degree of polymorphism to other individuals of the same species and within the genus. RESULTS: This study was performed by genotyping 137 SNPs through the SNPlex Genotyping System (Applied Biosystems Inc.) and by comparing the SNPlex sequencing results across 35 (of the 137) regions from 69 grape accessions. A heterozygous state transferability of 31.5% across the unrelated cultivars of V. vinifera, of 18.8% across the wild forms of V. vinifera, of 2.3% among non-vinifera Vitis species, and of 0% with Muscadinia rotundifolia was found. In addition, mean allele frequencies were used to evaluate SNP informativeness and develop useful subsets of markers. CONCLUSION: Using SNPlex application and corroboration from the sequencing analysis, the informativeness of SNP markers from the heterozygous grape cultivar Pinot Noir was validated in V. vinifera (including cultivars and wild forms), but had a limited application for non-vinifera Vitis species where a resequencing strategy may be preferred, knowing that homology at priming sites is sufficient. This work will allow future applications such as mapping and diversity studies, accession identification and genomic-research assisted breeding within V. vinifera.


Assuntos
Genoma de Planta , Polimorfismo de Nucleotídeo Único , Vitis/genética , DNA de Plantas/genética , Genótipo , Heterozigoto , Análise de Sequência de DNA/métodos
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