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1.
PLoS One ; 15(7): e0236343, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730349

RESUMO

Using pooled DNA genotyping to estimate the proportional contributions from multiple families in a pooled sample is of particular interest for selective breeding in aquaculture. We compared different pooled libraries with separate 2b-RAD sequencing of Litopenaeus vannamei individuals to assess the effect of different population structures (different numbers of individuals and families) on pooled DNA sequencing, the accuracy of parent sequencing of the DNA pools and the effect of SNP numbers on pooled DNA sequencing. We demonstrated that small pooled DNA genotyping of up to 53 individuals by 2b-RAD sequencing could provide a highly accurate assessment of population allele frequencies. The accuracy increased as the number of individuals and families increased. The allele frequencies of the parents from each pool were highly correlated with those of the pools or the corresponding individuals in the pool. We chose 500-28,000 SNPs to test the effect of SNP number on the accuracy of pooled sequencing, and no linear relationship was found between them. When the SNP number was fixed, increasing the number of individuals in the mixed pool resulted in higher accuracy of each pooled genotyping. Our data confirmed that pooled DNA genotyping by 2b-RAD sequencing could achieve higher accuracy than that of individual-based genotyping. The results will provide important information for shrimp breeding programs.


Assuntos
DNA/genética , Técnicas de Genotipagem , Sequenciamento de Nucleotídeos em Larga Escala , Penaeidae/genética , Mapeamento por Restrição , Alelos , Animais , Frequência do Gene/genética , Genética Populacional , Polimorfismo de Nucleotídeo Único/genética , Reprodutibilidade dos Testes
2.
PLoS One ; 15(6): e0233800, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32497070

RESUMO

Several studies suggest the relation of DNA methylation to diseases in humans and important phenotypes in plants drawing attention to this epigenetic mark as an important source of variability. In the last decades, several methodologies were developed to assess the methylation state of a genome. However, there is still a lack of affordable and precise methods for genome wide analysis in large sample size studies. Methyl sensitive double digestion MS-DArT sequencing method emerges as a promising alternative for methylation profiling. We developed a computational pipeline for the identification of DNA methylation using MS-DArT-seq data and carried out a pilot study using the Eucalyptus grandis tree sequenced for the species reference genome. Using a statistic framework as in differential expression analysis, 72,515 genomic sites were investigated and 5,846 methylated sites identified, several tissue specific, distributed along the species 11 chromosomes. We highlight a bias towards identification of DNA methylation in genic regions and the identification of 2,783 genes and 842 transposons containing methylated sites. Comparison with WGBS, DNA sequencing after treatment with bisulfite, data demonstrated a precision rate higher than 95% for our approach. The availability of a reference genome is useful for determining the genomic context of methylated sites but not imperative, making this approach suitable for any species. Our approach provides a cost effective, broad and reliable examination of DNA methylation profile on MspI/HpaII restriction sites, is fully reproducible and the source code is available on GitHub (https://github.com/wendelljpereira/ms-dart-seq).


Assuntos
Análise Custo-Benefício , Metilação de DNA/genética , Eucalyptus/genética , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Folhas de Planta/genética , Análise de Sequência de DNA/métodos , Árvores/genética , Cromossomos de Plantas/genética , Enzimas de Restrição do DNA/genética , Elementos de DNA Transponíveis/genética , Genes de Plantas/genética , Técnicas de Genotipagem/economia , Sequenciamento de Nucleotídeos em Larga Escala/economia , Projetos Piloto , Reprodutibilidade dos Testes , Mapeamento por Restrição , Análise de Sequência de DNA/economia , Sulfitos/farmacologia
3.
PLoS One ; 15(6): e0234635, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32530959

RESUMO

The entire genome of Helicoverpa armigera single nucleopolyhedrovirus (HearNPV-TR) was sequenced, and compared to genomes of other existing isolates. HearNPV-TR genome is 130.691 base pairs with a 38.9% G+C content and has 137 open reading frames (ORFs) of ≥ 150 nucleotides. Five homologous repeated sequences (hrs) and two baculovirus repeated ORFs (bro-a and bro-b) were identified. Phylogenetic analysis showed that HearNPV-TR is closer to HaSNPV-C1, HaSNPV-G4, HaSNPV-AU and HasNPV. However, there are significant differences in hr3, hr5 regions and in bro-a gene. Pairwise Kimura-2 parameter analysis of 38 core genes sequences of HearNPV-TR and other Helicoverpa NPVs showed that the genetic distances for these sequences were below 0.015 substitutions/site. Genomic differences as revealed by restriction profiles indicated that hr3, hr5 regions and bro-a gene may play a role in the virulence of HearNPV-TR.


Assuntos
Genoma Viral , Mariposas/virologia , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/isolamento & purificação , Análise de Sequência de DNA , Animais , DNA Circular/genética , Genes de Insetos , Fases de Leitura Aberta/genética , Filogenia , Mapeamento por Restrição , Turquia
4.
J Vis Exp ; (157)2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32225139

RESUMO

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5' splice site with an upstream 3' splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.


Assuntos
Elementos Alu/genética , RNA Circular/genética , Sequência de Bases , DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Exorribonucleases/metabolismo , Genes Reporter , Genoma , Humanos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Mapeamento por Restrição
5.
J Vet Diagn Invest ; 32(1): 112-117, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32013802

RESUMO

Bovine herpesvirus 1 (BoHV-1) causes several clinical syndromes in cattle worldwide. There are 3 subtypes of BoHV-1: 1.1, 1.2a, and 1.2b. Several molecular methods are commonly used in the detection and characterization of BoHV-1. Among them, restriction endonuclease analysis (REA) and single-nucleotide polymorphism (SNP) analysis of the complete viral genome allow classification of BoHV-1 into different subtypes. However, developing countries need simpler and cheaper screening assays for routine testing. We designed a standard multiplex PCR followed by a REA assay allowing straightforward subclassification of all BoHV-1 isolates tested into 1.1, 1.2a, and 1.2b subtypes based on the analysis of fragment length polymorphism. Our standard multiplex PCR-REA was used to analyze 33 field strains of BoHV-1 isolated from various tissues. The assay confirmed the subtype identified previously by REA. In addition, non-polymorphic or undigested fragments were sequenced in order to confirm the mutation affecting the RE HindIII site. Our PCR-REA method is an affordable and rapid test that will subtype all BoHV-1 strains.


Assuntos
Doenças dos Bovinos/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/classificação , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , DNA Viral/análise , Genoma Viral , Infecções por Herpesviridae/diagnóstico , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 1/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/veterinária , Mutação , Mapeamento por Restrição
6.
Genome Biol ; 21(1): 11, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937349

RESUMO

Hi-C is a popular technique to map three-dimensional chromosome conformation. In principle, Hi-C's resolution is only limited by the size of restriction fragments. However, insufficient sequencing depth forces researchers to artificially reduce the resolution of Hi-C matrices at a loss of biological interpretability. We present the Hi-C Interaction Frequency Inference (HIFI) algorithms that accurately estimate restriction-fragment resolution Hi-C matrices by exploiting dependencies between neighboring fragments. Cross-validation experiments and comparisons to 5C data and known regulatory interactions demonstrate HIFI's superiority to existing approaches. In addition, HIFI's restriction-fragment resolution reveals a new role for active regulatory regions in structuring topologically associating domains.


Assuntos
Algoritmos , Cromossomos , DNA/metabolismo , Genoma , Mapeamento por Restrição , Animais , Humanos , Camundongos
7.
J Vet Diagn Invest ; 31(5): 696-703, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31477001

RESUMO

Equid alphaherpesvirus 1 (EHV-1) infections can have a major impact on the horse industry and equine welfare by causing abortion or respiratory or neurologic disease. A single nucleotide polymorphism (A2254→G2254) in open reading frame (ORF) 30, encoding the catalytic subunit of the DNA polymerase, has been shown to be a strong predictive marker for neuropathogenicity. Given that a previously established real-time PCR (rtPCR) protocol yielded unsatisfactory results concerning determination of the EHV-1 genotype, we developed and evaluated a new conventional PCR protocol enabling identification of the genotype by sequencing and restriction enzyme analysis (REA). Thirty samples from horses with signs typical for EHV-1 infection were tested by rtPCR and our new conventional PCR. The results showed that compared to rtPCR, the conventional PCR protocol combined with sequencing and REA was more reliable concerning unambiguous determination of the EHV-1 genotype. Results of our new assay confirmed previous findings, according to which the non-neuropathogenic genotype A2254 is predominantly found in animals with fever, respiratory signs, and abortions or perinatal mortality, whereas the neuropathogenic genotype G2254 is primarily detected in animals suffering from neurologic disease. In some samples, results pointed towards coinfection with both genotypes. Further studies are required in order to elucidate the significance of infections with genotype A2254 and G2254 in neurologic and non-neurologic cases, respectively.


Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/classificação , Doenças dos Cavalos/virologia , Doenças do Sistema Nervoso/veterinária , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , DNA Polimerase Dirigida por DNA/genética , Feminino , Genótipo , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Cavalos , Doenças do Sistema Nervoso/virologia , Fases de Leitura Aberta , Gravidez , Reação em Cadeia da Polimerase em Tempo Real/métodos , Mapeamento por Restrição/veterinária
8.
Korean J Parasitol ; 57(4): 417-422, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31533409

RESUMO

From October 2015 to August 2018, tapeworm proglottids were obtained from 10 patients who were residents of Daegu and Gyeongbuk provinces and had a history of raw beef consumption. Most of them had no overseas travel experience. The gravid proglottids obtained from the 10 cases had 15-20 lateral uterine branches. A part of internal transcribed spacer 1 (ITS1) DNA of the 10 cases, amplified by polymerase chain reaction (PCR) and digested with AleI restriction enzyme, produced the same band pattern of Taenia saginata, which differentiated from T. asiatica and T. solium. Sequences of ITS1 and cytochrome c oxidase subunit 1 (cox1) showed higher homology to T. saginata than to T. asiatica and T. solium. Collectively, these 10 cases were identified as T. saginata human infections. As taeniasis is one of the important parasitic diseases in humans, it is necessary to maintain hygienic conditions during livestock farming to avoid public health concerns.


Assuntos
DNA Espaçador Ribossômico/análise , Taenia saginata/isolamento & purificação , Teníase/diagnóstico , Adulto , Idoso , Animais , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , República da Coreia , Mapeamento por Restrição , Homologia de Sequência , Taenia saginata/classificação , Taenia saginata/genética , Teníase/parasitologia , Adulto Jovem
9.
Proc Biol Sci ; 286(1911): 20191311, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31530141

RESUMO

The Palaearctic butterfly Melitaea didyma stands out as one of the most striking cases of intraspecific genetic differentiation detected in Lepidoptera: 11 partially sympatric mitochondrial lineages have been reported, displaying levels of divergence of up to 7.4%. To better understand the evolutionary processes underlying the diversity observed in mtDNA, we compared mtDNA and genome-wide SNP data using double-digest restriction site-associated DNA sequencing (ddRADseq) results from 93 specimens of M. didyma ranging from Morocco to eastern Kazakhstan. We found that, between ddRADseq and mtDNA results, there is a match only in populations that probably remained allopatric for long periods of time. Other mtDNA lineages may have resulted from introgression events and were probably affected by Wolbachia infection. The five main ddRADseq clades supported by STRUCTURE were parapatric or allopatric and showed high pairwise FST values, but some were also estimated to display various levels of gene flow. Melitaea didyma represents one of the first cases of deep mtDNA splits among European butterflies assessed by a genome-wide DNA analysis and reveals that the interpretation of patterns remains challenging even when a high amount of genomic data is available. These findings actualize the ongoing debate of species delimitation in allopatry, an issue probably of relevance to a significant proportion of global biodiversity.


Assuntos
Borboletas/genética , DNA Mitocondrial/análise , Especiação Genética , Genoma de Inseto , Filogenia , Polimorfismo de Nucleotídeo Único , África do Norte , Animais , Evolução Biológica , Europa (Continente) , Fluxo Gênico , Cazaquistão , Mapeamento por Restrição
10.
Nucleic Acids Res ; 47(19): e122, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31418018

RESUMO

Genome-wide profiling of copy number alterations and DNA methylation in single cells could enable detailed investigation into the genomic and epigenomic heterogeneity of complex cell populations. However, current methods to do this require complex sample processing and cleanup steps, lack consistency, or are biased in their genomic representation. Here, we describe a novel single-tube enzymatic method, DNA Analysis by Restriction Enzyme (DARE), to perform deterministic whole genome amplification while preserving DNA methylation information. This method was evaluated on low amounts of DNA and single cells, and provides accurate copy number aberration calling and representative DNA methylation measurement across the whole genome. Single-cell DARE is an attractive and scalable approach for concurrent genomic and epigenomic characterization of cells in a heterogeneous population.


Assuntos
Enzimas de Restrição do DNA/genética , Mapeamento por Restrição/métodos , Análise de Célula Única/métodos , Variações do Número de Cópias de DNA/genética , Metilação de DNA/genética , Epigenômica/métodos , Genoma Humano/genética , Genômica/métodos , Humanos , Análise de Sequência de DNA/métodos
11.
PLoS One ; 14(8): e0220981, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31393947

RESUMO

To obtain genetic information about the germplasm of tea (Camellia sinensis L.) in Japan, 167 accessions including 138 var. sinensis (96 Japanese var. sinensis and 42 exotic var. sinensis) and 29 Assam hybrids were analyzed using single nucleotide polymorphisms (SNPs) markers identified by double-digest restriction-site-associated DNA sequencing (ddRAD-seq) analysis. Approximately 10,000 SNPs were identified by ddRAD-seq and were mapped across the whole genome. The 167 tea accessions were classified into three genetic subgroups: (1) Japanese var. sinensis; (2) Japanese and exotic var. sinensis; (3) Assam hybrids and exotic var. sinensis. Leaf morphology varied widely within each genetic subgroups. The 96 Japanese var. sinensis were classified into four genetic subgroups as follows; two subgroups of Shizuoka (the largest tea production region) landraces, Uji (most ancient tea production region) landraces, and the pedigree of 'Yabukita', the leading green tea cultivar in Japan. These results indicated that the SNP markers obtained from ddRAD-seq are a useful tool to investigate the geographical background and breeding history of Japanese tea. This genetic information revealed the ancestral admixture situation of the 'Yabukita' pedigree, and showed that the genome structure of 'Yabukita' is clearly different from those of other Japanese accessions.


Assuntos
Melhoramento Vegetal , Polimorfismo de Nucleotídeo Único/genética , Mapeamento por Restrição , Sementes/genética , Análise de Sequência de DNA , Chá/genética , Mapeamento Cromossômico , Ecótipo , Genética Populacional , Folhas de Planta/anatomia & histologia , Característica Quantitativa Herdável
12.
Mol Phylogenet Evol ; 139: 106528, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31176966

RESUMO

The bark beetle genus Dendroctonus contains some of the most economically important pests of conifers worldwide. Despite many attempts, there is no agreement today on the phylogenetic relationships within the genus, which limits our understanding of its evolutionary history. Here, using restriction-site associated DNA (RAD) markers from 70 specimens representing 17 species (85% of the known diversity) we inferred the phylogeny of the genus, its time of origin and biogeographic history, as well as the evolution of key ecological traits (host plants, larval behavior and adults' attack strategies). For all combinations of tested parameters (from 6444 to 23,570 RAD tags analyzed), the same, fully resolved topology was inferred. Our analyses suggest that the most recent common ancestor (mrca) of all extant Dendroctonus species was widely distributed across eastern Palearctic and western Nearctic during the early Miocene, from where species dispersed to other Holarctic regions. A first main inter-continental vicariance event occurred during early Miocene isolating the ancestors of D. armandi in the Palearctic, which was followed by the radiation of the main Dendroctonus lineages in North America. During the Late Miocene, the ancestor of the 'rufipennis' species group colonized north-east Palearctic regions from western North America, which was followed by a second main inter-continental vicariance event isolating Pleistocene populations in Asia (D. micans) and western North America (D. murrayanae and D. punctatus). The present study supports previous hypotheses explaining intercontinental range disjunctions across the Northern Hemisphere by the fragmentation of a continuous distribution due to climatic cooling, host range fragmentation and geological changes during the late Cenozoic. The reconstruction of ancestral ecological traits indicates that the mrca bored individual galleries and mass attacked the boles of pines. The gregarious feeding behavior of the larvae as well as the individual attack of the base of trees have apparently independently evolved twice in North America (in the 'rufipennis' and the 'valens' species groups), which suggests a higher adaptive potential than previously thought and may be of interest for plant protection and biodiversity conservation in a rapidly changing world.


Assuntos
Besouros/classificação , Besouros/genética , Marcadores Genéticos/genética , Filogenia , Animais , Ásia , Biodiversidade , América do Norte , Mapeamento por Restrição , Análise de Sequência de DNA
13.
J Reprod Dev ; 65(4): 305-312, 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31061296

RESUMO

Age-associated methylation changes in genomic DNA have been recently reported in spermatozoa, and these changes can contribute to decline in fertility. In a previous study, we analyzed the genome-wide DNA methylation profiles of bull spermatozoa using a human DNA methylation microarray and identified one CpG site (CpG-1) that potentially reflects age-related methylation changes. In the present study, cryopreserved semen samples from a Japanese Black bull were collected at five different ages, which were referred to as JD1-5: 14, 19, 28, 54, and 162 months, respectively, and were used for genome-wide DNA methylation analysis and in vitro fertilization (IVF). Distinct age-related changes in methylation profiles were observed, and 77 CpG sites were found to be differently methylated between young and adult samples (JD1-2 vs. JD4-5). Using combined bisulfite restriction analysis (COBRA), nine CpG sites (including CpG-1) were confirmed to exhibit significant differences in their age-dependent methylation levels. Eight CpG sites showed an age-dependent increase in their methylation levels, whereas only one site showed age-dependent hypomethylation; in particular, these changes in methylation levels occurred rapidly at a young age. COBRA revealed low methylation levels in some CpG regions in the majority of the IVF blastocyst-stage embryos derived from spermatozoa at JD2-5. Interestingly, bulls with different ages did not show differences in their methylation levels. In conclusion, our findings indicated that methylation levels at nine CpG sites in spermatozoa changed with increasing age and that some CpG regions were demethylated after fertilization. Further studies are required to determine whether age-dependent different methylation levels in bull spermatozoa can affect fertility.


Assuntos
Envelhecimento/genética , Blastocisto/metabolismo , Ilhas de CpG/genética , Metilação de DNA , Desenvolvimento Embrionário/genética , Espermatozoides/metabolismo , Fatores Etários , Animais , Bovinos , Células Cultivadas , Técnicas de Química Combinatória/métodos , Metilação de DNA/genética , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Feminino , Fertilização In Vitro/veterinária , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Mapeamento por Restrição/métodos , Análise de Sequência de DNA/métodos , Sulfitos/química
14.
Ticks Tick Borne Dis ; 10(4): 911-917, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31054919

RESUMO

Hyalomma species (Acari: Ixodidae) are vectors of several human and animal pathogens. However, due to their similar morphological properties, classification of related Hyalomma species is often challenging. Here we describe a combined approach for molecular characterization of six Hyalomma species: H. aegyptium, H. dromedarii, H. excavatum, H. impeltatum, H. marginatum and H. turanicum. This procedure was developed using a combination of PCR amplification of four molecular markers, followed by sequencing and species-specific restriction analysis. Segments from the following genes were used as markers: 12S rRNA, 16S rRNA, Cytochrome C oxidase subunit 1 (COX1), and Cytochrome B (CytB). Phylogenetic analysis based on the amplified sequences was consistent with the morphology-based classification. It revealed relative close proximity of H. excavatum, H. marginatum and H. turanicum, and close proximity of H. aegyptium and H. dromedarii to each other. H. impeltatum was examined using the COX1 and CytB markers, and in both cases was located on a separate clade from the other five species. Digestion of the amplified products using specific restriction enzymes enabled clear distinction between the six species. This report is the first to describe CytB marker sequences of the studied species, and the first to describe COX1 marker sequences of H. aegyptium, H. excavatum, H. impeltatum and H. turanicum. The information obtained in this study may therefore be useful for future combined morphological-molecular Hyalomma characterization.


Assuntos
DNA Mitocondrial/genética , Ixodidae/classificação , Animais , Citocromos b/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Marcadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico/genética , RNA Ribossômico 16S/genética , Mapeamento por Restrição , Especificidade da Espécie
15.
J Bras Pneumol ; 45(2): e20180278, 2019 Apr 25.
Artigo em Inglês, Português | MEDLINE | ID: mdl-31038648

RESUMO

OBJECTIVE: Pulmonary nontuberculous mycobacterial infections are caused by nontuberculous mycobacteria (NTM), the microbiological diagnosis of which involves the isolation and identification of the same species in at least two sputum samples, one BAL fluid sample, or one lung biopsy sample. The objective of the present study was to determine the frequency at which the various NTM species are identified among selected individuals and in potential cases of pulmonary nontuberculous mycobacterial infection. METHODS: This was a retrospective analysis of the data on species isolated from respiratory specimens collected from 2,843 individuals between 2011 and 2014. Potential NTM infection cases were identified on the basis of the international microbiological criteria adopted in the state of São Paulo. RESULTS: A total of 50 species were identified using the molecular method PCR-restriction enzyme analysis. Samples collected from 1,014 individuals were analyzed in relation to the microbiological criteria, and 448 (44.18%) had a presumptive diagnosis of pulmonary nontuberculous mycobacterial infection, the species identified most frequently being, in descending order, Mycobacterium kansasii, M. abscessus, M. intracellulare, M. avium, and M. szulgai. CONCLUSIONS: Although various NTM species were identified among the individuals studied, those presumptively identified most frequently on the basis of the microbiological criteria adopted in the state of São Paulo were the ones that are most commonly associated with pulmonary nontuberculous mycobacterial infection worldwide or in specific geographic regions.


Assuntos
Infecções por Mycobacterium não Tuberculosas/diagnóstico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas/isolamento & purificação , Brasil/epidemiologia , Feminino , Humanos , Pulmão/microbiologia , Masculino , Infecções por Mycobacterium não Tuberculosas/epidemiologia , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Estudos Retrospectivos
16.
Genes (Basel) ; 10(4)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991756

RESUMO

The sex of an animal influences its economic traits, especially in species displaying sexual dimorphism. The Chinese soft-shelled turtle, Pelodiscus sinensis, is an economically important aquatic species that shows significant male sexual dimorphism, with a large body size, faster growth, a thick and wide calipash, and lower body fat. In this study, ten male and ten female turtles were subjected to restriction site-associated DNA sequencing (RAD-seq) using the Hi-Seq 4000 sequencing platform to isolate female-specific DNA fragments. We identified 5967 bp and 6532 bp fragments using genome walking. Three female-specific markers designed from these two fragments were confirmed to separate the sexes of Pelodiscus sinensis perfectly. One of the female-specific markers showed dosage association in female and male individuals. Individuals from different populations (n = 296) were used to validate that the female-specific markers could identify the genetic sex of Pelodiscus sinensis with 100% accuracy. The results of the present study demonstrated that RAD-seq was useful to develop sex-related markers in animals, and verified that the sex determination system of Pelodiscus sinensis belonged to the ZZ/ZW heterogametic system. Importantly, the developed markers could lead to a method for sex-controlled breeding in the Chinese soft-shelled turtle.


Assuntos
Marcadores Genéticos , Mapeamento por Restrição/veterinária , Análise de Sequência de DNA/veterinária , Tartarugas/genética , Animais , Tamanho Corporal , Feminino , Dosagem de Genes , Estudo de Associação Genômica Ampla , Masculino , Caracteres Sexuais
17.
Mol Phylogenet Evol ; 136: 196-205, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30999037

RESUMO

The West Indian avifauna has provided fundamental insights into island biogeography, taxon cycles, and the evolution of avian behavior. Our interpretations, however, should rely on robust hypotheses of evolutionary relationships and consistent conclusions about taxonomic status in groups with many endemic island populations. Here we present a phylogenetic study of the West Indian thrashers, tremblers, and allies, an assemblage of at least 5 species found on 29 islands, including what is considered the Lesser Antilles' only avian radiation. We improve on previous phylogenetic studies of this group by using double-digest restriction site-associated DNA sequencing (ddRAD-seq) to broadly sample loci scattered across the nuclear genome. A variety of analyses, based on either nucleotide variation in 2223 loci recovered in all samples or at 13,282 loci confidently scored as present or absent in all samples, converged on a single well-supported phylogenetic hypothesis. Results indicate that the resident West Indian taxa form a monophyletic group, exclusive of the Neotropical-Nearctic migratory Gray Catbird Dumetella carolinensis, which breeds in North America; this outcome differs from earlier studies suggesting that Gray Catbird was nested within a clade of island resident species. Thus, our findings imply a single colonization of the West Indies without the need to invoke a subsequent 'reverse colonization' of the mainland by West Indian taxa. Additionally, our study is the first to sample both endemic subspecies of the endangered White-breasted Thrasher Ramphocinclus brachyurus. We find that these subspecies have a long history of evolutionary independence with no evidence of gene flow, and are as genetically divergent from each other as other genera in the group. These findings support recognition of R. brachyurus (restricted to Martinique) and the Saint Lucia Thrasher R. sanctaeluciae as two distinct, single-island endemic species, and indicate the need to re-evaluate conservation plans for these taxa. Our results demonstrate the utility of phylogenomic datasets for generating robust systematic hypotheses.


Assuntos
Sequência Conservada , Passeriformes/classificação , Passeriformes/genética , Filogenia , Filogeografia , Animais , Sequência de Bases , DNA Mitocondrial/genética , Mapeamento por Restrição , Análise de Sequência de DNA , Especificidade da Espécie , Índias Ocidentais
18.
J Vis Exp ; (146)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30985753

RESUMO

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3' region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.


Assuntos
Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único/genética , Receptores de Tromboxano A2 e Prostaglandina H2/genética , Sequência de Bases , Genótipo , Humanos , Polimorfismo de Fragmento de Restrição/genética , Mapeamento por Restrição
19.
Mol Phylogenet Evol ; 137: 64-75, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31018164

RESUMO

The genus Taeniopoda Stål (Romaleidae) is a group of Nearctic-Neotropical grasshoppers whose systematics has been largely neglected. A recent phylogenetic study based on morphology and mitochondrial and nuclear markers failed to resolve the species boundaries in this genus and showed a lack of reciprocal exclusivity between T. eques (Burmeister) and T. tamaulipensis Rehn. Here we assessed the species limits and phylogenetic relationships in Taeniopoda based on 3RAD data, and evaluated the presence of gene flow and niche overlap between the above two species using clustering and ecological niche modelling (ENM) analyses to determine their taxonomic status. We performed de novo assembly of different 3RAD data sets with distinct parameters settings to explore whether they impact the recovered relationships. Ten species were consistently delimited, with T. picticornis and T. stali regarded as conspecific and the populations of T. auricornis from Guatemala representing a separate species. We maintained the specific status of T. eques and T. tamaulipensis, though our results suggest that they represent a ring species since their genetic composition appear to change gradually following a "loop form" along their geographical distribution. The phylogenomic analyses confirmed the paraphyly of Taeniopoda with respect to Romalea and recovered three major clades. Similar to previous studies, the relationships of our examined matrices were highly congruent despite their different levels of missing data. However, the similarity threshold and minimum number of samples that must share a locus for it to be retained impact the amount of loci and missing data of the matrices. This study demonstrates the utility of 3RAD to detect gene flow and to resolve species limits and phylogenetic relationships among closely related taxa.


Assuntos
Classificação , Gafanhotos/classificação , Mapeamento por Restrição , Análise de Sequência de DNA , Clima Tropical , Animais , Teorema de Bayes , Análise por Conglomerados , Geografia , Gafanhotos/genética , Filogenia , Análise de Componente Principal , Especificidade da Espécie
20.
Mol Phylogenet Evol ; 136: 21-28, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30914398

RESUMO

The orchid genus Nigritella is closely related to Gymnadenia and has from time to time been merged with the latter. Although Nigritella is morphologically distinct, it has been suggested that the separating characters are easily modifiable and subject to rapid evolutionary change. So far, molecular phylogenetic studies have either given support for the inclusion of Nigritella in Gymnadenia, or for their separation as different genera. To resolve this issue, we analysed data obtained from Restriction-site associated DNA sequencing, RADseq, which provides a large number of SNPs distributed across the entire genome. To analyse samples of different ploidies, we take an analytical approach of building a reduced genomic reference based on de novo RADseq loci reconstructed from diploid accessions only, which we further use to map and call variants across both diploid and polyploid accessions. We found that Nigritella is distinct from Gymnadenia forming a well-supported separate clade, and that genetic diversity within Gymnadenia is high. Within Gymnadenia, taxa characterized by an ITS-E ribotype (G. conopsea s.str. (early flowering) and G. odoratissima), are divergent from taxa characterized by ITS-L ribotype (G. frivaldii, G. densiflora and late flowering G. conopsea). Gymnigritella runei is confirmed to have an allopolyploid origin from diploid Gymnadenia conopsea and tetraploid N. nigra ssp. nigra on the basis of RADseq data. Within Nigritella the aggregation of polyploid members into three clear-cut groups as suggested by allozyme and nuclear microsatellite data was further supported.


Assuntos
Orchidaceae/genética , Filogenia , Mapeamento por Restrição , Análise de Sequência de DNA , Geografia , Funções Verossimilhança , Análise de Componente Principal
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