Extração de DNA de materiais de arquivo e fontes escassas para utilização em reação de polimerização em cadeia (PCR)

Este trabalho visou a comparação de cinco métodos diferentes de extração de DNA de materiais de arquivo (tecidos incluídos em parafina, esfregaços de sangue periférico - corados e não corados com Leishman, lâminas com mielogramas, gotas de sangue em Guthrie Card) e de fontes escassas (células bucais, um e três bulbos capilares e 2 mL de urina), para que fossem avaliadas a facilidade de aplicação e a facilidade de amplificação deste DNA pela técnica da reação de polimerização em cadeia (PCR). Os métodos incluíram digestão por proteinase K, seguida ou não por purificação com fenol/clorofórmio; Chelex 100® (BioRad); Insta Gene® (BioRad) e fervura em água estéril. O DNA obtido foi testado para amplificação de três fragmentos gênicos: Brain-derived neutrophic factor (764 pb), Factor V Leiden (220 pb) e Abelson (106 pb). de acordo com o comprimento do fragmento gênico estudado, da fonte potencial de DNA e do método de extração utilizado, os resultados caracterizaram o melhor caminho para padronização de procedimentos técnicos a serem incluídos no manual de Procedimentos Operacionais Padrão do Laboratório de Biologia Molecular do Hemocentro - HC - Unesp - Botucatu.

Novel polymorphic microsatellite markers in section Caulorrhizae (Arachis, Fabaceae)

This work reports the characterization of 11 polymorphic microsatellite loci in section Caulorrhizae. The primer pairs were designed from Arachis pintoi and showed full transferability to Arachis repens species. These new markers were used to evaluate the genetic diversity in germplasm (accessions and cultivars) of section Caulorrhizae. This new set of markers detected greater gene diversity than morphological and molecular markers such as AFLP (amplified fragment length polymorphism) and RAPD (rapid analysis of polymorphic DNA) previously used in this germplasm.

Genetic relationships among Arachis species based on AFLP

Amplified Fragment Length Polymorphism (AFLP) was used to establish the genetic relationships among 20 species from seven of the nine sections of genus Arachis. The level of polymorphism among nine accessions of the cultivated peanut, A. hypogaea L., was also evaluated. Three combinations of primers were used to amplify the AFLPs. The fragments were separated in 6% denaturing acrylamide gels. A total of 408 fragments were analyzed. An average of 135.3 fragments per primer combination were scored, and the largest number of fragments was 169 using primer combination Eco RI - ACC / Mse I - CTG, while the lowest was 108, with Eco RI - ACT / Mse I - CTT. In general, the genetic relationships established using AFLPs agreed with the classification established using morphology and crossability data. The results indicated that AFLPs are good markers for establishing the relationships among Arachis species. The polymorphism detected in A. hypogaea by this method was higher than the one found with other markers, like RAPDs and RFLPs. However, our data suggest that the polymorphism detected be using AFLP with only three primer combinations is still too low to be used for any kind of genetic study in this species.

Genetic diversity of Stryphnodendron adstringens (Mart.) Coville determined by AFLP molecular markers

Bark extracts of Stryphnodendron adstringens (Mart) Coville a Leguminosae species, well known in Brazil as barbatimao, are popularly used as healing agent. The objective of this work was to determine the genetic diversity of S. adstringens populations and to correlate genetic distances to the production of tannins. S. adstringens accessions from populations found in Cerrado regions in the states of Goias, Minas Gerais and São Paulo were analyzed using the AFLP (Amplified Fragment Length Polymorphism) technique. A total of 236 polymorphic bands were scored and higher proportion of genetic diversity was found inter populations (70.9%), rather than intra populations (29.1%). F-ST value was found to be significantly greater than zero (0.2906), demonstrating the complex genetic structure of S. adstringens populations. Accessions collected in Cristalina, GO, showed higher percentage of polymorphic loci (87.3%) and the highest genetic diversity. The lowest genetic variability was detected among accessions from the population growing in Caldas Novas, GO. The genetic distance among populations was estimated using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), which grouped populations into 3 clusters. Moreover, chemotypes with tannin concentration above 40% showed higher genetic similarity. AFLP analysis proved to be an efficient gene mapping technique to determine the genetic diversity among remaining populations of S. adstringens. Obtained results may be employed to implement further strategies for the conservation of this medicinal plant. (C) 2011 Elsevier Ltd. All rights reserved.

Genetic diversity of Rhizoctonia solani AG-3 from potato and tobacco in North Carolina

Anastomosis group 3 (AG-3) of Rhizoctonia solani (teleomorph = Thanatephorus cucumeris) is frequently associated with diseases of potato (AG-3 PT) and tobacco (AG-3 TB). Although isolates of R. solani AG-3 from these two Solanaceous hosts are somatically related based on anastomosis reaction and taxonomically related based on fatty acid, isozyme and DNA characters, considerable differences are evident in their biology, ecology, and epidemiology. However, genetic diversity among field populations of R. solani AG-3 PT and TB has not been documented. In this study, the genetic diversity of field populations of R. solani AG-3 PT and AG-3 TB in North Carolina was examined using somatic compatibility and amplified fragment length polymorphism (AFLP) criteria. A sample of 32 isolates from potato and 36 isolates from tobacco were paired in all possible combinations on PDA plus activated charcoal and examined for their resulting somatic interactions. Twenty-eight and eight distinct somatic compatibility groups (SCG) were identified in the AG-3 PT and AG-3 TB samples, respectively. AFLP analyses indicated that each of the 32 AG-3 PT isolates had a distinct AFLP phenotype, whereas 28 AFLP phenotypes were found among the 36 isolates of AG-3 TB. None of the AG-3 PT isolates were somatically compatible or shared a common AFLP phenotype with any AG-3 TB isolate. Clones (i.e., cases where two or more isolates were somatically compatible and shared the same AFLP phenotype) were identified only in the AG-3 TB population. Four clones from tobacco represented 22% of the total population. All eight SCG from tobacco were associated with more than one AFLP phenotype. Compatible somatic interactions between AG-3 PT isolates occurred only between certain isolates from the same field (two isolates in each of four different fields), and when this occurred AFLP phenotypes were similar but not identical.

Variabilidade genética intrapopulacional em Myracrodruon urundeuva Fr. All. por marcador AFLP

Levels of genetic variability for in situ and ex situ genetic conservation were estimated in a population of Myracrodruon urundeuva using the PCR (polymerase chain reaction) technique with the AFLP (Amplified fragment-length polymorphism) genetic marker. Seeds for progeny tests were collected from 30 open-pollination trees (matrices) at Paulo de Faria Ecological Station - SP. From this genetic material, three progeny tests were installed on the Teaching and Research Farm of Ilha Solteira Faculty of Engineering - University of São Paulo State (UNESP), which is located in Selvlria - MS, Brazil. The analysis by genetic marker was conducted with three combinations of different starters EcoRl-Msel, resulting in a total number of 137 polymorphic bands, thus forming a table of binary data. These data were used for the analysis of genetic divergence and distance between progenies. High levels of genetic divergence were observed among families. Based on the Analysis of Molecular Variance (AMOVA), it was shown that 16.2% of genetic diversity is found among progenies and 83.8% within progenies, which suggests deviances of random matings. The grouping of progenies, based on genetic distances, suggests that progenies deriving from trees which are close to each other tend to be more similar. This, in turn, indicates that the population originating the seeds may be genetically structured.

QTL identification for tolerance to fruit set in tomato by FAFLP markers

This study aimed to detect quantitative trait loci (QTL) by fALP (Fluorescent Amplified Fragment Length Polymorphism) markers associated to the trait tomato fruit set at high temperatures. A biparental cross between line Jab-95 (heat-tolerant) and cultivar Caribe (heat-susceptible) was made. A total of 192 plants of the F2 generation were evaluated, generating 172 polymorphic markers through six primer combinations previously identified by the Bulked Segregant Analysis technique. To construct the genetic map, 106 of the 172 markers that segregated in the expected Mendelian segregation proportion (3:1) were used. The map covered 1191.46 cM of the genome. Six trait-linked QTL were identified in the analysis of simple markers and three others by the interval-mapping methodology. These results could be highly useful in improvement programs, since heat-tolerant plants can be selected rapidly, which improves tomato fruit set.

Characterization and genome organization of a repetitive element associated with the nucleolus organizer region in leporinuselongatus (anostomidae: Characiformes)

Chromosome mapping and studies of the genomic organization of repetitive DNA sequences provide valuable insights that enhance our evolutionary and structural understanding of these sequences, as well as identifying chromosomal rearrangements and sex determination. This study investigated the occurrence and organization of repetitive DNA sequences in Leporinus elongatus using restriction enzyme digestion and the mapping of sequences by chromosomal fluorescence in situ hybridization (FISH). A 378-bp fragment with a 54.2% GC content was isolated after digestion with the SmaI restriction enzyme. BLASTN search found no similarity with previously described sequences, so this repetitive sequence was named LeSmaI. FISH experiments were conducted using L. elongatus and other Anostomidae species, i.e. L. macrocephalus,L. obtusidens, L. striatus, L. lacustris, L. friderici, Schizodon borellii, S. isognathus, and Abramites hypselonotus which detected signals that were unique to male and female L. elongatus individuals. Double-FISH using LeSmaI and 18S rDNA showed that LeSmaI was located in a nucleolus organizer region (NOR) in the male and female metaphases of L. elongatus. This report also discusses the role of repetitive DNA associated with NORs in the diversification of Anostomidae species karyotypes. Copyright © 2012 S. Karger AG, Basel.

Interclonal Variations in the Molecular Karyotype of Trypanosoma cruzi: Chromosome Rearrangements in a Single Cell-Derived Clone of the G Strain

Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure. © 2013 Lima et al.

Perfil de citocinas no soro e na secreção cervical de mulheres com lesão intraepitelial de baixo grau, lesão intraepitelial de alto grau e carcinoma epidermóide invasor

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Similaridade genética em acessos de goiabeiras e araçazeiros: análises químicas e bioquímicas dos frutos

Pós-graduação em Ciências Biológicas (Botânica) - IBB

Micropropagação e uso de marcadores moleculares na determinação do sexo do mamoeiro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caracterização genotípica de amaostras de Toxoplasma gondii isoladas de ovinos de abatedouro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Revisiting AFLP fingerprinting for an unbiased assessment of genetic structure and differentiation of taurine and zebu cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Diversidade de populações de Phyllosticta spp. de goiabeiras e de mangueiras em diferentes ambientes

Pós-graduação em Agronomia (Produção Vegetal) - FCAV