Optimal single-embryo mass spectrometry fingerprinting

In pre-implantation embryos, lipids play key roles in determining viability, cryopreservation and implantation properties, but often their analysis is analytically challenging because of the few picograms of analytes present in each of them. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) allows obtaining individual phospholipid profiles of these microscopic organisms. This technique is sensitive enough to enable analysis of individual intact embryos and monitoring the changes in membrane lipid composition in the early stages of development serving as screening method for studies of biology and biotechnologies of reproduction. This article introduces an improved, more comprehensive MALDI-MS lipid fingerprinting approach that considerably increases the lipid information obtained from a single embryo. Using bovine embryos as a biological model, we have also tested optimal sample storage and handling conditions before the MALDI-MS analysis. Improved information at the molecular level is provided by the use of a binary matrix that enables phosphatidylcholines, sphingomyelins, phosphatidylserines, phosphatidylinositols and phosphoethanolamines to be detected via MALDI(±)-MS in both the positive and negative ion modes. An optimal MALDI-MS protocol for lipidomic monitoring of a single intact embryo is therefore reported with potential applications in human and animal reproduction, cell development and stem cell research. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.

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)

A trade off between separation, detection and sustainability in liquid chromatographic fingerprinting

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

Green chromatographic fingerprinting: An environmentally friendly approach for the development of separation methods for fingerprinting complex matrices

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

HPLC-DAD method for metabolic fingerprinting of the phenotyping of sugarcane genotypes

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Acetone as a greener alternative to acetonitrile in liquid chromatographic fingerprinting

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

Genetic differentiation between sympatric populations of Bacillus cereus and Bacillus thuringiensis

Little is known about genetic exchanges in natural populations of bacteria of the spore-forming Bacillus cereus group, because no population genetics studies have been performed with local sympatric populations. We isolated strains of Bacillus thuringiensis and B. cereus from small samples of soil collected at the same time from two separate geographical sites, one within the forest and the other at the edge of the forest. A total of 100 B. cercus and 98 B. thuringiensis strains were isolated and characterized by electrophoresis to determine allelic composition at nine enzymatic loci. We observed genetic differentiation between populations of B. cereus and B. thuringiensis. Populations of a given Bacillus species-B. thuringiensis or B. cereus-were genetically more similar to each other than to populations of the other Bacillus species. Hemolytic activity provided further evidence of this genetic divergence, which remained evident even if putative clones were removed from the data set. Our results suggest that the rate of gene flow was higher between strains of the same species, but that exchanges between B. cereus and B. thuringiensis were nonetheless possible. Linkage disequilibrium analysis revealed sufficient recombination for B. cereus populations to be considered panmictic units. In B. thuringiensis, the balance between clonal proliferation and recombination seemed to depend on location. Overall, our data indicate that it is not important for risk assessment purposes to determine whether B. cereus and B. thuringiensis belong to a single or two species. Assessment of the biosafety of pest control based on B. thuringiensis requires evaluation of the extent of genetic exchange between strains in realistic natural conditions.

Análise da variabilidade genética de acessos dePsidium spp. (Myrtaceae) avaliados quanto à reação a Meloidogyne enterolobii

This study aimed to characterize molecular of 13 accessions of Psidium spp. (Myrtaceae) that was been identified for the reaction to rootknot guava nematode. The DNA extraction of the samples was carried according to the protocol of Shillito & Saul (1988). The molecular markers type fAFLP, were obtained from fAFLP Regular Plant Genomes Fingerprinting Kit' (Applied Biosystems from Brasil Ltda.) and were tested 24 selectives combinations of primers, of which 18 showed amplification that produced 272 polymorphic markers. To the analysis of the markers were employed the softwares GeneScan (ABI Prism versao 1.0) and Genotyper (ABI Prism version 1.03), and the data collected were transformed into a binary matrix that was analyzed in the software PAUP (Phylogenetic Analysis Using Parcimony - version 3.01). Were calculated genetic distance index intra and interespecific between the genotipes. It was found that the AFLP markers were efficient in the discrimination between accessions, as well as in showing genetic similarity among accessions identified as resistant to the nematode Meloidogyne enterolobii, which could be discussed in the future.

Caracterização molecular e diversidade genética de diferentes variedades de abacate por marcadores microssatélites

Pertencente à família Lauraceae, o abacateiro compreende três raças hortícolas: antilhana, guatemalense e mexicana. Os marcadores moleculares são uma ferramenta rápida e eficaz para estudos genômicos, uma vez que detectam o polimorfismo diretamente ao nível do DNA e não sofrem qualquer tipo de influência ambiental. Com base nesse polimorfismo, é possível fazer inferências sobre as relações entre o genótipo e o fenótipo dos indivíduos, o que, em última análise, permite aumentar a eficiência dos programas de melhoramento. Diante o exposto, o objetivo foi investigar a diversidade genética entre sete variedades de abacate a partir de 5 lócus de marcadores moleculares microssatélites (SSR). Nas amostras de abacateiros avaliadas, encontrou-se um total de 18 alelos, com uma média de 3,6 alelos por lócus. O dendrograma gerado a partir de análise de agrupamento UPGMA agrupou, separadamente do resto dos genótipos, a cultivar Geada da raça Antilhana, possivelmente por esta variedade ser uma raça pura, e o restante foi agrupado em dois grandes grupos das raças, a Guatemalense e a Mexicana. Os genótipos das sete variedades de abacate apresentam diversidade genética nos cinco lócus de marcadores moleculares microssatélites (SSR) avaliados, o que indica que são materiais promissores para utilização em futuros programas de melhoramento.

A naturally occurring recombinant isolate of Lettuce mosaic virus

LMV-Common and LMV-Most are two seed-borne types of Lettuce mosaic virus (LMV), genus Potyvirus. LMV-Most, but not LMV-Common, overcomes the resistance afforded to lettuce by two recessive genes, mo1(1) and mo1(2). An RT-PCR-based assay thought to be specific for LMV-Most also amplified LMV-Tn2, previously typified as LMV-Common. The sequence of selected regions along the genome indicated that LMV-Tn2 is a natural recombinant between LMV-Most and LMV-Common isolates, with a putative recombination site located within the P3 coding region. This is the first evidence of a naturally occurring LMV recombinant isolate.

Specific detection of Lettuce mosaic virus isolates belonging to the Most type

Lettuce mosaic virus (LMV)-Most isolates can infect and are seed-borne in cultivars containing the mol gene. A reverse transcription and polymerase chain reaction (RT-PCR)-based test was developed for the specific detection of LMV-Most isolates. Based on the complete genome sequences of three LMV isolates belonging respectively to the Most type, the Common type and neither of these two types, three different assays were compared: (i) presence of a diagnostic restriction site in the region of the genome encoding the variable N-terminus of the capsid protein, in the 3' end of the genome, (ii) RT-PCR using primers designed to amplify a cDNA corresponding to a portion of the P1 coding region, in the 5' end of the genome and (iii) RT-PCR using primers designed to amplify a central region of the genome. The assays were performed against a collection of 21 isolates from different geographical origins and representing the molecular variability of LMV. RT-PCR of the central region of the genome was preferred because its results are expected to be less affected by natural recombination between LMV isolates, and it allows sensitive detection of LMV-Most in situations of single as well as mixed contamination. (C) 2004 Elsevier B.V. All rights reserved.

Seleção para aumento de produtividade e qualidade de frutos em abobrinha 'Piramoita' comparando dois métodos de melhoramento

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

Genotyping, serotyping and determination of mating-type of Cryptococcus neoformans clinical isolates from São Paulo State, Brazil

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