Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data

<p>Abstract</p> <p>Background</p> <p>Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes.</p> <p>Results</p> <p>By the use of two distinct algorithms, implemented by <it>geNorm </it>and <it>NormFinder</it>, we have assessed the gene expression of nine candidate reference genes in cotton: <it>GhACT4, GhEF1α5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhβTUB3 </it>and <it>GhUBQ14</it>. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of <it>GhPP2A1 </it>and <it>GhUBQ14 </it>genes were the most stable across all samples and also when distinct plants organs are examined. <it>GhACT4 </it>and <it>GhUBQ14 </it>present more stable expression during flower development, <it>GhACT4 </it>and <it>GhFBX6 </it>in the floral verticils and <it>GhMZA </it>and <it>GhPTB </it>during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development.</p> <p>Conclusion</p> <p>We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of <it>GhUBQ14 </it>and <it>GhPP2A1 </it>housekeeping genes as superior references for normalization of gene expression measures in different cotton plant organs; <it>GhACT4 </it>and <it>GhUBQ14 </it>for flower development, <it>GhACT4 </it>and <it>GhFBX6 </it>for the floral organs and <it>GhMZA </it>and <it>GhPTB </it>for fruit development. We also provide the primer sequences whose performance in qPCR experiments is demonstrated. These genes will enable more accurate and reliable normalization of qPCR results for gene expression studies in this important crop, the major source of natural fiber and also an important source of edible oil. The use of bona fide reference genes allowed a detailed and accurate characterization of the temporal and spatial expression pattern of two MADS-box genes in cotton.</p

Identification and evaluation of new reference gene in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data.

Disponível em:.Acesso em: 6 jan. 2011

Method development for the analysis of volatile compounds in olive oil.

Olive oil consists mainly of triglycerides, in about 97 to 99% by weight. The minor compounds are a complex mixture of polar, apolar and amphiphilic substances, such as tocopherols, phenolic compounds, sterols, chlorophyll, carotenoids, terpene acids, monoglycerides and diglycerides, free fatty acids and volatile compounds. These volatiles are the compounds directly responsible for the aroma of the oil. Extra virgin olive oil has a complex aroma with more than 100 volatile compounds identified, among aldehydes, alcohols, esters, hydrocarbons, ketones and furans. The objective of this study was to develop an analytical method for volatile compounds in olive oils using solid-phase microextraction (SPME), gas chromatography coupled to mass spectrometry (GC-MS) and gas chromatography with flame ionization detection (GC-FID). For the SPME, different parameters like flask size (4, 10 and 20 mL), sampling temperatures (40 and 60 °C), headspace conditioning (10 and 60 min) and fiber exposure times (15 and 40 min) were tested. For GC analyses two different internal standards, methyl octanoate and tetradecane, were tested, as well as sub-ambient oven temperatures with liquid nitrogen. A 1 g of sample and a divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PMDS) fiber were used in all the tests. Analytical curves from the FID data were constructed for linearity evaluation, whereas internal standard was used for quantification ofcompounds such as 3-hexenol, 2-hexanal and limonene. Identification was performed based on mass spectra, co-injection of standards and retention indices data. The best conditions for SPME analysis were sample temperature of 40 °C, 10 min of headspace conditioning and fiber exposure for 40 min, in a 4 mL flask. For the chromatographic analyzes, tetradecane was chosen as the internal standard. Oven temperature program with cryofocusing led to a much better separation and, therefore, better quantitation and identification of the morevolatile compounds.De 15 a 16 maio 2019. Trabalhos apresentados de forma oral

Analysis of volatile compounds in extra virgin olive oil from Rio Grande do Sul by SPME/GC-MS.

De 15 a 16 maio 2019. Trabalhos apresentados de forma oral

Rapid diagnosis of coccidioidomycosis by nested PCR assay of sputum

Coccidioidomycosis is a deep infection caused by two dimorphic fungi, Coccidioides immitis and Coccidioides posadasii. Diagnosis of the disease requires culture of suspicious clinical samples on mycological media. However, as these species are virulent pathogens, handling of their cultures is a high-risk activity, and is limited to Biosafety Level 3 laboratories. This study describes the direct detection of C. posadasii DNA in an inappropriate sputum sample by PCR amplification of the highly specific Ag2/PRA antigen gene. the results obtained suggest that direct detection of the Ag2/PRA sequence in sputum is an excellent method for rapid and specific diagnosis of coccidioidomycosis.Fed Univ Ceara, Med Mycol Specialized Ctr, Fortaleza, Ceara, BrazilFed Univ Ceara, Postgrad Program Med Sci, Fortaleza, Ceara, BrazilState Univ Ceara, Postgrad Program Vet Sci, Fortaleza, Ceara, BrazilState Univ Ceara, Dept Sci Biol, Fortaleza, Ceara, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilWeb of Scienc

Plant growth forms dictate adaptations to the local climate

Adaptive radiation is a significant driver of biodiversity. Primarily studied in animal systems, mechanisms that trigger adaptive radiations remain poorly understood in plants. A frequently claimed indicator of adaptive radiation in plants is growth form diversity when tied to the occupation of different habitats. However, it remains obscure whether morphological adaptations manifest as growth form diversity per se or as its constituent traits. We use the classic Aeonium radiation from the Canary Islands to ask whether adaptation across climatic space is structured by growth form evolution. Using morphological sampling with site-associated climate in a phylogenetic context, we find that growth forms dictate adaptations to the local environment. Furthermore, we demonstrate that the response of specific traits to analogous environments is antagonistic when growth forms are different. This finding suggests for the first time that growth forms represent particular ecological functions, allowing the co-occurrence of closely related species, being a product of divergent selection during evolution in sympatry.info:eu-repo/semantics/publishedVersio

Genome size variation within Crithmum maritimum: Clues on the colonization of insular environments

Angiosperms present an astonishing diversity of genome sizes that can vary intra- or interspecifically. The remarkable new cytogenomic data shed some light on our understanding of evolution, but few studies were performed with insular and mainland populations to test possible correlations with dispersal, speciation, and adaptations to insular environments. Here, patterns of cytogenomic diversity were assessed among geographic samples (ca. 114) of Crithmum maritimum (Apiaceae), collected across the Azores and Madeira archipelagos, as well as in adjacent continental areas of Portugal. Using flow cytometry, the results indicated a significant intraspecific genome size variation, spanning from reduced sizes in the insular populations to larger ones in the mainland populations. Moreover, there was a tendency for an increase in genome size along the mainland populations, associated with lower temperatures, higher precipitation, and lower precipitation seasonality. However, this gradient might be the result of historic phylogeographical events associated with previous dispersal and extinction of local populations. Overall, our findings provided evidence that smaller genome sizes might play a critical role in the colonization of islands, corroborating other studies that argue that organisms with smaller genomes use fewer resources, having a selective advantage under insular environments. Although further studies are needed to improve our understanding of the mechanisms underlying genome size evolution on islands, conservation strategies must be promoted to protect the rich cytogenomic diversity found among C. maritimum populations, which occur in coastal areas that are particularly threatened by human activity, pollution, invasive species, and climate changes.info:eu-repo/semantics/publishedVersio

Inter- and intra-island speciation and their morphological and ecological correlates in Aeonium (Crassulaceae), a species-rich Macaronesian radiation

Background and Aims The most species-rich and ecologically diverse plant radiation on the Canary Islands is the Aeonium alliance (Crassulaceae). In island radiations like this, speciation can take place either within islands or following dispersal between islands. Aiming at quantifying intra- and inter-island speciation events in the evolution of Aeonium, and exploring their consequences, we hypothesized that (1) intra-island diversification resulted in stronger ecological divergence of sister lineages, and that (2) taxa on islands with a longer history of habitation by Aeonium show stronger ecological differentiation and produce fewer natural hybrids. Methods We studied the biogeographical and ecological setting of diversification processes in Aeonium with a fully sampled and dated phylogeny inferred using a ddRADseq approach. Ancestral areas and biogeographical events were reconstructed in BioGeoBEARS. Eleven morphological characters and three habitat characteristics were taken into account to quantify the morphological and ecological divergence between sister lineages. A co-occurrence matrix of all Aeonium taxa is presented to assess the spatial separation of taxa on each island. Key Results We found intra- and inter-island diversification events in almost equal numbers. In lineages that diversified within single islands, morphological and ecological divergence was more pronounced than in lineages derived from inter-island diversification, but only the difference in morphological divergence was significant. Those islands with the longest history of habitation by Aeonium had the lowest percentages of co-occurring and hybridizing taxon pairs compared with islands where Aeonium arrived later. Conclusions Our findings illustrate the importance of both inter- and intra-island speciation, the latter of which is potentially sympatric speciation. Speciation on the same island entailed significantly higher levels of morphological divergence compared with inter-island speciation, but ecological divergence was not significantly different. Longer periods of shared island habitation resulted in the evolution of a higher degree of spatial separation and stronger reproductive barriers.info:eu-repo/semantics/publishedVersio

Atributos morfológicos de Luvissolo no Núcleo de Desertificação de Cabrobó no Estado de Pernambuco.

No Brasil, a região semiárida ocupa uma área de 969.589,4 km², parte das áreas localizadas na região semiárida do Nordeste brasileiro estão susceptíveis à desertificação. Essas áreas susceptíveis a desertificação são conhecidas como Núcleos de Desertificação identificadas como: Núcleo de desertificação de Gilbués (PI), de Irauçuba (CE), do Seridó (PB) e de Cabrobó (PE). No Núcleo de desertificação de Cabrobó, o município de Itacuruba está inserido na unidade geoambiental da Depressão Sertaneja. Os Luvissolos são de grande ocorrência nas áreas cristalinas sertanejas associados ao relevo suavemente ondulado. O presente trabalho tem como objetivo de contribuir com a caracterízação morfológica de Luvissolo localizado na Núcleo de Desertificação de Cabrobó do estado de Pernambuco. Foi realizada a abertura de uma trincheira e posteriormente, a caracterização morfológica do perfil de acordo com o Manual de Descrição e Coleta de Solo no Campo. No Núcleo de Desertificação de Cabrobó, inserido na Depressão sertaneja, o Luvissolo apresentou revestimento pedregoso na superfície, o que dificulta o seu manejo