Estimation of Nuclear Genome Size of Three Species of Camponotus (Mayr, 1861) (Hymenoptera: Formicidae: Formicinae) and Their Cytogenetic Relationship

The chromosome variability among ant species is remarkable, and the processes generating such variation are still under discussion since polyploidy has been observed in some distinct taxa. The chromosome number of species belonging to the Camponotus, subgenera Myrmothrix and Myrmobrachys, are highly different, whereas, the first subgenus has double the number of chromosomes of the second. In order to test the hypothesis of chromosome number doubling through polyploidy, the genome sizes of Camponotus (Myrmothrix) rufipes, Camponotus (Myrmothrix) renggeri and Camponotus (Myrmobrachys) crassus were estimated by flow cytometry. The chromosome number of specimens from the nests studied was also defined. No significant variation was noted in the genome size among them. The mean haploid genome size value (1C) of workers for the three species was 286.16 Mpb (0.29 pg). The polyploidy hypothesis can be ruled out as an evolutionary step linking the karyotype variations among the three studied species since the genome size of C. crassus with 2n = 20 chromosomes was the same as that of C. rufipes and C. renggeri with 2n = 40. The lack of variation in the amount of DNA between the related species C. rufipes and C. renggeri also demonstrate that flow cytometry is not an adequate approach to distinguish them. Our results highlight the importance of combining distinct methods, DNA quantification, and cytogenetics from the same colony. Understanding the path of chromosome evolution of three species with distinct degrees of relatedness should provide further information in enriching our knowledge about the Minimum Interaction Theory

Detection of diploid males in a natural colony of the cleptobiotic bee Lestrimelitta sp (Hymenoptera, Apidae)

When working at quantifying the genome size of stingless bees, it was observed that males of Lestrimelitta sp possessed the same amount of nuclear DNA as the females. Thus, we used flow cytometry (FCM) and cytogenetic analysis to confirm the ploidy of these individuals. The males analyzed proved to be diploid, since, through cytometric analysis, it was demonstrated that the mean genome size of both males and females was the same (C = 0.463 pg), and, furthermore, cytogenetic analysis demonstrated that both had 2n = 28 chromosomes

Chromosome specific probe construction from a single microdissected chromosome

Sondas cromossomo-específicas referem-se a um conjunto de sequências de DNA marcadas com fluorescência, específicas de um par cromossômico, visualizadas através da hibridização em preparações citogenéticas. Tais sondas possibilitam a visualização altamente sensível e exclusiva de cromossomos individuais, ampliando a resolução diferencial de cariótipos. Elas podem ser empregadas em análises de organização do genoma, genômica comparativa, estudos evolutivos e filogenéticos, auxiliar na identificação de pares de homólogos, comparação cariotípica e obtenção de mapas físicos com marcas que auxiliem em programas de melhoramento genético. Técnicas de microdissecção de cromossomos, juntamente com métodos de isolamento e amplificação do DNA microdissectado, representam ferramentas úteis para serem aplicadas na construção dessas sondas cromossomo-específicas. Um fator considerado limitante no uso da microdissecção é a necessidade de coletar em torno de 20 cópias de um determinado cromossomo. Isso implica na necessidade de reconhecimento prévio do alvo e a certeza na identificação. A utilização de um único cromossomo resolve essa questão e possibilita a construção de sondas sem necessidade de identificação prévia, além de reduzir os esforços relativos à microdissecção. Considerando o potencial informativo dessas sondas e o desafio de construí-las a partir de um único cromossomo, o presente trabalho foi realizado com objetivo de padronizar metodologias para construção de sondas cromossomo-específicas a partir de um cromossomo isolado por microdissecção. Para tanto, foram realizadas adaptações das técnicas para obtenção de cromossomos mitóticos e meióticos adequados para caracterização cariotípica e aplicação das técnicas de microdissecção e hibridização in situ fluorescente. Foram utilizadas metáfases provenientes de cultura de linfócitos humanos para padronização de (i) métodos de microdissecção; (ii) enzimas adequadas e condições de amplificação de um cromossomo por DOP-PCR; (iii) enzimas para a marcação fluorescente das sondas por random primer e (iv) condições de estringência para a hibridização in situ fluorescente (FISH). Após essas padronizações iniciais, as metodologias foram empregadas em milho (Zea mays) e pimentão (Capsicum annuum) para primeiros testes envolvendo espécies vegetais. Foram obtidas sondas cromossomo-específicas para o par 2 do cariótipo humano, para o par cromossômico 1 de milho e para pimentão (provavelmente o par 7 ou 8). Esses resultados demonstraram que, apesar da construção de sondas cromossomo-específicas ser uma metodologia extensa e desafiadora, a padronização de cada etapa possibilitou a marcação de um par específico a partir da coleta de um único cromossomo para humano, milho e pimentão. A microdissecção, a transferência do material para o microtubo e a comparação de diferentes parâmetros na amplificação por DOP-PCR foram consideradas como as etapas mais críticas do processo. A qualidade das preparações cromossômicas foi um fator crucial para aplicação das metodologias de microdissecção e FISH. Esse trabalho revela estratégias baseadas na microdissecção de um único cromossomo que possibilitaram a produção de sondas cromossomo-específicas para diferentes organismos, como humano, milho e pimentão. As etapas padronizadas poderão direcionar os procedimentos de construção de sondas cromossomo-específicas para todos os cromossomos dos cariótipos das espécies utilizadas nesse trabalho, bem como poderão ser aplicadas em outras plantas.Chromosome-specific probes are DNA sequences, fluorescently labelled specifically for a chromosome pair. Such probes can be applied for analysis in evolutionary and phylogenetic studies, for karyotype comparison and to obtain physical maps that help in plant breeding programs. Techniques of chromosome microdissection, along with methods of isolation and amplification of microdissected DNA, represent useful tools to be employed in the construction of chromosome-specific probes. A considered limiting factor in the use of microdissection is the necessity to collect around 20 copies of a particular chromosome. This implies the need of prior recognition of the target and the conviction in identification. The use of a single chromosome solves this issue and allows the construction of probes without prior identification, besides reducing the efforts on microdissection. Considering the potential of these informative probes, this study aimed to compare and standardize methods of classical and molecular cytogenetics to build probes. Therefore, technical adjustments were made to obtain mitotic and meiotic chromosomes suitable for karyotype characterization and application in microdissection techniques and in situ hybridization. Metaphases from human lymphocytes were used to standardization of (i) microdissection methods; (ii) chromosomes amplification; (iii) DNA fluorescent labelling and (iv) stringency conditions for fluorescence in situ hybridization (FISH). After the initial protocol standardization, the methodologies were used in maize (Zea mays) and sweat peppers (Capsicum annuum). A chromosome-specific probe for pair 2 of human chromosomes, a probe to chromosome 1 of corn and a probe for sweat pepper (probably the pair 7 or 8) were obtained. Although chromosome-specific probes construction involves many steps, the standardization of each process stages allowed, in our conditions, to obtain a specific probe from a single chromosome for humans, corn and sweat pepper. The microdissection and the comparison of different DOP-PCR parameters were considered as the most critical steps of the process. Human and plant chromosomes showing preserved morphology and structure provided a suitable template DNA for probes construction. The quality of chromosome preparation was a key factor for the microdissection and application of FISH methodologies. This work reveals strategies based on microdissection of a single chromosome that made possible the production of chromosome-specific probes for different organisms, such as human, corn and pepper. Standardized steps may guide the procedures of chromosome-specific probes construction for all chromosomes of karyotypes of the species used in this work, and it may be applied to other plant.Conselho Nacional de Desenvolvimento Científico e Tecnológic

Determination of the AT GC base composition by flow cytometry in bees

A determinação do tamanho genômico e da composição de bases AT e GC é considerada uma informação crucial dentro das análises que visam à caracterização do material genético de um indivíduo, ou mesmo em estudos de biologia molecular e de interpretações filogenéticas e evolutivas. Por ser relativamente rápida, precisa e econômica, a citometria de fluxo tem se destacado como ferramenta para quantificação do conteúdo de DNA e caracterização da composição de bases em diversos grupos de plantas e animais, incluindo insetos. Considerando os insetos himenópteros, até o momento cerca de 100 indivíduos tiveram seu conteúdo de DNA determinado, e 46 % destes são abelhas sem ferrão. Apenas uma espécie de abelha, a Apis melífera, teve sua composição de bases AT e GC determinadas até o momento e esses dados foram obtidos por meio do sequenciamento genômico. Esse trabalho buscou detalhar o procedimento para a quantificação de DNA e desenvolver metodologia para a determinação da composição de bases AT e GC por citometria de fluxo em abelhas. Objetivou-se então contribuir com o aumento do número de espécies com conteúdo de DNA determinado e disponibilizar uma metodologia eficiente para caracterização do genoma. As espécies de abelhas empregadas foram Scaptotrigona xantotricha, Trigona hyalinata e Partamona rustica. Os gânglios de cada pupa foram utilizados para preparação da suspensão nuclear analisada no citômetro de fluxo. A S. xantotricha (2C = 0,88 picogramas) foi utilizada como padrão interno nas análises de determinação do conteúdo de DNA nuclear total e da proporção de bases AT e GC. Os histogramas gerados na citometria de fluxo apresentaram coeficientes de variação abaixo de 5,0 % e possibilitaram a determinação da quantidade absoluta de DNA nuclear e da composição de bases AT e GC. P. rustica e T. hyalinata apresentaram 1,15 e 1,07 picogramas de DNA, respectivamente. S. xantotricha apresentou 61,32 % de bases AT e 38,68 % de bases GC; P. rustica apresentou AT = 62,82 % e GC = 37,18 %; T. hyalinata apresentou AT = 62,40 % e GC = 37,60 %. Esse trabalho detalha o protocolo para quantificação do DNA por meio da citometria de fluxo, contribuindo para o aumento do número de espécies com conteúdo de DNA determinado. Ainda, disponibiliza metodologia para a eterminação da composição de bases AT e GC em abelhas também empregando a citometria de fluxo. Essa nova técnica pode ser aplicada a outras espécies de abelhas ou até mesmo a outros insetos himenópteros, a fim de fornecer dados relevantes para análises de caracterização do genoma.The genomic size determination and AT GC base composition should be considered an important factor in the analysis to genetic material characterization, or even in studies of molecular biology and phylogenetic interpretations. Flow cytometry has emerged as a tool for quantification of DNA content and characterization of base composition in many plant and animal groups, including insects. Considering the hymenopterous, about 100 species had their DNA content determined, and 46 % of them are stingless bees. This work aimed to detail the procedure for DNA quantification and to develop methodology for determining the AT and GC base composition by flow cytometry in bees. It will increase the number of species with DNA content determined and provide an effective genomic characterization procedure. Scaptotrigona xantotricha (2C = 0.88 picograms) was used as internal standard to determinate the DNA content and base composition of Trigona hyalinata and Partamona rustica. The histograms generated in the flow cytometry showed coefficients of variation below 5.0 % and enabled the determination of the nuclear DNA content and AT GC composition. P. rustica and T. hyalinata showed 1.15 and 1.07 picograms of DNA, respectively. S. xantotricha presented 61.32 % of bases AT and 38.68 % of GC, P. rustica presented AT = 62.82 % and GC = 37.18 %, T. hyalinata presented AT = 62.40 % and GC = 37.60 %. This study details the protocol for DNA quantification by flow cytometry, contributing to increasing the number of species of DNA content determined. Also provides methodology for determining the base composition AT and GC bees using flow cytometry. This new technique can be applied to other bee species or even other hymenopteran insects, in order to provide relevant data for genomic characterization analysis.Conselho Nacional de Desenvolvimento Científico e Tecnológic

Reflexões de um grupo de professores acerca do melhoramento genético humano a partir de discussões de textos de divulgação científica

RESUMO: A possibilidade da manipulação genética humana levanta sérios questionamentos éticos. Nesse contexto objetiva-se analisar os sentidos produzidos em discursos de professores a partir da leitura e reflexão de textos de divulgação científica acerca do tema. A pesquisa foi realizada por meio da aplicação de questionários e vídeo gravação das falas de 17 professores durante um curso de formação continuada. Os dados constituídos foram organizados e analisados mediante Análise de Discurso Francesa. Os resultados apontaram que a formação discursiva e ideológica dos professores e as reflexões suscitadas durante o curso contribuíram para os docentes compreenderem os diferentes sentidos produzidos pelos discursos das atuais técnicas de manipulação genética humana e suas aproximações ideológicas com o “velho discurso eugênico” e suas implicações éticas

Genome size variation in

The stingless bees of the genus Melipona comprise a group with approximately 40 Neotropical species. Despite their ecological and economic importance, the size of the genomes of these species remains poorly known. Thus, the present study measured the DNA content of 15 Melipona species. The mean genome size (1C) of the females ranged from 0.27 pg to 1.38 pg, with increments of, approximately, 0.12 pg. It was possible to recognize two groups of species: the first presented relatively low DNA content (average = 0.29 pg), while the second showed high DNA content (average = 0.98 pg). This result corroborates the cytogenetic classification of these species into two groups, one of them comprising species with a low heterochromatin content ( 50%). Amongst the groups with low and high DNA content, there was no significant correlation between the DNA content and the size of the bees. The data obtained may aid in the selection of species which are suitable for sequencing projects, besides providing an overview of the diversity in the genome size of the Melipona genus

Flow cytometry and cytogenetic tools in eucalypts: genome size variation × karyotype stability

The eucalypts comprise a group of woody plants used in commercial forest plantations owing to their high growth rates, adaptability to various ecological conditions and multiple applications. Despite the enormous amount of molecular data available for eucalypts, a basic understanding of the nature of its genome still requires information regarding the DNA amount in the genus. In this work, we estimated the genome size and base composition of 25 eucalypt species. With a comparative karyotype approach, we aimed to identify possible chromosomal alterations correlated with the genome size variation. Classical cytogenetic and genomic in situ hybridization experiments were conducted for this purpose. The studied species showed genome size ranging from 2C = 0.91 (Corymbia intermedia) to 2C = 1.37 pg (Eucalyptus paniculata) and AT/CG ratios varying from AT = 61.3 (Eucalyptus urophylla) to AT = 62.85% (C. intermedia). Comparative karyotype analysis revealed no remarkable differences in chromosome number (2n = 22) or morphology among eucalypt species despite considerable differences in nuclear DNA content. The genome in situ hybridization method did not distinguish non-homologous chromosomal regions of Eucalyptus baileyana and Corymbia citriodora, despite the difference of 0.45 pg between their genome sizes. The results found in the present work corroborate the consideration of small and dispersed DNA changes as the main cause of genome size variation in eucalypts

Genome size diversity in stingless bees (Hymenoptera: Apidae, Meliponini)

The first studies on the genome size of stingless bee species showed a range from 0.27 pg (Melipona subnitida and Melipona quadrifasciata) to 1.38 pg (Melipona capixaba). Considering this variation, we quantified the DNA content of 26 species of Meliponini, in order to provide input for future comparative studies in this tribe. Haploid genome size (1C) estimates, using flow cytometry analyses (FCM), ranged from 0.26 ± 0.003 pg (Paratrigona subnuda) to 0.98 ± 0.023 pg (Melipona flavolineata), with an average of 0.54 ± 0.17 pg. FCM analyses also demonstrated a small difference in the haploid genome size between males and females of the same species, with the males generally having a smaller genome than females. Our data also evidenciated that variations in the genome size of stingless bees do not correlate with changes in chromosome number and that in some genera the DNA content is more variable than in others

Estimation of nuclear genome size of the genus Mycetophylax Emery, 1913: Evidence of no whole-genome duplication in Neoattini

Genome size estimates and their evolution can be useful for studying the phylogenetic relationships and taxonomy of a particular group. In the present study, the genome sizes of the three species that comprise the Mycetophylax genus were estimated by flow cytometry (FCM). There was little variation in genome size among them. The mean haploid genome size value of male and female individuals of Mycetophylax morschi was 312.96 Mbp (0.32 pg) and that of Mycetophylax conformis and Mycetophylax simplex females were 312.96 Mbp (0.32 pg) and 381.42 Mbp (0.39 pg), respectively. At first glance, this variation could be related with the heterochromatin content. Our results, together with other previous reports, have contributed to our knowledge about Attini genome size and will be useful to improve the understanding of the evolution of this tribe. It will help select potential model species in Attini for future genomic and sequencing projects.L’estimation de la taille des génomes et leur évolution peuvent être utiles pour comprendre les relations phylogénétiques et taxonomiques d’un groupe particulier. Dans cette étude, les tailles des génomes de trois espèces de fourmis du genre Mycetophylax ont été estimées par cytométrie en flux (CMF). Ces trois espèces présentent peu de variation de taille du génome. La valeur moyenne de la taille du génome haploïde mâle et femelle de Mycetophylax morschi était de 312,96 Mbp (0,32 pg) et celle des femelles de Mycetophylax conformis et Mycetophylax simplex de 312,96 Mbp (0,32 pg) et de 381,42 Mbp (0,39 pg), respectivement. Cette différence, à première vue, peut être liée à la teneur en hétérochromatine. Ces résultats, ainsi que d’autres études, contribuent à améliorer nos connaissances sur la taille du génome des Attini et peuvent être utiles pour la compréhension de l’évolution de cette tribu. Ils aideront à sélectionner de potentielles espèces modèles chez les Attini pour de futurs projets de séquençage et génomique

Mitotic metaphase cells of <i>G</i>. <i>assimilis</i> (a-f) and <i>E</i>. <i>surinamensis</i> (g-j), demonstrating examples of the hybridization patterns of different microsatellite-motif probes.

<p>The sex chromosomes and the signals for each type of microsatellite motif are shown in the images. Note the intense signals along the chromosomal arms of both species, although band-like signals are also evident in distinct chromosomes and in distinct positions in <i>E</i>. <i>surinamensis</i> cells. One chromosome is missing in (b). Bar = 5 μm.</p