Variabilidad genética del ADN mitocondrial de Vultur gryphus (cóndor andino) en Cusco y Apurímac a partir de plumas de muda / Genetic variability of mitochondrial DNA of Vultur gryphus (Andean Condor) in Cusco and Apurimac from molting feathers

Resumen La variabilidad genética intrapoblacional de Vultur gryphus (cóndores andinos) de las regiones de Cusco y Apurímac fue evaluada mediante amplificación y secuenciación del ADN mitocondrial correspondientes a la región control y subunidad ribosomal 12S (D-Loop-ARNr12S), y a los genes Citocromo Oxidasa subunidad I (COI) y NADH deshidrogenasa subunidad II (ND2). El ADN se extrajo a partir de cálamos de plumas de muda de ejemplares en cautiverio y silvestres. Se analizaron los principales índices de diversidad genética como son: la diversidad haplotípica, la diversidad nucleotídica, el número promedio de diferencias nucleotídicas y el número de sitios polimórficos. La tasa de éxito de amplificación mediante PCR fue de 100% para las tres regiones de ADN analizadas. Se secuenció 600 pb de la región D-Loop-ARNr12S caracterizándose cuatro haplotipos, 704 pb del gen COI caracterizándose seis haplotipos y 1090 pb del gen ND2 caracterizándose cinco haplotipos. El gen COI presentó el mayor valor de diversidad haplotípica (Hd = 0.468), la región del gen D-Loop-ARNr12S presentó el mayor índice de diversidad nucleotídica (π = 0.00086), mientras que el gen COI presentó el mayor número promedio de diferencias nucleotídicas (K = 0.52615). Los resultados muestran bajos niveles de variabilidad genética en los genes mitocondriales de los cóndores andinos de la zona de estudio, que indicarían una población con estructura genética homogénea.

Abstract The intrapopulation genetic variability of Vultur gryphus (Andean condors) from Cusco and Apurimac regions was evaluated by amplification and sequencing of mitochondrial DNA corresponding to the control region and 12S ribosomal subunit (D-Loop-RNAr12S), Cytochrome Oxidase subunit I (COI) genes and NADH dehydrogenase subunit II (ND2) gene. DNA was extracted from the calamus of feathers recollected from captive and wild specimens. The main indices of genetic diversity such as the haplotype diversity, the nucleotide diversity, the average number of nucleotide differences and the number of polymorphic sites were analyzed. The PCR amplification success rate was 100% for the three mitochondrial amplified sequences. Four haplotypes were identified from the 600 bp sequenced of D-Loop-RNAr12S region; six haplotypes from the 704 bp sequenced of the COI gene; five haplotypes from the 1090 bp sequenced of the ND2 gene. The COI gene presented the highest haplotype diversity (Hd = 0.468), the D-Loop-RNAr12S region presented the highest index of nucleotide diversity (π = 0.00086), while the COI gene presented the highest average number of nucleotide differences (K = 0.52615). The results show low levels of genetic variability in the mitochondrial genes of the Andean Condor in the study area, indicating a population with a homogeneous genetic structure.

The most important fishery resource in the Amazon, the migratory catfish Brachyplatystoma vaillantii (Siluriformes: Pimelodidae), is composed by an unique and genetically diverse population in the Solimões-Amazonas River System

The migratory catfish Brachyplatystoma vaillantii is one of the most important fishery resources in the Amazon. Intense capture occurs associated to its life cycle. In order to know the genetic status, we sequenced the mitochondrial DNA control region from 150 individuals of B. vaillantii, collected in five fishing landing locations, covering the length of the Solimões-Amazonas River in Brazil. Genetic diversity parameters suggest there is no genetic differentiation between the five localities. Population's expansion indicated by R 2 and Fu's Fs tests was also confirmed by the high number of unique haplotypes found. The Analyses of molecular variance indicated that nearly all variability was contained within locations (99.86%), and estimates of gene flow among B. vaillantii were high (F ST = 0.0014). These results suggest that Brachyplatystoma vaillantii forms a panmitic population along the Solimões-Amazonas River and, has greater genetic variability than other species of the Brachyplatystoma genus available so far. Although the influence of different tributaries on B. vaillantii migration patterns remains uncertain, a single population in the main channel should be consider in future policies for management of this resource. However, since the species' life cycle uses habitats in several countries, its management and conservation depend greatly of internationally joined efforts.(AU)

O bagre migrador, Brachyplatystoma vaillantii, é um dos mais importantes recursos pesqueiros da Amazônia. Intensa captura ocorre associada ao seu ciclo de vida. Para conhecer seu status genético, sequenciamos a região de controle do DNA mitocondrial de 150 indivíduos, coletados em cinco locais de desembarque pesqueiro, abrangendo toda a extensão do rio Solimões-Amazonas no Brasil. Os parâmetros de diversidade genética sugerem que não existe diferenciação genética entre as cinco localidades amostradas. A expansão populacional indicada pelos testes R 2 e Fs de Fu, também foi confirmada pelo elevado número de haplótipos únicos encontrados. A análise de variância molecular indicou que quase toda a variabilidade estava contida nas localidades (99,86%), e as estimativas de fluxo gênico desta espécie eram altas (F ST = 0,0014). Esses resultados sugerem que Brachyplatystoma vaillantii forma uma população panmítica ao longo do rio Solimões-Amazonas com maior variabilidade genética que outras espécies do gênero Brachyplatystoma disponíveis no momento. Embora a influência dos diferentes afluentes na migração de B. vaillantii permaneça incerta, em futuras políticas de gestão deste recurso deve-se considerá-lo como uma única população no canal principal. Entretanto, uma vez que seu ciclo de vida abrange habitats em vários países, seu manejo e conservação dependem muito de esforços internacionais em conjunto.(AU)

Comparative analysis of camelid mitochondrial genomes

Camelus dromedarius has played a pivotal role in both culture and way of life in the Arabian peninsula, particularly in arid regions where other domestic animals cannot be easily domesticated. Although, the mitochondrial genomes have recently been sequenced for several camelid species, wider phylogenetic studies are yet to be performed. The features of conserved gene elements, rapid evolutionary rate, and rare recombination make the mitochondrial genome a useful molecular marker for phylogenetic studies of closely related species. Here we carried out a comparative analysis of previously sequenced mitochondrial genomes of camelids with an emphasis on C. dromedarius, revealing a number of noticeable findings. First, the arrangement of mitochondrial genes in C. dromedarius is similar to those of the other camelids. Second, multiple sequence alignment of intergenic regions shows up to 90% similarity across different kinds of camels, with dromedary camels to reach 99%. Third, we successfully identified the three domains (termination-associated sequence, conserved domain and conserved sequence block) of the control region structure. The phylogenetic tree analysis showed that C. dromedarius mitogenomes were significantly clustered in the same clade with Lama pacos mitogenome. These findings will enhance our understanding of the nucleotide composition and molecular evolution of the mitogenomes of the genus Camelus, and provide more data for comparative mitogenomics in the family Camelidae.

BK virus characterisation among HIV-1-Infected individuals and its association with immunosuppression

Purpose: BK virus (BKV) is an opportunistic pathogen which causes significant morbidity and mortality in individuals who are immunodeficient. We aimed to quantitate and characterise BKV and to correlate with the degree of immunosuppression among human immunodeficiency virus (HIV)-1-infected individuals. Methods: BKV DNA detection was carried out using an in-house quantitative real-time polymerase chain reaction on paired whole-blood and urine samples collected from 187 antiretroviral therapy (ART)-naïve HIV-1-infected individuals and 93 healthy individuals who served as controls. Sequencing was performed for a proportion of high BK viral load (VL) samples to observe non-coding control region (NCCR) rearrangements. Results: BKV positivity in urine was 25.6% among HIV-infected individuals and 10.7% in control individuals (P = 0.03). The BK VL showed a significant negative correlation with CD4+ T-cell counts, a positive correlation with WHO clinical staging and no significant correlation with HIV-1 VL. Of 42 BKVs from urine samples sequenced, two showed rearrangements without clinically severe disease or high VL. Their NCCR and VP1 sequence-based genotyping revealed genotype I. In a small subset of individuals (n = 8) on ART who were being followed up, six individuals showed either decrease or complete clearance of virus with ART. Conclusion: There was a higher frequency of BK viruria in HIV-1-infected individuals than among healthy controls and the positivity correlated with the degree of immunosuppression. There was no association of high VL with NCCR rearrangements in urine.

Effect of HPV-16 LCR internal meCpG site demethylation on HPV positive head and neck tumor cells / 中国耳鼻咽喉头颈外科

OBJECTIVE To investigate the effect of HPV-16 LCR internal meCpG site demethylation on HPV positive head and neck tumor cells. METHODS After 5-azoxy-2-deoxycytidine(5-Aza-2'-dc) demethylation treatment, the methylation status of CpG sites in the long regulatory region of UM-SCC47, CaSki and SiHa tumor cells was detected by BSP assay. The expression of HPV 16 E6 and E7 mRNA was detected by real-time PCR. The effects of 5-Aza-2'-dc on cell growth and apoptosis were detected. Using Lipolnfectamine RNAiMAX to transfect siRNA, the expression of E6 and E7 was silenced, and then the changes of biological state of cell proliferate inhibition, apoptosis and cycle were detected. RESULTS Cells treated with 5-Aza-2, -dc at a concentration of 0.5 μmol/L for 96 h had the best demethylation. After demethylation of 5-Aza-2'-dc, the expression levels of E6 and E7 mRNA in UM-SCC47 cells and CaSki cells were significantly decreased compared with the control group(t=1.356, 2.623, P=0.031, 0.005; t=1.798, 2.015, P=0.011, 0.009). The expression levels of E6 and E7 mRNA in SiHa cells were not significantly differences compared with the control group(t=1.591, 1.153, P=1.105, 0.753); After treated with 5-Aza-2'-dc, cells proliferation were significantly inhibited. After E6 and E7 expression was silenced, the growth of all three kinds of cells was inhibited. The apoptosis of all three kinds of cells were significantly increased, cells arrested in S phase and G2/M phase were significantly increased compared with the control groups, the difference were statistically significant. CONCLUSION After demethylation, the response of HPV-16 positive tumor cells with different methylation status in the long regulatory region is completely different, indicating that in the HPV-16 positive tumors, there may be some information about the methylation status in the long regulatory region Different tumor characteristics and mechanism of cancer.

Regulation of HPV transcription

Human papillomavirus infection is associated with the development of malignant and benign neoplasms. Approximately 40 viral types can infect the anogenital mucosa and are categorized into high- and low-risk oncogenic human papillomavirus, depending on their association with the development of cervical carcinoma. High-risk human papillomavirus 16 and 18 are detected in 55% and 15% of all invasive cervical squamous cell carcinomas worldwide, respectively. Low-risk human papillomavirus 6 and 11 are responsible for 90% of genital warts and are also associated with the development of recurrent respiratory papillomatosis. Human papillomavirus preferentially infects mitotic active cells of the basal layer from both mucosal and cutaneous epithelium through microabrasions. The viral life cycle synchronizes with the epithelial differentiation program, which may be due, in part, to the binding of differentially expressed cellular transcription factors to the long control region throughout the various epithelial layers. This review aimed to summarize the current knowledge regarding the mechanisms by which viral gene expression is regulated and the influence of human papillomavirus heterogeneity upon this phenomenon. A better understanding of the regulatory mechanisms may elucidate the particularities of human papillomavirus-associated pathogenesis and may provide new tools for antiviral therapy.

Análisis de haplotipos de la tortuga cabezona Caretta caretta (Testudines: Cheloniidae) en dos playas del Caribe colombiano / Analysis of haplotypes of the loggerhead turtle, Caretta caretta (Testudines: Cheloniidae), on two beaches of the Colombian Caribbean / Análise de haplotipos da tartaruga cabeçona, Caretta caretta (Testudines: Cheloniidae) em duas praias do Caribe colombiano

Resumen Introducción. La tortuga Caretta caretta habita los mares tropicales y subtropicales. Es una especie en vía de extinción que anida las playas en Colombia y hace extensas migraciones. Los haplotipos mitocondriales de esta tortuga se han utilizado para estudios de genética poblacional, filogeografía y estado de las especies con el objetivo de desarrollar planes de conservación de la especie. Objetivo. Identificar haplotipos mitocondriales en tortugas cabezonas anidantes del Caribe colombiano. Materiales y Métodos. Se recolectaron muestras de sangre periférica de esta especie en dos sitios del Caribe colombiano: Don Diego (playa de anidación) y la Isla San Martin de Pajarales (localidad de alimentación). El ADN total fue extraído a partir de las células sanguíneas, y utilizado para amplificar por PCR la región control mitocondrial (398 pb). Estos productos fueron purificados y secuenciados. Se realizó un alineamiento básico buscando regiones de similitud local entre las secuencias obtenidas y las descritas previamente para la especie. Se hicieron análisis filogenéticos utilizando los criterios de máxima parsimonia (MP) y máxima verosimilitud (ML). Resultados. Se identificaron tres haplotipos, CC-A1 y CC-A2 comúnmente encontrados en poblaciones reproductivas de México, el Mediterráneo y el sudeste de Estados Unidos, y un nuevo haplotipo CC-SM1 en la playa Don Diego (Magdalena). Los árboles filogenéticos muestran relación de una porción de los individuos anidantes y de forrajeo de las agregaciones del Caribe colombiano con las súper-agregaciones del Atlántico y el Mediterráneo, sugiriendo que estas podrían ser algunas de las fuentes importantes de individuos presentes en Colombia. Conclusiones. Es necesario estudiar una muestra más grande para poder confirmar hipótesis planteadas. Se identificó un nuevo haplotipo denominado CC-SM1. Este es el primer estudio sobre haplotipos mitocondriales de C. caretta realizado en Colombia.

Abstract Introduction. The Caretta caretta turtle inhabits tropical and subtropical seas. It is an endangered species that nests on Colombian beaches and makes long migrations. The mitochondrial haplotypes of this turtle have been used for population genetics, phylogeography and species status studies with the aim of developing species conservation plans. Objective. Identify mitochondrial haplotypes in nesting loggerhead turtles from the Colombian Caribbean. Materials and Methods. Peripheral blood samples from this species were collected in two sites of the Colombian Caribbean: Don Diego (nesting beach) and San Martin de Pajarales island (feeding ground). The total DNA was extracted from blood cells and used to amplify the mitochondrial control region by PCR (398 bp). These products were purified and sequenced. A basic alignment was performed looking for local similarity regions between the sequences obtained and those previously described for the species. Phylogenetic analyses were conducted by using the maximum parsimony (MP) and maximum likelihood (ML) criteria. Results. Three haplotypes were identified: CC-A1 and CC-A2, which are commonly found in breeding populations in Mexico, the Mediterranean and southeast U.S.; and a new haplotype, CC-SM1, on the Don Diego beach (Magdalena). The phylogenic trees show a relationship between a portion of the nesting and feeding individuals from the Colombian Caribbean aggregations and the Atlantic and Mediterranean super-aggregations, suggesting that these could be some of the important sources for individuals inhabiting Colombia. Conclusions. It is necessary to study a larger sample to be able to confirm the proposed hypotheses. A new haplotype called CC-SM1was identified. This is the first study on C. caretta mitochondrial haplotypes conducted in Colombia.

Resumo Introdução. A tartaruga Caretta caretta habita os mares tropicais e subtropicais. É uma espécie em via de extinção que enraíza as praias na Colômbia e faz extensas migrações. Os haplotipos mitocondriais desta tartaruga se há utilizado para estudos de genética populacional, fílogeografía e estado das espécies com o objetivo de desenvolver planos de conservação da espécie. Objetivo. Identificar haplotipos mitocondriais em tartarugas cabeçonas enraizada no Caribe colombiano. Materiais e Métodos. Se coletaram amostras de sangue periférica desta espécie em dois lugares do Caribe colombiano: Don Diego (praia de enraizamento) e a Ilha San Martin de Pajarales (localidade de alimentação). O DNA total foi extraído a partir das células sanguíneas, e utilizado para amplificar por PCR a região controle mitocondrial (398 pb). Estes produtos foram purificados e sequenciados. Se realizou um alinhamento básico buscando regiões de semelhança local entre as sequências obtidas e as descritas previamente para a espécie. Se fez análise filogenéticos utilizando os critérios de máxima parcimônia (MP) e máxima verossimilitude (ML). Resultados. Se identificaram três haplotipos, CC-A1 e CC-A2 comumente encontrados nas populações reprodutivas do México, o Mediterráneo e o sudeste de Estados Unidos, e um novo haplotipo CC-SM1 na praia Don Diego (Magdalena). As árvores filogenéticos mostram relação de uma porção dos indivíduos enraizados e de forragem das agregações do Caribe colombiano com as super-agregações do Atlântico e o Mediterrâneo, sugerindo que estas poderiam ser algumas das fontes importantes de indivíduos presentes na Colômbia. Conclusões. É necessário estudar uma amostra maior para poder confirmar hipótese proposta. Se identificou um novo haplotipo denominado CC-SM1. Este é o primeiro estudo sobre haplotipos mitocondriais de C. caretta realizado na Colômbia.

Genetic differentiation in the populations of red piranha, Pygocentrus nattereri Kner (1860) (Characiformes: Serrasalminae), from the river basins of northeastern Brazil / Diferenciação genética em populações da piranha vermelha, Pygocentrus nattereri Kner (1860) (Characiformes: Serrasalminae), em bacias hidrográficas do nordeste do Brasil

Abstract The red piranha, Pygocentrus nattereri, is an important resource for artisanal and commercial fisheries. The present study determines the genetic differentiation among P. nattereri populations from the northeastern Brazilian state of Maranhão. The DNA was isolated using a standard phenol-chloroform protocol and the Control Region was amplified by PCR. The PCR products were sequenced using the didesoxyterminal method. A sequence of 1039 bps was obtained from the Control Region of 60 specimens, which presented 33 polymorphic sites, 41 haplotypes, һ =0.978 and π =0.009. The neutrality tests (D and Fs) were significant (P < 0.05) for most of the populations analyzed. The AMOVA indicated that most of the molecular variation (72%) arises between groups. The fixation index was highly significant (FST = 0.707, P < 0.00001). The phylogenetic analyses indicated that the specimens represented a monophyletic group. Genetic distances between populations varied from 0.8% to 1.9%, and were <0.5% within populations. The degree of genetic differentiation found among the stocks of P. nattereri indicates the need for the development of independent management plans for the different river basins in order to preserve the genetic variability of their populations.

Resumo A piranha vermelha, Pygocentrus nattereri, é um recurso importante para pesca artesanal e comercial. O presente estudo determinou a diferenciação genética entre populações de P. nattereri no nordeste do estado brasileiro do Maranhão. O DNA foi isolado utilizando o protocolo de Fenol-clorofórmio e a Região Controle foi amplificada por PCR. Os produtos da PCR foram sequenciados usando o método didesoxiterminal. Uma sequência de 1039 pbs foi obtida da Região Controle de 60 espécimes, que apresentaram 33 sítios polimórficos, 41 haplótipos, һ= 0.978 e π= 0.009. Os testes de neutralidade (D and Fs) foram significativos (P < 0.05) para a maioria das populações analisadas. A AMOVA indicou que a maior parte da variação molecular (72%) surge entre os grupos. O índice de fixação foi altamente significativo (FST = 0.707, P = < 0.00001). As análises filogenéticas indicaram que os espécimes representam um grupo monofilético. Distâncias genéticas entre as populações variaram de 0.8% a 1.9%, e de <0.5% dentro das populações. O grau de diferenciação genética encontrada entre os estoques de P. nattereri indicam a necessidade para o desenvolvimento de planos de manejo independentes para as diferentes bacias hidrográficas, a fim de preservar a variabilidade genética dessas populações.

Phylogeographic pattern of Jenynsia multidentata (cyprinodontiformes: anablepidae) in the southern boundary of the Brazilian subregion, Argentina

The Atlantic drainage basins are located in the southern Pampean area, in the southernmost part of the Brazilian subregion. Tectonic and paleoclimatic phenomena, occurring during the Quaternary, have currently isolated these basins from the present hydrographic system. Their ichthyofaunal composition is similar to that of hydric systems located further northward. Jenynsia multidentata is a species with wide distribution in the Atlantic drainage basins, as well as in those Northern basins. Here we investigated the phylogeographic pattern of Jenynsia multidentata and analyzed its relationships with the paleoclimatic and geologic history of the region. The analysis of the population's assemblage showed moderate genetic diversity, demographic equilibrium and marked genetic structure in the populations that occupy the extremes of the distributional range. The populations in the center of the range did not present genetic diversity, sharing a single haplotype. These results allow inferring that the presence of Jenynsia multidentata in the area results from historical demographic processes that are independent but complementary. In turn, these processes could arise from paleoclimatic changes occurred during the Quaternary.

Las cuencas de pendiente atlántica del área pampeana Austral son el límite meridional de la subregión Brasílica. Fenómenos tectónicos y paleoclimáticos ocurridos durante el Cuaternario han dejado aisladas a estas cuencas del diseño hidrográfico actual. La composición íctica de estás cuencas es similar a la de los sistemas hídricos ubicados más al norte. Jenynsia multidentata es una especie con un amplia distribución en las cuencas de pendiente atlántica, como así también, en las cuencas ubicadas al norte. En el presente trabajo se investigó el patrón filogeográfico de Jenynsia multidentata, y se examinó la relación entre este patrón y la historia paleoclimática y geológica de la región. En el análisis del conjunto de poblaciones se encontró evidencia de una moderada diversidad genética, equilibrio demográfico, y una marcada estructura poblacional, en las poblaciones que ocupan los extremos de la distribución. Mientras que en las poblaciones que ocupan el centro de la distribución no se encontró diversidad genética presentando un único haplotipo. De los resultados obtenidos se infiere que la presencia de Jenynsia multidentata en el área es el resultado de procesos demográficos históricos independientes pero complementarios. A su vez, estos procesos podrían surgir de los cambios paleoclimáticos ocurridos durante el Cuaternario.

Ecologia molecular de ostras (Crassostrea spp. )do Atlântico Tropical / Molecular ecology of oysters (Crassostrea spp. ) from Tropical Atlantic

Crassostrea (Sacco, 1897) é o gênero mais importante do mundo de ostras de cultivo e consiste de 34 espécies distribuídas pelas regiões tropicais e temperadas do globo. C. gasar e C. rhizophorae são as duas espécies nativas que estão distribuídas ao longo de toda a costa do Brasil até o Caribe. C. gasar também ocorre na costa da Africa. Ainda que sua distribuição seja extensa e com disponibilidade abundante, o cultivo de ostras nativas no Brasil ainda é incipiente e a delimitação correta dos estoques mantém-se incerta. O sucesso do desenvolvimento da malacocultura, que é recomendada internacionalmente como forma sustentável de aquicultura, depende da resolução desses problemas. Assim, com o objetivo de determinar geneticamente seus estoques no Atlântico como também estimar sua história demográfica, dois diferentes marcadores moleculares foram empregados: sequências de DNA da região controle mitocondrial e loci de microssatélites espécie-especifícos, desenvolvidos no presente estudo. Foram sequenciados fragmentos da região controle de um total de 930 indivíduos de C. gasar e C. rhizophorae coletados em 32 localidades que incluíram o Caribe, a Guiana Francesa, a costa brasileira e a África. Também foram realizadas genotipagens de 1178 indivíduos, e ambas as espécies, com 9 e 11 loci de microssatélites para C. gasar e C. rhizophorae, respectivamente. Os dados genéticos foram analisados através de diferentes abordagens (índices de estruturação (FST) e de (Jost D), análise molecular de variância (AMOVA), análise espacial molecular de variância (SAMOVA), Bayesian Skyline Plots (BSP), análise fatorial de correspondência (AFC) e análise de atribuição Bayesiana (STRUCTURE)). Os resultados indicaram um padrão geral de estruturação, onde dois diferentes estoques foram detectados para ambas as espécies: grupos do norte e do sul, onde o Rio de Janeiro seria a região limitante entre os dois estoques. Os maiores valores dos índices de estruturação foram encontrados ...

Crassostrea (Sacco, 1897) is the most important genus of cultivated oysters in the world and consisting of 34 species distributed by tropical and temperate regions of the globe. C. gasar and C. rhizophorae are the two native species which have wide distribution along the entire Brazilian coast up to the Caribbean. C. gasar also occurs on coast of Africa. Despite its extensive distribution and abundant availability, cultivation of those oysters in Brazil is incipient, and the correct delimitation of the existing stocks is still uncertain. The successful development of malacoculture which is recommended internationally as an environmentally sustainable form of aquaculture depends on the resolution of these issues. Thus, in order to genetically determinate their stocks in the Atlantic and to estimate their demographic history, two different molecular markers were employed: sequences of the mitochondrial DNA control region and species-specific microsatellite loci, developed in the present study. We have sequenced a fragment of the mitochondrial control region from a total of 930 individuals of C. gasar and C. rhizophorae collected in 32 localities including the Caribbean, French Guyana, Brazilian coast and Africa. We have also genotyped 1178 individuals of both species with 9 and 11 loci of microsatellites for C. gasar and C. rhizophorae, respectively. Genetic data were analyzed with different approaches (fixation (FST) and differentiation (Jost D) indices, analysis of molecular variance (AMOVA), spatial analysis of molecular variance (SAMOVA), Bayesian Skyline Plots (BSP), factorial correspondence analysis (AFC) and Bayesian attribution analysis (STRUCTURE)). The results indicated a general structure pattern, where two different stocks were detected for both species: north and south groups, where Rio de Janeiro would be the limited region between them. Higher values of fixation indices were found for C. gasar, indicating that this species would be more ...

Estrutura populacional e filogeografia de Panulirus argus (Latreille, 1804) / Population structure and phylogeography of Panulirus argus (Latreille, 1804)

Panulirus argus (Latreille, 1804) é uma das principais espécies de lagosta no Atlântico, sendo um dos maiores recursos pesqueiros do Atlântico Ocidental, onde apresenta um alto valor comercial. A forte explotação da espécie resulta em uma grande pressão sobre suas populações. Recentemente, foi descoberto que sob o binômio P. argus estão contidas duas espécies crípticas que ocorrem em alopatria, uma na região do Caribe e outra na costa brasileira. Esta tese tem como objetivo estudar como se estruturam geneticamente as populações dessas duas espécies, com o propósito de fornecer mais informações para a determinação de estoques e um correto manejo das espécies, e analisar os processos históricos evolutivos que moldaram suas histórias demográficas. Para tal, foram estudados dois marcadores mitocondriais (região controle e o gene da Citocromo Oxidase I) e loci de microssatélites de indivíduos de 7 regiões do Caribe (Florida, Bahamas, Turks e Caicos, Porto Rico, Cuba, Colômbia e Venezuela) e 11 estados do Brasil (Pará, Maranhão, Piauí, Ceará, Rio Grande do Norte, Pernambuco, Alagoas, Bahia, Espírito Santo, Rio de Janeiro e São Paulo). Dentro de cada espécie foram observadas duas linhagens mitocondriais diferentes, que co-ocorriam, de maneira homogênea, ao longo de suas distribuições. Hipotetiso que essas linhagens foram formadas a partir de um evento de vicariância com contato secundário ou como consequência de um efeito gargalo seguido de expansão. As duas linhagens são evidentes nas sequências da região controle mitocondrial, mas no gene da COI foram evidentes apenas em P. cf. argus do Caribe. As linhagens do Brasil se separaram há aproximadamente 233 - 288 mil anos e cada uma sofreu expansão em tempos diferentes, a primeira se expandiu há 100 mil anos e a segunda linhagem há 50 mil anos. As linhagens do Caribe se separaram cerca de 1 milhão de anos atrás e possuem o mesmo tempo de expansão, 50 mil anos. Os microssatélites não revelaram subdivisão populacional ...

Panulirus argus (Latreille, 1804) is one of the main lobster species in the Atlantic, and one of the largest fisheries in the western Atlantic, with a high commercial value. The heavy exploitation of the species results in much pressure on its populations. Recently, it was discovered that under the name P. argus there are two cryptic species that occur in allopatry, one in the Caribbean and the other on the coast of Brazil. This thesis studies the population genetic structure of those two species with the purpose of providing more information to delimitate stocks for fisheries management, and for understanding the historical processes that have shaped their evolutionary demographic histories. For this, we analysed two mitochondrial markers (control region and the Cytochrome Oxidase I gene) and microsatellite markers of individuals from 7 localities in the Caribbean (Florida, Bahamas, Turks and Caicos, Puerto Rico, Cuba, Colombia, and Venezuela) and 11 States of Brazil (Pará, Maranhão, Piauí, Ceará, Rio Grande do Norte, Pernambuco, Alagoas, Bahia, Espírito Santo, Rio de Janeiro, and São Paulo). Within each species two different mitochondrial lineages were observed. They occurred throughout their distributions, and it is hypothesized that they were formed from a vicariance event with secondary contact or are the result of a genetic bottleneck followed by expansion. The lineages of P. cf. argus from Brazil were only observed in the mitochondrial control region and were separated approximately 233-288 thousand years ago, and each lineage underwent expansion at different times: the first expanded 100,000 years ago and the second 50,000 years ago. The lineages of the Caribbean species were found for the two mitochondrial markers. They were separated about 1 million years ago and have had the same expansion time, 50,000 years. Microsatellites revealed no population subdivision for either species, but the molecular markers together suggest a differential gene...

Rapid and Simple Screening of Mitochondrial DNA in Koreans by the Analysis of Highly Variable Control Region SNPs / 대한법의학회지

Human mitochondrial DNA (mtDNA) is generally used to identify highly degraded forensic samples, particularly when the extracted DNA is not sufficient for nuclear DNA analysis. However, direct sequencing, the most widely used mtDNA analysis method, is laborious and time-consuming, and precludes the simultaneous analysis of many samples. Here, we describe a rapid and simple screening method for mtDNA analysis in Koreans using single base extension (SBE) methods. Sixteen highly polymorphic mtDNA SNPs from the control region were selected, and a multiplex SBE system was constructed to analyze them. Because the developed system consists of two duplex PCRs, which produce small amplicons with fewer than 270 bp, it works well with highly degraded samples such as old skeletal remains. Using this multiplex SBE system, 145 different haplotypes were expected to be observed from 593 unrelated Koreans. Seventy-three haplotypes were expected to be observed only once, and the most frequent haplotype was expected to occur 80 times. Since the mean number of pairwise differences was estimated to be 4.55, the developed system could be useful to exclude samples that do not match evidence and reference samples. Therefore, the multiplex SBE system used in this study will be a useful tool to analyze many samples simultaneously and to efficiently screen out non-matching mtDNA sequences in forensic casework.

The complete mitochondrial genome of the small yellow croaker and partitioned Bayesian analysis of Sciaenidae fish phylogeny

To understand the phylogenetic position of Larimichthys polyactis within the family Sciaenidae and the phylogeny of this family, the organization of the mitochondrial genome of small yellow croaker was determined herein. The complete, 16,470 bp long, mitochondrial genome contains 37 mitochondrial genes (13 protein-coding, 2 ribosomal RNA and 22 transfer RNA genes), as well as a control region (CR), as in other bony fishes. Comparative analysis of initiation/termination codon usage in mitochondrial protein-coding genes of Percoidei species, indicated that COI in Sciaenidae entails an ATG/AGA codon usage different from other Percoidei fishes, where absence of a typical conserved domain or motif in the control regions is common. Partitioned Bayesian analysis of 618 bp of COI sequences data were used to infer the phylogenetic relationships within the family Sciaenidae. An improvement in harmonic mean -lnL was observed when specific models and parameter estimates were assumed for partitions of the total data. The phylogenetic analyses did not support the monophyly of Otolithes, Argyrosomus, and Argyrosominae. L. polyactis was found to be most closely related to Collichthys niveatus, whereby, according to molecular systematics studies, the relationships within the subfamily Pseudosciaenidae should be reconsidered.

Populations analysis of the Brazilian Sharpnose Shark Rhizoprionodon lalandii (Chondrichthyes: Carcharhinidae) on the São Paulo coast, Southern Brazil: inferences from mt DNA sequences

Sharks of the genus Rhizoprionodon can be considered some of the most important predators along the trophic coastal marine ecosystems and represent an important economic resource for the small-scale fisheries, especially on the Brazilian coastline. In order to analyze the population structure of the shark Rhizoprionodon lalandii of São Paulo, Southeastern coast of Brazil, levels of genetic diversity were identified by nucleotide sequence analyses of the mitochondrial DNA control region. The results obtained from this study present moderate values of haplotype diversity and low nucleotide diversity. Although the AMOVA tests (ΦST = 0.08394, P < 0.01) had shown slightly differences among the studied samples, evidence for the occurrence of population structuring was not found, which may be a general feature of sharks living in coastal areas.

Tubarões do gênero Rhizoprionodon são considerados predadores de grande importância ao longo da cadeia trófica nos ecossistemas costeiros e marinhos, também representando um importante recurso econômico para a pesca, especialmente no litoral brasileiro. A fim de analisar a estrutura populacional do tubarão Rhizoprionodon lalandii no litoral de São Paulo, sudeste do Brasil, foram identificados os níveis de diversidade genética a partir da análise de sequências nucleotídicas da região controladora do DNA mitocondrial. Os dados obtidos neste estudo apresentam valores moderados de diversidade haplotípica e baixos índices de diversidade nucleotídica. Embora os testes de AMOVA (ΦST = 0,08394, P < 0,01) tenham revelado uma pequena diferença entre as amostras estudadas, evidências sobre a ocorrência de estruturação populacional não foram encontradas o que pode representar uma característica geral para tubarões vivendo em áreas costeiras.

Heteroplasmy of Mitochondrial DNA Shown in Korean and Mongolian / 체질인류학회지

As characterization of mitochondrial DNA (mtDNA) shows maternal inheritance and exists as more than thousands copies per cell, it is widely used for population genetics and forensic scientific field. However, mitochondrial DNA study has difficulties because heteroplasmy of mtDNA is being reported from coding and control region. In this study, we have analyzed 200 samples to examine heteroplasmy in mitochondrial DNA of Korean and Mongolian. The control region and coding region in mtDNA of blood from Koreans and Mongolians were analyzed with PCR amplication and sequencing. As a result, several heteroplasmy was observed from total 10 positions including 5 positions in coding region and 5 positions in control region, respectively. Moreover, it showed more than one heteroplasmy in coding region from 6 samples in Korean and 17 samples in Mongolian. Interestingly, heteroplasmy at 5178 position was shown in 6 samples among 23 samples. Considering that the position is important for deciding haplogroup D, we suggest that additional analysis on 4883 position needs for correct haplogrouping. Beside, we also found heteroplasmy in the other positions of 204, 4853, or 16249. Therefore, we suggest that it is required of combinatory analysis on several key nucleotide positions to obtain good results when determining mitochondrial haplogroups.

Genetic characterisation of populations of the critically endangered Goliath grouper (Epinephelus itajara, Serranidae) from the Northern Brazilian coast through analyses of mtDNA

The Goliath grouper (Epinephelus itajara) is one of the most endangered species of fish of the subfamily Epinephelinae. Slow to develop and mature, and dependent on mangrove habitats for breeding, the species also suffers intense harvesting, which has reduced drastically in numbers in many areas. To contribute to the understanding of the characteristics of E. itajara populations, we conducted a molecular genetics study of the species, focusing on populations from the Northern Brazilian coast. The mtDNA control region (D-loop) of 116 individuals from five localities (Bragança, Ajuruteua, Parnaíba, Fortaleza and Natal) was analysed, and a sequence of 499 base pairs identified. Analyses of the sequences indicated that genetic variability was generally lower in E. itajara than in other endangered species of the genus. AMOVA found no significant grouping structure among the populations. Nested Clade Analysis revealed a significant association between genetic variability and geographic distribution among only three populations (Ajuruteua, Parnaíba and Natal). Genetic diversity was higher in populations from the Amazon region, which may be related to the better conservation of mangrove habitats in this area. Therefore, the present study could be used for the implementation of conservation and management measures in order to protect and consolidate these populations.

Sequence and analysis of the mitochondrial DNA control region in the sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae)

This study aimed at the sequence and analysis of the mtDNA control region (CR) of the Diatraea saccharalis. The genome PCR amplification was performed using the complementary primers to the flanking regions of Bombyx mori CR mitochondrial segment. The sequencing revealed that the amplified product was 568 bp long, which was smaller than that observed for B. mori (725 bp). Within the amplified segment, a sequence with 338 nucleotides was identified as the control region, which displayed a high AT content (93.5%). The D. saccharalis mtDNA CR multiple sequence alignment analysis showed that this region had high similarity with the Lepidoptera Cydia pomonella.

A broca da cana, Diatraea saccharalis pertence à família dos lepidópteros. A presença da larva pode ser extremamente destrutiva, chegando a inviabilizar a atividade canavieira, causando prejuízos consideráveis à agroindústria sucro-alcooleira. Atualmente a broca da cana vem sendo extinta da plantação por métodos de controle biológico, entretanto a evolução desses programas depende de maiores conhecimentos básicos da biologia molecular deste inseto. O estudo do segmento do genoma mitocondrial denominado região controle é amplamente utilizado em análises genéticas e filogenéticas em insetos. O objetivo desse trabalho foi sequenciar e analisar a região controle do genoma mitocondrial de Diatraea saccharalis. Esse segmento apresentou 338 nucleotídeos, menor que o observado em Bombyx mori, com conteúdo de 93,5% de A/T. As analises realizadas mostraram que Diatraea saccharalis apresenta 76% de similaridade com Cydia pomonella.

Sequence change in the HS2-LCR and Gg-globin gene promoter region of sickle cell anemia patients

The fetal hemoglobin (HbF) levels and betaS-globin gene haplotypes of 125 sickle cell anemia patients from Brazil were investigated. We sequenced the Gg- and Ag-globin gene promoters and the DNase I-2 hypersensitive sites in the locus control regions (HS2-LCR) of patients with HbF level disparities as compared to their ßS haplotypes. Sixty-four (51.2 percent) patients had CAR/Ben genotype; 36 (28.8 percent) Ben/Ben; 18 (14.4 percent) CAR/CAR; 2 (1.6 percent) CAR/Atypical; 2 (1.6 percent) Ben/Cam; 1 (0.8 percent) CAR/Cam; 1 (0.8 percent) CAR/Arab-Indian, and 1 (0.8 percent) Sen/Atypical. The HS2-LCR sequence analyses demonstrated a c.-10.677G>A change in patients with the Ben haplotype and high HbF levels. The Gg gene promoter sequence analyses showed a c.-157T>C substitution shared by all patients, and a c.-222_-225del related to the Cam haplotype. These results identify new polymorphisms in the HS2-LCR and Gg-globin gene promoter. Further studies are required to determine the correlation between HbF synthesis and the clinical profile of sickle cell anemia patients.

Complete sequence and organization of the mitochondrial genome of Cyclemys atripons (Testudines, Geoemydidae)

The Black Bridged Leaf Turtle, Cyclemys atripons (Testudines; Cryptodira; Geoemydidae), is a poorly known species within the genus Cyclemys. We determined the complete nucleotide sequence of the Cyclemys atripons mitochondrial genome (mtDNA) and found it to be 16,500 base pairs (bp) in length, with the genome organization, gene order and base composition being identical to that of the typical vertebrate. However, unlike for most turtle mtDNA so far reported, an extra base was not found in the NADH3 gene. The C. atripons control region of mtDNA was 981 bp long. Comparisons with three other geoemydids showed that the C. atripons control region contained a highly variable region at the 3' end composed of AT enriched tandem repeats containing a fifteen-unit 5'-A (AT)3-3' variable number of tandem repeats (VNTRs).

Amplificação por PCR da região controle do DNA mitocondrial de raças de bombyx mori / Bombyx mori mitocondrial DNA control region amplification by PCR / Amplificación por pcr de la región control del dna mitocondrial de razas de Bombyx mori

A sericicultura é uma importante atividade agroindustrial no Brasil, sendo o Estado do Paraná responsável por aproximadamente 90% de toda a produção nacional. A localização de marcadores genéticos para o bicho-da-seda (Bombyx mori L.) é importante para a diferenciação intra e interespecífica e para o uso em melhoramento genético da espécie, sendo o DNA mitocondrial (DNAmt) um dos marcadores genéticos mais utilizados no estudo de insetos. O objetivo deste trabalho foi amplificar a região controle do DNA mitocondrial de quatro raças de Bombyx mori, pela técnica de Polymerase Chain Reaction (PCR). Obteve-se a amplificação de um fragmento de aproximadamente 750 pb referente à região controle, demonstrando a eficiência da metodologia empregada para a amplificação desta região específica do DNAmt de Bombyx mori.

Sericulture is an important agro industrial activity in Brazil, mainly in Paraná State, that is responsible for 90% of the national production. For the genetic improvement of silkworm (Bombyx mori L.), it is necessary to develop techniques to amplify molecular markers in order to use it on genetic analysis. Mitochondrial DNA (mtDNA) has been widely used as a molecular marker for insects and provides suitable markers for studies on genetic variability and molecular characterization. The control region is the major non-coding region of animal mtDNA and has been responsible for providing important evolutionary data about many insect groups. The aim of this paper was to amplify the mtDNA control region of four Bombyx mori strains by polymerase chain reaction (PCR). A 750 bp fragment including the control region was amplified showing the efficiency of the methodology to amplify specific regions of the Bombyx mori mtDNA.

La sericicultura es una importante actividad agroindustrial en Brasil, siendo el Estado del Paraná responsable por aproximadamente 90% de toda la producción nacional. La localización de marcadores genéticos para el gusano de seda (Bombyx mori L.) es importante para la diferenciación intra e interespecífica y para el uso en mejoramiento genético de la especie, siendo el DNA mitocondrial (DNAmt) uno de los marcadores genéticos más utilizados en el estudio de insectos. El objetivo de esta investigación fue amplificar la región control del DNA mitocondrial de cuatro razas de Bombyx mori, por la técnica de Polymerase Chain Reaction (PCR). Se obtuvo la amplificación de un fragmento de aproximadamente 750 pb referente a la región control, demostrando la eficiencia de la metodología empleada para la amplificación de esta región específica del DNAmt de Bombyx mori.