Isolamento e caracterização de marcadores de DNA associados ao sexo e segmentos de DNA repetitivo em espécies do gênero Brycon (Characidae, Bryconinae)
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Data
2011-04-29
Autores
Silva, Eder Marques da [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Bryconinae compreende um grupo de peixes com um grande número de espécies distribuídas em sistemas hidrográficos da América Central e da América do Sul. Análises cromossômicas baseadas em técnicas citogenéticas clássicas revelaram que todas as espécies estudadas até o momento apresentam cariótipos similares sem a presença de cromossomos sexuais heteromórficos. Visando ampliar os dados genéticos acerca das relações entre as espécies deste gênero e sobre a evolução cromossômica deste grupo, o objetivo do presente estudo foi isolar e caracterizar fragmentos de DNA associados a um determinado sexo e repetições de DNA satélite em Brycon cephalus e B. orbignyanus. Desta forma, uma análise de marcadores RAPD (“Random Amplified Polymorphic DNA”) e uma dinâmica de digestão enzimática de DNA total foram realizadas. Sessenta e seis primers decâmeros foram utilizados em PCR revelando um fragmento de RAPD, composto por 535 pb, presente em fêmeas de B. cephalus. Dados de hibridização em membrana (“dot-blot”) e de um marcador SCAR (“Sequence Characterized Amplified Region”) desenvolvido confirmaram a especificidade deste fragmento a fêmeas desta espécie e revelaram sua ausência em duas outras espécies do gênero - B. orbignyanus e B. lundii.Digestões enzimáticas de DNA de B. cephalus e B. orbignyanus não evidenciaram diferenças entre machos e fêmeas. Por outro lado, as digestões realizadas com EcoRV, EcoRI, NotI, XbaI e XhoI em B. cephalus levaram à identificação de fragmentos de aproximadamente 250-300 pb em ambos os sexos que provavelmente correspondem a repetições de DNA satélite, denominadas de SatBc1, SatBc2, SatBc3, SatBc4 e SatBc5, respectivamente. A enzima HindIII permitiu a visualização de duas bandas de restrição com cerca de 650 e 850 pb (denominadas de SatBo1 e SatBo2) em machos e fêmeas de B. orbignyanus...
Bryconinae stands for a fish group with several species that have been reported in South and Central America hydrographic systems. Chromosome analyses based on classical cytogenetic techniques have revealed that all species so far studied present similar karyotypes with no heteromorphic sex chromosomes. In order to improve genetic data on the species relationships and chromosome evolution, the purpose of the present study was to isolate and characterize sex-associated DNA fragments and satellite repeats in Brycon cephalus and B. orbignyanus. For this purpose, a random amplified polymorphic DNA (RAPD) assay and a genomic DNA restriction digestion analysis were performed. Sixty six decamer primers were used in PCR revealing a RAPD fragment, composed by 535 bp, present in females of B. cephalus. Data on dot-blot hybridization and on a designed SCAR (Sequence Characterized Amplified Region) marker confirmed its female-specificity in this species and revealed its absence in two other species of the genus - B. orbignyanus and B. lundii. Genomic DNA restriction digestions in B. cephalus and B. orbignyanus resulted in no differences between sexes of both species. However, EcoRV, EcoRI, NotI, XbaI and XhoI DNA digestions in B. cephalus led to the identification of fragments of approximately 250-300 bp in both sexes that probably correspond to satellite DNA repeats, denominated SatBc1, SatBc2, SatBc3, SatBc4 and SatBc5, respectively.The HindIII enzyme permitted the visualization of two restriction bands of around 650 and 850 bp (named as SatBo1 and SatBo2) in males and females of B. orbignyanus. Characterization of the SatBc1 fragment isolated from B. cephalus indicated that its monomeric unit is constituted by 255 bp and 52.9% AT rich. The obtained RAPD data can lead to the supposition that B. cephalus may present heteromorphic sex chromosomes that should be in an early phase... (Complete abstract click electronic access below)
Bryconinae stands for a fish group with several species that have been reported in South and Central America hydrographic systems. Chromosome analyses based on classical cytogenetic techniques have revealed that all species so far studied present similar karyotypes with no heteromorphic sex chromosomes. In order to improve genetic data on the species relationships and chromosome evolution, the purpose of the present study was to isolate and characterize sex-associated DNA fragments and satellite repeats in Brycon cephalus and B. orbignyanus. For this purpose, a random amplified polymorphic DNA (RAPD) assay and a genomic DNA restriction digestion analysis were performed. Sixty six decamer primers were used in PCR revealing a RAPD fragment, composed by 535 bp, present in females of B. cephalus. Data on dot-blot hybridization and on a designed SCAR (Sequence Characterized Amplified Region) marker confirmed its female-specificity in this species and revealed its absence in two other species of the genus - B. orbignyanus and B. lundii. Genomic DNA restriction digestions in B. cephalus and B. orbignyanus resulted in no differences between sexes of both species. However, EcoRV, EcoRI, NotI, XbaI and XhoI DNA digestions in B. cephalus led to the identification of fragments of approximately 250-300 bp in both sexes that probably correspond to satellite DNA repeats, denominated SatBc1, SatBc2, SatBc3, SatBc4 and SatBc5, respectively.The HindIII enzyme permitted the visualization of two restriction bands of around 650 and 850 bp (named as SatBo1 and SatBo2) in males and females of B. orbignyanus. Characterization of the SatBc1 fragment isolated from B. cephalus indicated that its monomeric unit is constituted by 255 bp and 52.9% AT rich. The obtained RAPD data can lead to the supposition that B. cephalus may present heteromorphic sex chromosomes that should be in an early phase... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Genetica animal, DNA, Animal genetics
Como citar
SILVA, Eder Marques da. Isolamento e caracterização de marcadores de DNA associados ao sexo e segmentos de DNA repetitivo em espécies do gênero Brycon (Characidae, Bryconinae). 2011. 96 f. Dissertação (mestrado) - Universidade Estadual Paulista, Instituto de Biociências de Botucatu, 2011.