Identificação e mapeamento de famílias de DNA repetitivo em Characidium sp. aff. C. vidali (Teleostei, Characiformes) e sua atuação na evolução dos cromossomos B
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2019-02-25
Autores
Nobile, Maria Lígia Marques de Oliveira
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Universidade Estadual Paulista (Unesp)
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Characidium é um grupo de peixes amplamente distribuídos pela região Neotropical, embora seja considerado o mais especioso dentro de Crenuchidae, do ponto de vista citogenético o número de espécies investigadas ainda é baixo, o que dificulta a caracterização quanto a organização cromossômica do gênero. Em relação ao número diploide, as espécies de Characidium conservaram um cariótipo com 2n = 50 cromossomos, do tipo metacêntricos e submetacêntricos (com exceções), o que resulta em uma macroestrutura homogênea para o grupo. Porém, investigações utilizando sequências repetitivas têm contribuído para ilustrar que a organização microestrutural cromossômica pode diferir entre as espécies, refletindo o hábito destes peixes constituírem populações pequenas e isoladas em cabeceiras de riachos. Adicionalmente, algumas espécies de Characidium também foram descritas portando cromossomos B em seus cariótipos, e a utilização de ferramentas citomoleculares têm contribuído para explorar quanto a origem e evolução destes componentes cariotípicos. Neste sentido, o objetivo do presente estudo foi agregar técnicas citomoleculares com resultados de sequenciamento massivo, para tentar compreender a ocorrência de cromossomos B no genoma de Characidium sp. aff. C. vidali. Os resultados obtidos mostraram que i) o mapeamento físico de diferentes sondas de DNA repetitivo contribuíram não apenas para caracterizar o cariótipo da espécie em estudo, como também adicionaram mais informações quanto a organização e evolução da microestrutura cromossômica dos peixes pertencente à Characidium; ii) Characidium sp. aff. C. vidali mostrou sítios teloméricos intersticiais (ITS e double-ITS), e como a espécie apresenta o número diploide padrão do grupo (2n = 50 cromossomos), não há indícios de rearranjos cromossômicos estruturais, e tais marcações podem ser evidências de mecanismos de reparo do próprio genoma, evitando possíveis quebras e modificações na estrutura cromossômica da espécie; iii) a sonda telomérica também marcou a porção terminal dos cromossomos B de Characidium sp. aff. C. vidali, e como todas as células de todos os espécimes analisados apresentaram supranumerários, foi proposto que nessa população, os cromossomos B já estão bem estabelecidos; iv) os cromossomos B de Characidium sp. aff. C. vidali tiveram origem intraespecífica, e os resultados obtidos após pintura cromossômica com a sonda CvBp revelam que ao longo de sua evolução, estes supranumerários acumularam sequências que não são mais compartilhadas com os cromossomos do complemento A da própria espécie; v) os dados obtidos com as marcações de quatro microssatélites e pintura cromossômica (com a sonda CvBp), evidenciaram uma relação entre os cromossomos B Characidium sp. aff. C. vidali e um par de cromossomos acrocêntricos presentes em C. serrano, C. pterostictum e C. timbuiense, e somado a isso o padrão de marcação dos sítios teloméricos, assim como análises de filogenia e distância genética mostram que essas espécies representam um grupo monofilético; vi) tais evidências permitiram propor que os supranumerários presentes em Characidium sp. aff. C. vidali apresentam relação com um genoma ancestral ao grupo monofilético; vii) o mapeamento de sequências satélites obtidas por sequenciamento massivo, não apresentou marcações nos cromossomos B da espécie em estudo. Dessa maneira, o presente trabalho reforça a proposição de que Characidium é um interessante modelo para estudos citogenéticos, com ênfase sobre origem e evolução de cromossomos supranumerários.
Characidium is a group of fish widely distributed in the Neotropical region, although it is considered the most specious within Crenuchidae, from the cytogenetic point of view the number of species investigated is still low, which makes it difficult to characterize the chromosomal organization of the genus. In relation to the diploid number, Characidium species retained a karyotype with 2n = 50 chromosomes, metacentric and submetacentric (with exceptions), resulting in a homogeneous macrostructure for the group. However, investigations using repetitive sequences have contributed to illustrate that the chromosomal microstructural organization may differ between species, reflecting the habit of these fish constituting small and isolated populations in headwaters of streams. In addition, some species of Characidium have also been described carrying B chromosomes in their karyotypes, and the use of cyto-molecular tools has contributed to explore the origin and evolution of these karyotype components. In this sense, the objective of the present study was to aggregate cyto-molecular techniques with massive sequencing results to try to understand the occurrence of B chromosomes in the genome of Characidium sp. aff. C. vidali. The results showed that i) the physical mapping of different repetitive DNA probes contributed not only to characterize the karyotype of the species under study, but also added more information about the organization and evolution of the chromosomal microstructure of fish belonging to Characidium; ii) Characidium sp. aff. C. vidali showed interstitial telomeric sites (ITS and double-ITS), and since the species shows the standard diploid number of the group (2n = 50 chromosomes), there are no indications of structural chromosome rearrangements, and such markings may be evidence of mechanisms of repair of the genome itself, avoiding possible breaks and modifications in the chromosome structure of the species; iii) the telomeric probe also marked the terminal portion of the B chromosomes of Characidium sp. aff. C. vidali, and as all the cells of all the analyzed specimens presented supernumerary, it was proposed that in this population, the B chromosomes are already well established; iv) the B chromosomes of Characidium sp. aff. C. vidali were originated intraspecific, and the results obtained after chromosome painting with the CvBp probe reveal that throughout their evolution, these supernumerary accumulated sequences that are no longer shared with the complement chromosomes of the species itself; v) the data obtained with the four microsatellite and chromosome painting (with the CvBp probe), showed a relationship between the B chromosomes Characidium sp. aff. C. vidali and a pair of acrocentric chromosomes present in C. serrano, C. pterostictum and C. timbuiense, and in addition to the marking pattern of telomeric sites, as well as analyzes of phylogeny and genetic distance show that these species represent a group monophyletic; vi) such evidence allowed us to propose that the supernumeraries present in Characidium sp. aff. C. vidali present relationship with an ancestral genome to the monophyletic group; (vii) the mapping of satellite sequences obtained by massive sequencing did not show B chromosomes of the species under study. In this way, the present work reinforces the proposition that Characidium is an interesting model for cytogenetic studies, with emphasis on origin and evolution of supernumerary chromosomes.
Characidium is a group of fish widely distributed in the Neotropical region, although it is considered the most specious within Crenuchidae, from the cytogenetic point of view the number of species investigated is still low, which makes it difficult to characterize the chromosomal organization of the genus. In relation to the diploid number, Characidium species retained a karyotype with 2n = 50 chromosomes, metacentric and submetacentric (with exceptions), resulting in a homogeneous macrostructure for the group. However, investigations using repetitive sequences have contributed to illustrate that the chromosomal microstructural organization may differ between species, reflecting the habit of these fish constituting small and isolated populations in headwaters of streams. In addition, some species of Characidium have also been described carrying B chromosomes in their karyotypes, and the use of cyto-molecular tools has contributed to explore the origin and evolution of these karyotype components. In this sense, the objective of the present study was to aggregate cyto-molecular techniques with massive sequencing results to try to understand the occurrence of B chromosomes in the genome of Characidium sp. aff. C. vidali. The results showed that i) the physical mapping of different repetitive DNA probes contributed not only to characterize the karyotype of the species under study, but also added more information about the organization and evolution of the chromosomal microstructure of fish belonging to Characidium; ii) Characidium sp. aff. C. vidali showed interstitial telomeric sites (ITS and double-ITS), and since the species shows the standard diploid number of the group (2n = 50 chromosomes), there are no indications of structural chromosome rearrangements, and such markings may be evidence of mechanisms of repair of the genome itself, avoiding possible breaks and modifications in the chromosome structure of the species; iii) the telomeric probe also marked the terminal portion of the B chromosomes of Characidium sp. aff. C. vidali, and as all the cells of all the analyzed specimens presented supernumerary, it was proposed that in this population, the B chromosomes are already well established; iv) the B chromosomes of Characidium sp. aff. C. vidali were originated intraspecific, and the results obtained after chromosome painting with the CvBp probe reveal that throughout their evolution, these supernumerary accumulated sequences that are no longer shared with the complement chromosomes of the species itself; v) the data obtained with the four microsatellite and chromosome painting (with the CvBp probe), showed a relationship between the B chromosomes Characidium sp. aff. C. vidali and a pair of acrocentric chromosomes present in C. serrano, C. pterostictum and C. timbuiense, and in addition to the marking pattern of telomeric sites, as well as analyzes of phylogeny and genetic distance show that these species represent a group monophyletic; vi) such evidence allowed us to propose that the supernumeraries present in Characidium sp. aff. C. vidali present relationship with an ancestral genome to the monophyletic group; (vii) the mapping of satellite sequences obtained by massive sequencing did not show B chromosomes of the species under study. In this way, the present work reinforces the proposition that Characidium is an interesting model for cytogenetic studies, with emphasis on origin and evolution of supernumerary chromosomes.
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Palavras-chave
Characidium, DNA repetitivo, cromossomo B, Sequenciamento de Nova Geração (NGS), repetitive DNA, B chromosome, Next Generation Sequencing (NGS)