Estudo da organização estrutural de elementos repetitivos isolados do genoma de espécies de Proceratophrys (Amphibia, Anura, Odontophrynidae)
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2019-02-26
Autores
Silva, Marcelo João da [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Dados citogenéticos de Proceratophrys ainda são escassos na literatura quando comparados a alguns outros grupos de anuros, e são restritos atualmente às espécies P. boiei, P. appendiculata e P. renalis, que possuem um número diploide de 2n = 22 cromossomos e Região Organizadora de Nucléolo (RON) localizada no par 8, além de um padrão incomum de distribuição da heterocromatina constitutiva nos cromossomos de P. boiei, que apresentam grandes blocos heterocromáticos, além de um sistema de cromossomos sexuais do tipo ZZ:ZW. A maioria dos genomas eucarióticos são compostos por grandes porções de sequências de DNAs repetitivos que estão localizadas principalmente na heterocromatina altamente compactada, e em muitos casos, é um dos componentes mais abundantes dos cromossomos sexuais. Nesse sentido, o grupo dos Proceratophrys torna-se interessante alvo para análises citogenéticas juntamente com a aplicação de ferramentas moleculares e genômicas, as quais podem contribuir no entendimento da evolução e diversidade de DNAs repetitivos, e com isso explorar discussões em relação a diferenciação de cromossomos sexuais. Portanto, o objetivo deste estudo foi analisar citogeneticamente a espécie P. boiei, sob o ponto de vista clássico e genômico, em busca de sequências repetitivas, avaliar a presença dessas sequências e buscar entender a sua organização no genoma dessa espécie. Ainda, o trabalho teve como objetivo descrever citogeneticamente outras espécies de Proceratophrys, aumentando a amostra de cariótipos descritos na literatura. Para isso, foram utilizadas técnicas convencionais e diferenciais de citogenética, sequenciamento genômico de P. boiei para busca de elementos repetitivos, através de análises de alto rendimento no software RepeatExplorer e outros programas de bioinformática, seguido de análises moleculares por meio da Reação em Cadeia da Polimerase (PCR) e Hibridação Fluorescente in situ (FISH). Por meio do sequenciamento foi possível estimar o tamanho do genoma de P. boiei em 1,6 Gpb, e com as análises, foi visto que 41% do genoma corresponde a sequências repetitivas, incluindo satDNAs, rDNA, elementos transponíveis e outros elementos repetitivos não caracterizados. Para esse trabalho, foram escolhidos três elementos repetitivos para serem explorados e localizados cromossomicamente em P. boiei, e a seleção foi feita levando-se em conta a maior abundância genômica. Foi possível constatar que essas sequências se tratam de DNAs satélites (satDNA) que foram nomeadas de PboSat1-176, PboSat2-173 e PboSat3-189. Os satDNAs PboSat1-176 e PboSat2-189 mapeados em P. boiei possuem localização cromossômica centromérica e pericentromérica, sugerindo o possível envolvimento destas repetições para a função centromérica. Estão presentes também no cromossomo sexual W, ocupando toda a área heterocromática, revelando a contribuição desta classe de DNA repetitivo para a quantidade e complexidade da heterocromatina constitutiva em P. boiei, podendo estar relacionados a diferenciação dos cromossomos sexuais nesta espécie. Já o satélite PboSat3-189 também teve localização centromérica, porém com um número de cópias bem menor em alguns pares de cromossomos, sugerindo que eventos de amplificação/deleção de sequências altamente dinâmicos estão ocorrendo aleatoriamente no genoma de P. boiei. No trabalho, também foi descrito pela primeira vez os cariótipos de P. schirchi, P. laticeps e P. melanopogon, que apresentam um número diploide de 2n = 22, além de detectar a presença de RONs no par de cromossomos 8 e no par 4 em P. melanopogon e revelar regiões de heterocromatina constitutiva no centrômero da maioria dos cromossomos destas espécies.
Cytogenetic data from Proceratophrys are still scarce in the literature when compared to some other groups of anurans, and are currently restricted to the species P. boiei, P. appendiculata and P. renalis, which have a diploid number of 2n = 22 chromosomes and the Nuclear Organizing Region (NOR) located in pair 8, in addition to an unusual pattern of distribution of constitutive heterochromatin in the P. boiei chromosomes, which present large heterochromatic blocks, as well as a system of ZZ:ZW sex chromosomes. Most eukaryotic genomes are composed of large portions of repetitive DNA sequences that are located primarily in highly compacted heterochromatin, and in many cases, is one of the most abundant components of sex chromosomes. In this sense, the Proceratophrys group becomes an interesting target for cytogenetic analysis along with the application of molecular and genomic tools, which may contribute to the understanding of the evolution and diversity of repetitive DNAs, and thereby explore discussions regarding the differentiation of sex chromosomes. Therefore, the objective of this study was to analyze the P. boiei species, from a classical and genomic point of view, in a search for repetitive sequences, evaluating the presence of these sequences and seeking to understand their organization in the genome of this species. In addition, the objective of this work was to describe cytogenetically other species of Proceratophrys, increasing the karyotype sample described in the literature. For this, we used conventional and differential cytogenetic techniques, genomic sequencing of P. boiei to search for repetitive elements, through high-throughput analyzes in the software RepeatExplorer and other bioinformatics programs, followed by molecular analyzes through Polymerase Chain Reaction (PCR) and Fluorescence in situ Hybridization (FISH). Through sequencing it was possible to estimate the size of the genome of P. boiei at 1.6Gpb, and with the analyzes, it was seen that 41% of the genome corresponds to repetitive sequences, including satDNAs, rDNA, transposable elements and other uncharacterized repeating elements. For this work, three repetitive elements were chosen to be explored and located chromosomally in P. boiei. This selection was made considering the greater genomic abundance, among other characteristics of similarity between them. After detailed analysis, it was possible to confirm that these sequences were satellite DNAs (satDNA) that were named PboSat1-176, PboSat2-173 and PboSat3-189. The PboSat1-176 and PboSat2-189 satDNAs mapped in P. boiei have centromeric and pericentromeric chromosomal location, suggesting the possible involvement of these replicates for the centromeric function. They are also present on the sexual chromosome W, occupying the entire heterochromatic area, revealing the contribution of this class of repetitive DNA to the quantity and complexity of the constitutive heterochromatin in P. boiei, which may be related to the sex chromosome differentiation in this species. The PboSat3-189 satellite was also centromeric in location but with a much smaller number of copies in some pairs of chromosomes, suggesting that highly dynamic sequence amplification / deletion events may be happening randomly in the P. boiei genome. In the work, the karyotypes of P. schirchi, P. laticeps and P. melanopogon, which present a diploid number of 2n = 22, were also described for the first time, as well as to detect the presence of RONs in chromosome pair 8 and in pair 4 in P. melanopogon and reveal regions of constitutive heterochromatin in the centromere of most of the chromosomes of these species.
Cytogenetic data from Proceratophrys are still scarce in the literature when compared to some other groups of anurans, and are currently restricted to the species P. boiei, P. appendiculata and P. renalis, which have a diploid number of 2n = 22 chromosomes and the Nuclear Organizing Region (NOR) located in pair 8, in addition to an unusual pattern of distribution of constitutive heterochromatin in the P. boiei chromosomes, which present large heterochromatic blocks, as well as a system of ZZ:ZW sex chromosomes. Most eukaryotic genomes are composed of large portions of repetitive DNA sequences that are located primarily in highly compacted heterochromatin, and in many cases, is one of the most abundant components of sex chromosomes. In this sense, the Proceratophrys group becomes an interesting target for cytogenetic analysis along with the application of molecular and genomic tools, which may contribute to the understanding of the evolution and diversity of repetitive DNAs, and thereby explore discussions regarding the differentiation of sex chromosomes. Therefore, the objective of this study was to analyze the P. boiei species, from a classical and genomic point of view, in a search for repetitive sequences, evaluating the presence of these sequences and seeking to understand their organization in the genome of this species. In addition, the objective of this work was to describe cytogenetically other species of Proceratophrys, increasing the karyotype sample described in the literature. For this, we used conventional and differential cytogenetic techniques, genomic sequencing of P. boiei to search for repetitive elements, through high-throughput analyzes in the software RepeatExplorer and other bioinformatics programs, followed by molecular analyzes through Polymerase Chain Reaction (PCR) and Fluorescence in situ Hybridization (FISH). Through sequencing it was possible to estimate the size of the genome of P. boiei at 1.6Gpb, and with the analyzes, it was seen that 41% of the genome corresponds to repetitive sequences, including satDNAs, rDNA, transposable elements and other uncharacterized repeating elements. For this work, three repetitive elements were chosen to be explored and located chromosomally in P. boiei. This selection was made considering the greater genomic abundance, among other characteristics of similarity between them. After detailed analysis, it was possible to confirm that these sequences were satellite DNAs (satDNA) that were named PboSat1-176, PboSat2-173 and PboSat3-189. The PboSat1-176 and PboSat2-189 satDNAs mapped in P. boiei have centromeric and pericentromeric chromosomal location, suggesting the possible involvement of these replicates for the centromeric function. They are also present on the sexual chromosome W, occupying the entire heterochromatic area, revealing the contribution of this class of repetitive DNA to the quantity and complexity of the constitutive heterochromatin in P. boiei, which may be related to the sex chromosome differentiation in this species. The PboSat3-189 satellite was also centromeric in location but with a much smaller number of copies in some pairs of chromosomes, suggesting that highly dynamic sequence amplification / deletion events may be happening randomly in the P. boiei genome. In the work, the karyotypes of P. schirchi, P. laticeps and P. melanopogon, which present a diploid number of 2n = 22, were also described for the first time, as well as to detect the presence of RONs in chromosome pair 8 and in pair 4 in P. melanopogon and reveal regions of constitutive heterochromatin in the centromere of most of the chromosomes of these species.
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Palavras-chave
Sequenciamento genômico, Citogenética, DNA satélite, RepeatExplorer, FISH, Genomic sequencing, Cytogenetics, Satellite DNA