Aplicação de técnicas de citogenética e genómica no estudo da evolução do cariótipo de cobras da Península Ibérica
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2022-07-26
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Uma das maiores evidências reveladas por projetos de sequenciação de genomas eucariotas foi o facto de estes possuírem uma elevada quantidade de sequências repetitivas de DNA, sendo o elevado dinamismo destes componentes associado à variação do tamanho do genoma entre espécies divergentes. Os elementos transponíveis (TEs) fazem parte desta fração genómica, com a capacidade de se movimentarem dentro do genoma. Estes podem ser divididos em duas classes baseadas nas caraterísticas estruturais e mecanismos de transposição: TEs de classe I, denominados como retrotransposões, ou TEs de classe II, conhecidos como transposões de DNA. Os retrotransposões possuem a capacidade de serem transferidos para indivíduos da mesma espécie ou até para espécies diferentes (transferência horizontal), podendo este mecanismo levar a um aumento do tamanho de genomas de algumas linhagens num curto espaço de tempo.
Dentro da ordem Squamata, os genomas das serpentes são muito variáveis quando nos referimos a atividade e diversidade genómica sendo as sequências repetitivas que contribuem principalmente para o tamanho do genoma e na maioria dos casos os LINEs e os SINEs são predominantes. O retrotransposão Bovine-B LINE (Bov-B LINE) pertence a uma das famílias de retrotransposões mais abundantes em vertebrados que demonstram evidências de transferência horizontal. Bov-B LINE foi originalmente identificado em Bos taurus (BTA), mas mais tarde foram descobertos retrotransposões Bov-B LINE altamente conservados em répteis escamados. Recentemente foi descoberto o retrotransposão Sauria SINE, encontrando-se distribuído em genomas da ordem Squamata.
No presente trabalho procedeu-se à análise dos retrotransposões Bov-B LINE e Sauria SINE em genomas da ordem Squamata presentes na Península Ibérica de forma a compreender melhor o seu envolvimento na regulação e evolução dos genomas das serpentes. Para tal, procedeu-se ao mapeamento in silico destes retrotransposões nos assemblys mais recentes de Naja naja (Nana_V5), Thamnophis elegans (rThaEle1.pri) e BTA (ARS-LIC_NZ_Jersey). Realizou-se também uma análise filogenética in silico das sequências de DNA ribossomal e dos retrotransposões Bov-B LINE e Sauria SINE em genomas de répteis escamados.
Recorrendo à técnica de DNA-FISH, mapeou-se fisicamente Bov-B LINE em cromossomas de BTA. Através da técnica RT-qPCR quantificaram-se o número de cópias deste retrotransposão nas espécies estudadas e também se analisaram as variantes com recurso a HRM.
O estudo in silico deste trabalho permitiu analisar as relações filogenéticas entre espécies da mesma ordem (Squamata), demostrando a importância dos dados de sequenciação na caraterização de TEs e revelando uma forma simples de mapear estas sequências em genomas sequenciados com assembly.
A análise in situ de Bov-B LINE permitiu reforçar os resultados in silico ao demonstrar que este retrotransposão se encontrava disperso por todo o cariótipo de BTA. Finalmente a quantificação do número de cópias deste retrotransposão e a análise das variantes levou a resultados importantes quanto à influência dos retrotransposões nos tamanhos dos genomas e à descoberta de sete variantes diferentes de Bov-B LINE.
A integração de todos os resultados obtidos ao longo deste trabalho permite evidenciar a importância que os retrotransposões podem ter na evolução e regulação dos genomas de serpentes.
One of the major aspects revealed by eukaryotic genome sequencing projects was the high content in repetitive DNA sequences, being that the high dynamism of these components is associated with the variation of genome size between divergent species. Transposable elements (TEs) are segments of DNA with the ability to move within a genome. They are divided into two classes based on structural characteristics and transposition mechanisms: class I TEs, referred to as retrotransposons, or class II TEs, known as DNA transposons. Retrotransposons have the capability to be transferred to individuals of the same species or even to different species (horizontal transfer), and this mechanism may lead to an increase in size of the genomes of some lineages in a short period of time. Within the Squamata order, the Serpentes genomes have great variability when referring to genomic activity and diversity with repetitive sequences are the main contributor to genome size and in most cases LINEs and SINEs are predominant. The Bovine-B LINE retrotransposon (Bov-B LINE) belongs to one of the most abundant retrotransposon families in vertebrates that demonstrate evidence of horizontal transfer. Bov-B LINE was originally identified in Bos taurus (BTA), but later highly conserved Bov-B LINE retrotransposons were discovered in squamate reptiles. The Sauria SINE retrotransposon was recently discovered and is distributed in genome of the Squamata order. In this work the Bov-B LINE and Sauria SINE retrotransposons were analysed in Squamata genomes present in the Iberian Peninsula in order to better understand their involvement in the regulation and evolution of Serpentes genomes. For this purpose, in silico mapping of these retrotransposons was carried out in the most recent assemblies of Naja naja (Nana_V5), Thamnophis elegans (rThaEle1.pri) and BTA (ARS-LIC_NZ_Jersey). An in silico phylogenetic analysis of ribosomal DNA sequences and Bov-B LINE and Sauria SINE retrotransposons was also performed in squamate reptile genomes. Using the DNA-FISH technique, Bov-B LINE was physically mapped onto BTA chromosomes. Using the RT-qPCR technique, the number of copies of this retrotransposon were quantified in the studied species and the variants were also analysed using HRM. The in silico study of this work allowed us to analyse the phylogenetic relationships between species of the same order (Squamata) showing the importance of sequencing data in the characterization of TEs and revealing a simple way to map these sequences in sequenced genomes with an assembly. The in situ analysis of Bov-B LINE reinforced the in silico results by demonstrating that this retrotransposon was dispersed throughout the entire BTA karyotype. Finally, the quantification of the number of copies of this retrotransposon and the analysis of the variants led to important results regarding the influence of retrotransposons on genome sizes and the discovery of seven different variants of Bov-B LINE. The integration of all the obtained results throughout this work makes it possible to highlight the importance that retrotransposons may have in the evolution and regulation of snake genomes.
One of the major aspects revealed by eukaryotic genome sequencing projects was the high content in repetitive DNA sequences, being that the high dynamism of these components is associated with the variation of genome size between divergent species. Transposable elements (TEs) are segments of DNA with the ability to move within a genome. They are divided into two classes based on structural characteristics and transposition mechanisms: class I TEs, referred to as retrotransposons, or class II TEs, known as DNA transposons. Retrotransposons have the capability to be transferred to individuals of the same species or even to different species (horizontal transfer), and this mechanism may lead to an increase in size of the genomes of some lineages in a short period of time. Within the Squamata order, the Serpentes genomes have great variability when referring to genomic activity and diversity with repetitive sequences are the main contributor to genome size and in most cases LINEs and SINEs are predominant. The Bovine-B LINE retrotransposon (Bov-B LINE) belongs to one of the most abundant retrotransposon families in vertebrates that demonstrate evidence of horizontal transfer. Bov-B LINE was originally identified in Bos taurus (BTA), but later highly conserved Bov-B LINE retrotransposons were discovered in squamate reptiles. The Sauria SINE retrotransposon was recently discovered and is distributed in genome of the Squamata order. In this work the Bov-B LINE and Sauria SINE retrotransposons were analysed in Squamata genomes present in the Iberian Peninsula in order to better understand their involvement in the regulation and evolution of Serpentes genomes. For this purpose, in silico mapping of these retrotransposons was carried out in the most recent assemblies of Naja naja (Nana_V5), Thamnophis elegans (rThaEle1.pri) and BTA (ARS-LIC_NZ_Jersey). An in silico phylogenetic analysis of ribosomal DNA sequences and Bov-B LINE and Sauria SINE retrotransposons was also performed in squamate reptile genomes. Using the DNA-FISH technique, Bov-B LINE was physically mapped onto BTA chromosomes. Using the RT-qPCR technique, the number of copies of this retrotransposon were quantified in the studied species and the variants were also analysed using HRM. The in silico study of this work allowed us to analyse the phylogenetic relationships between species of the same order (Squamata) showing the importance of sequencing data in the characterization of TEs and revealing a simple way to map these sequences in sequenced genomes with an assembly. The in situ analysis of Bov-B LINE reinforced the in silico results by demonstrating that this retrotransposon was dispersed throughout the entire BTA karyotype. Finally, the quantification of the number of copies of this retrotransposon and the analysis of the variants led to important results regarding the influence of retrotransposons on genome sizes and the discovery of seven different variants of Bov-B LINE. The integration of all the obtained results throughout this work makes it possible to highlight the importance that retrotransposons may have in the evolution and regulation of snake genomes.
Descrição
Dissertação de Mestrado em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Elementos transponíveis , Bov-B LINE