Microdissecção e sequenciamento completo de cromossomos B em peixes aplicados em estudos evolutivos e funcionais
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2016-02-19
Autores
Utsunomia, Ricardo [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
No presente estudo, duas populações da espécie de peixe Moenkhausia sanctaefilomenae, uma coletada no rio Batalha, Bauru-SP (bacia do rio Tietê) e outra coletada no rio Novo, Ocauçu-SP (bacia do rio Paranapanema), foram analisadas através de técnicas citogenéticas básicas e moleculares. Além disso, o sequenciamento na plataforma Illumina HiSeq2000 de ambos os genomas e de uma variante do cromossomo B microdissecado também foram realizados. Os resultados obtidos revelaram que todos os indivíduos da população do rio Batalha apresentam de 0 a 6 cromossomos B por célula, enquanto os indivíduos da população do rio Novo não mostram elementos supranumerários. Deve-se destacar que os cromossomos B encontrados nos indivíduos do rio Batalha podem ser divididos em 2 variantes, sendo uma eucromática (B1) e altamente frequente na população, e outra heterocromática (B2) e com uma baixa prevalência. A hibridação in situ fluorescente (FISH) de DNAs repetitivos evidenciou que ambas as variantes apresentam clusters de DNAr 18S, histona H3 e satDNAs MS3 e MS7, com diferenças apenas na abundância do DNAr 18S. A análise das sequências presentes nos cromossomos B revelam que a variante B1 é mais antiga que a variante B2, embora ambas pareçam ter derivado de um mesmo cromossomo. Além disso, a seleção parece estar relaxada em ambas as variantes para o gene de histona H3. Para um melhor conhecimento do conteúdo genômico do cromossomo B, uma biblioteca microdissecada da variante B1 foi sequenciada e os dados obtidos revelam um intenso acúmulo de elementos transponíveis neste cromossomo supranumerário, além de possíveis genes de cópia única, que estão presentes em diferentes grupos de ligação no genoma de Astyanax mexicanus. Estes resultados serão utilizados como base para estudos evolutivos e funcionais deste sistema de cromossomo B. A integralização de abordagens cromossômicas e moleculares foi utilizada na tentativa de melhor compreender questões relacionadas à biologia evolutiva de cromossomos supranumerários em peixes Neotropicais.
In the present study, two populations of the fish species Moenkhausia sanctaefilomenae, one collected at the Batalha river, Bauru-SP (Tietê river basin) and the other collected at the Novo river, Ocauçu-SP (Paranapanema river basin), were analyzed through classical and molecular cytogenetic techniques. Besides, Illumina HiSeq2000 sequencing was performed for both genomes and for one microdissected B-variant. Results obtained revealed that all the individuals from the Batalha river show from 0 to 6 B chromosomes per cell, while the specimens from Novo river did not show any supernumerary chromosomes. One must note that the B chromosomes found in the Batalha river might be classified into 2 variants, one euchromatic (B1) and present in all the population, and another heterochromatic (B2) with lower prevalence. Fluorescence in situ hybridization (FISH) using repetitive DNA probes revealed that both B-types showed 18S rDNA, H3 histone and MS3 and MS7 satDNAs. Interestingly, the only between-variant difference was the abundance of 18S rDNA. Sequence analysis revealed that the B1 variant is older than B2, although both variants seem to be derived from the same autosomal pair. Notably, selection seems to be relaxed for H3.3 histone genes located on the B chromosomes. For a better knowledge about the genomic content of B chromosomes, a microdissected library of B1 variant was sequenced and data obtained revealed an intense accumulation of transposable elements in this supernumerary, in addition to single copy genes, that are present in different linkage groups of Astyanax mexicanus genome. These results will be used for future evolutionary and functional studies about B chromosomes. The combined approach of chromosomal and molecular methods was used to better understand evolutionary issues of supernumerary chromosomes in Neotropical fish.
In the present study, two populations of the fish species Moenkhausia sanctaefilomenae, one collected at the Batalha river, Bauru-SP (Tietê river basin) and the other collected at the Novo river, Ocauçu-SP (Paranapanema river basin), were analyzed through classical and molecular cytogenetic techniques. Besides, Illumina HiSeq2000 sequencing was performed for both genomes and for one microdissected B-variant. Results obtained revealed that all the individuals from the Batalha river show from 0 to 6 B chromosomes per cell, while the specimens from Novo river did not show any supernumerary chromosomes. One must note that the B chromosomes found in the Batalha river might be classified into 2 variants, one euchromatic (B1) and present in all the population, and another heterochromatic (B2) with lower prevalence. Fluorescence in situ hybridization (FISH) using repetitive DNA probes revealed that both B-types showed 18S rDNA, H3 histone and MS3 and MS7 satDNAs. Interestingly, the only between-variant difference was the abundance of 18S rDNA. Sequence analysis revealed that the B1 variant is older than B2, although both variants seem to be derived from the same autosomal pair. Notably, selection seems to be relaxed for H3.3 histone genes located on the B chromosomes. For a better knowledge about the genomic content of B chromosomes, a microdissected library of B1 variant was sequenced and data obtained revealed an intense accumulation of transposable elements in this supernumerary, in addition to single copy genes, that are present in different linkage groups of Astyanax mexicanus genome. These results will be used for future evolutionary and functional studies about B chromosomes. The combined approach of chromosomal and molecular methods was used to better understand evolutionary issues of supernumerary chromosomes in Neotropical fish.
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Palavras-chave
Citogenética, Cromossomos supranumerários, FISH, DNA satélite