Prospecção de regiões informativas do genoma mitocondrial de Cervidae
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2021-10-22
Autores
Ricoldi, Marina Cabral [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A tribo Odocoileini é o grupo mais heterogêneo, com maiores problemas e controvérsias taxonômicas dos cervídeos. Ela é dividida em duas subtribos bem suportadas: Odocoileina e Blastocerina. Todos os cervídeos da região Neotropical são da tribo Odocoileini e suas espécies representantes destacam-se pela ampla variedade morfológica e de habitats. Dentre as espécies tem Mazama gouazoubira (veado-catingueiro) a qual possui uma grande amplitude em sua distribuição geográfica e na diversidade de habitats que ocupa. No Brasil pode ocupar quase todos os biomas, exceto a Amazônia. O veado-catingueiro apresenta ainda variações regionais, ecológicas e individuais de coloração, além de uma alta diversidade haplotípica. Por essa razão análises genéticas são importantes, já que, fornecem informações valiosas para taxonomia e para determinação da existência de estruturação populacional e, consequentemente, unidades evolutivamente significativas distintas. Para tanto, primers foram confeccionados a partir das sequências existentes (Genbank) e das produzidas no presente projeto em uma análise in silico das sequências das regiões Citocromo B, ND5, ND2 e D-loop (região controle) do DNA mitocondrial, visto que já se conhece o mitogenoma da espécie. As sequências foram alinhadas com as várias existentes para a mesma espécie e de espécies distintas, buscando-se áreas polimórficas flanqueadas por regiões conservadas. Prospectamos primers que permitam amplificação de pequenos fragmentos (150-250pb) informativos das diferentes regiões. Os primers foram utilizados para amplificação do mtDNA em amostras de fezes de veado-catingueiro, sendo testada a eficiência de amplificação para os diferentes marcadores. Também foram realizadas duas análises Máxima Verossimilhança e a Inferência Bayesiana para confirmação dos sinais filogenéticos e uma rede de haplotipos para analises intrapopulacionais das matrizes dos fragmentos separados e concatenadas. Nossos resultados mostraram que os marcadores testados possuem sinal filogenético relevante para estudos filogenéticos preliminares, identificação de gêneros e populações, principalmente quando a amostra contém pouco DNA de qualidade. Os fragmentos das regiões podem apresentar resultados de diversidade genética similares ao da região completa, a diversidade depende do local da região onde fragmento se encontra. Os amplicons menores tem maior facilidade de amplificação no material fecal e que sua concatenação pode gerar um marcador de importância para análises filogenéticas em cervídeos.
The Odocoileini tribe is the most heterogeneous group, with the greatest taxonomic problems and controversies among deer. It is divided into two well-supported subtribes: Odocoilein and Blastocerin. All deer in the Neotropical region are from the Odocoileini tribe and their representative species stand out for their wide morphological and habitat variety. Among the species there is Mazama gouazoubira (brocket deer) which has a wide range of geographic distribution and the diversity of habitats it occupies. In Brazil it can occupy almost all biomes, except for the Amazon. The brocket deer also presents regional, ecological and individual color variations, in addition to a high haplotype diversity. For this reason genetic analyzes are important, as they provide valuable information for taxonomy and for determining the existence of population structure and, consequently, distinct evolutionarily significant units. For this purpose, primers were made from existing sequences (Genbank) and those produced in this project in an in silico analysis of sequences from the Cytochrome B, ND5, ND2 and D-loop (control region) regions of the mitochondrial DNA, since the mitogenome of the species is known. The sequences were aligned with the several existing ones for the same species and from different species, looking for polymorphic areas flanked by conserved regions. We are looking for primers that allow the amplification of small fragments (150-250bp) that are informative from different regions. The primers were used for mtDNA amplification in brocket deer feces samples, and the amplification efficiency for the different markers was tested. Two Maximum Likelihood analyzes and Bayesian Inference were also performed to confirm the phylogenetic signals and a haplotype network for intrapopulation analyzes of the matrices of the separated and concatenated fragments. Our results showed that the tested markers have a relevant phylogenetic signal for preliminary phylogenetic studies, identification of genera and populations, especially when the sample contains little quality DNA. The fragments of the regions can show similar genetic diversity results to the complete region, the diversity depends on the location of the region where the fragment is found. Smaller amplicons are easier to amplify in fecal material and their concatenation can generate an important marker for phylogenetic analysis in deer.
The Odocoileini tribe is the most heterogeneous group, with the greatest taxonomic problems and controversies among deer. It is divided into two well-supported subtribes: Odocoilein and Blastocerin. All deer in the Neotropical region are from the Odocoileini tribe and their representative species stand out for their wide morphological and habitat variety. Among the species there is Mazama gouazoubira (brocket deer) which has a wide range of geographic distribution and the diversity of habitats it occupies. In Brazil it can occupy almost all biomes, except for the Amazon. The brocket deer also presents regional, ecological and individual color variations, in addition to a high haplotype diversity. For this reason genetic analyzes are important, as they provide valuable information for taxonomy and for determining the existence of population structure and, consequently, distinct evolutionarily significant units. For this purpose, primers were made from existing sequences (Genbank) and those produced in this project in an in silico analysis of sequences from the Cytochrome B, ND5, ND2 and D-loop (control region) regions of the mitochondrial DNA, since the mitogenome of the species is known. The sequences were aligned with the several existing ones for the same species and from different species, looking for polymorphic areas flanked by conserved regions. We are looking for primers that allow the amplification of small fragments (150-250bp) that are informative from different regions. The primers were used for mtDNA amplification in brocket deer feces samples, and the amplification efficiency for the different markers was tested. Two Maximum Likelihood analyzes and Bayesian Inference were also performed to confirm the phylogenetic signals and a haplotype network for intrapopulation analyzes of the matrices of the separated and concatenated fragments. Our results showed that the tested markers have a relevant phylogenetic signal for preliminary phylogenetic studies, identification of genera and populations, especially when the sample contains little quality DNA. The fragments of the regions can show similar genetic diversity results to the complete region, the diversity depends on the location of the region where the fragment is found. Smaller amplicons are easier to amplify in fecal material and their concatenation can generate an important marker for phylogenetic analysis in deer.
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Palavras-chave
Filogenia, Genoma, Genética de populações