Construção de um denso mapa genético e associação genômica para peso corporal e sexo em Piaractus mesopotamicus
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2019-11-29
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Universidade Estadual Paulista (Unesp)
Resumo
Estudos genéticos com foco no aprimoramento de espécies de peixes Neotropicais são raros e, nesse sentido, uma espécie alvo é o pacu (Piaractus mesopotamicus), uma vez que é considerada uma das espécies de peixe de grande interesse econômico, com grande produção e valor comercial para a piscicultura brasileira. O presente trabalho teve como objetivo desenvolver ferramentas genéticas e genômicas através da genotipagem dos marcadores genéticos do tipo SNPs (Single Nucleotide Polymorphisms) para desenvolvimento de um mapa de ligação para esta espécie e, dessa maneira identificar regiões correlacionadas com características de peso corporal e sexo. Inicialmente foram formadas 14 famílias, sendo os indivíduos marcados com pit-tags e alocados em 3 tanques distintos. Os indivíduos foram pesados e tiveram o sexo identificado; no entanto, apenas 3 famílias foram genotipadas (família1, família2 e família3). Na etapa seguinte, foi realizada a extração do DNA e a elaboração de uma biblioteca genômica para cada família através da metodologia Restriction-Site Associated DNA-Sequencing (RADseq). As bibliotecas foram sequenciadas na plataforma Hiseq 4000 Illumina, resultando em uma média de 35.697.700 de sequências brutas de cada família. Um total de 11.240 marcadores SNP foram prospectados e filtrados, sendo que destes 6.437 foram utilizados na construção do primeiro mapa genético da espécie, que possui 2.814,92cM com intervalo médio de 0.44. Esses mesmos marcadores também foram utilizados para desenvolver o primeiro GWAS (Estudo de Associação Ampla do Genoma) prospectado com indivíduos genotipados e não genotipados, totalizando 399 amostras. A análise do GWAS foi realizada por meio da abordagem ssGBLUP e explicou 3,21% para o peso corporal e 2,89% para o sexo. A partir da identificação dos loci foi realizado o Blast e a busca pelos genes candidatos, sendo identificados genes para o peso corporal envolvidos com fatores de crescimento, regulação e metabolismo do osso, codificação do colágeno, proliferação e crescimento celular. Do mesmo modo foi verificado que os genes candidatos para identificação sexual estavam relacionados à espermatogênese, diferenciação sexual, células germinativas e expressão do hormônio anti-Mülleriano (AMH) em células de Sertoli. Considera-se que as ferramentas genéticas e genômicas desenvolvidas neste trabalho poderão auxiliar em investigações sobre a evolução da espécie, organização do genôma, identificação de QTLs (loci de característica quantitativa) e em programas de melhoramento genético para o pacu e espécies aparentadas.
Genetic studies focusing on the improvement of neotropical fish species are rare and, in this sense, one target species is pacu (Piaractus mesopotamicus), as it is considered one of the most produced fish species and commercial value for Brazilian fish farming. The present work aimed to develop genetic and genomic tools through the genotyping of SNPs (Single Nucleotide Polymorphisms) genetic markers for the development of a linkage map for this species, and thus identify regions correlated with body weight and sex. Initially, 14 families were formed, individuals were tagged with Pit-tags and allocated in 3 distinct tanks. The subjects were weighed and had the gender identified; however, only 3 families were genotyped (family1, family2 and family3). In the next step, DNA extraction and the elaboration of a genomic library for each family were performed through the Restriction-Site Associated DNA-Sequencing (RADseq) methodology. The libraries were sequenced on the platform Hiseq 4000 Illumina, resulting in an average of 35.697.700 raw sequences from each family. A total of 11.240 SNP markers were prospected and filtered, of which 6.437 were used to construct the first genetic map of the species, which has 2.814,92cM with an average interval of 0.44. These same markers were also used to develop the first GWAS (Genome Wide Association Study) with genotyped and non-genotyped individuals, totaling 399 samples. The GWAS analysis was performed using the ssGBLUP approach and explained 3,21% for body weight and 2,89% for sex. Based on the loci identification, the Blast was performed and the candidate genes were searched, and genes for body weight involved with bone growth, regulation and metabolism, collagen coding, proliferation and cell growth factors were identified. Similarly, it was found that candidate genes for sexual identification were related to spermatogenesis, sexual differentiation, germ cells and anti-Müllerian hormone (AMH) expression in Sertoli cells. It is considered that the genetic and genomic tools developed in this work can assist in investigations on species evolution, genome organization, QTL identification (quantitative trait loci) and breeding programs for pacu and related species.
Genetic studies focusing on the improvement of neotropical fish species are rare and, in this sense, one target species is pacu (Piaractus mesopotamicus), as it is considered one of the most produced fish species and commercial value for Brazilian fish farming. The present work aimed to develop genetic and genomic tools through the genotyping of SNPs (Single Nucleotide Polymorphisms) genetic markers for the development of a linkage map for this species, and thus identify regions correlated with body weight and sex. Initially, 14 families were formed, individuals were tagged with Pit-tags and allocated in 3 distinct tanks. The subjects were weighed and had the gender identified; however, only 3 families were genotyped (family1, family2 and family3). In the next step, DNA extraction and the elaboration of a genomic library for each family were performed through the Restriction-Site Associated DNA-Sequencing (RADseq) methodology. The libraries were sequenced on the platform Hiseq 4000 Illumina, resulting in an average of 35.697.700 raw sequences from each family. A total of 11.240 SNP markers were prospected and filtered, of which 6.437 were used to construct the first genetic map of the species, which has 2.814,92cM with an average interval of 0.44. These same markers were also used to develop the first GWAS (Genome Wide Association Study) with genotyped and non-genotyped individuals, totaling 399 samples. The GWAS analysis was performed using the ssGBLUP approach and explained 3,21% for body weight and 2,89% for sex. Based on the loci identification, the Blast was performed and the candidate genes were searched, and genes for body weight involved with bone growth, regulation and metabolism, collagen coding, proliferation and cell growth factors were identified. Similarly, it was found that candidate genes for sexual identification were related to spermatogenesis, sexual differentiation, germ cells and anti-Müllerian hormone (AMH) expression in Sertoli cells. It is considered that the genetic and genomic tools developed in this work can assist in investigations on species evolution, genome organization, QTL identification (quantitative trait loci) and breeding programs for pacu and related species.
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Palavras-chave
Pacu, Linkage map, GWAS, RADseq