Genetic Applications in the Conservation of Neotropical Freshwater Fish

Neotropical fish correspond to approximately 30% of all fish species worldwide. The diversity of fish species found in Neotropical basins reflects variations in life-history strategies and exhibition of particular morphological, physiological and ecological attributes. These attributes are mainly related to different forms of feeding, life maintenance and reproduction. Today, fish populations are being threatened by anthropogenic actions that are having a visible impact on the natural state of continental aquatic ecosystems. The main causes are overfishing, non-native species introduction, reservoir-dam systems, mining, pollution and deforestation. The biology and population dynamics of the species are still unclear due to lack of research. Genetic tools can be useful resources for the conservation of Neotropical fish species in several ways. Molecular genetic markers are considered powerful tools to identify cryptic and hybrid fish and also allow the evaluation of the genetic variability and structure of populations of Neotropical ichthyofauna. Several analyses of molecular markers have been performed on Neotropical fish, including allozyme analysis, restriction fragment length polymorphisms in regions of DNA (RFLP), randomly amplified polymorphic DNA (AFLP), randomly amplified polymorphic DNA (RAPD), microsatellites, single nucleotide polymorphisms (SNPs) and mitochondrial DNA (mtDNA) markers. In order to analyse a high number of markers, next generation sequencing has allowed researchers to generate a large amount of genomic information that can be applied to the conservation of Neotropical fish

Genomic selection signatures in farmed Colossoma macropomum from tropical and subtropical regions in South America

Tambaqui or cachama (Colossoma macropomum) is one of the most important neo-tropical freshwater fish used for aquaculture in South America, and its production is concentrated at low latitudes (close to the Equator, 0°), where the water tempera-ture is warm. Therefore, understanding how selection shapes genetic variations and structure in farmed populations is of paramount importance in evolutionary biology. High- throughput sequencing to generate genome-wide data for fish species allows for elucidating the genomic basis of adaptation to local or farmed conditions and un-covering genes that control the phenotypes of interest. The present study aimed to detect genomic selection signatures and analyze the genetic variability in farmed pop-ulations of tambaqui in South America using single- nucleotide polymorphism (SNP) markers obtained with double-digest restriction site-associated DNA sequencing. Initially, 199 samples of tambaqui farmed populations from different locations (lo-cated in Brazil, Colombia, and Peru), a wild population (Amazon River, Brazil), and the base population of a breeding program (Aquaculture Center, CAUNESP, Jaboticabal, SP, Brazil) were genotyped. Observed and expected heterozygosity was 0.231–0.350 and 0.288– 0.360, respectively. Significant genetic differentiation was observed using global FST analyses of SNP loci (FST = 0.064, p< 0.050). Farmed populations from Colombia and Peru that differentiated from the Brazilian populations formed distinct groups. Several regions, particularly those harboring the genes of significance to aq-uaculture, were identified to be under positive selection, suggesting local adapta-tion to stress under different farming conditions and management practices. Studies aimed at improving the knowledge of genomics of tambaqui farmed populations are essential for aquaculture to gain deeper insights into the evolutionary history of these fish and provide resources for the establishment of breeding programsConselho Nacional de Desenvolvimento Científico e Tecnológico, Grant/Award Number: 140740/2016–3, 311559/2018– 2 and 422670/2018-9; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; Fundação de Amparo à Pesquisa do Estado de São Paulo, Grant/Award Number: 2016/18294–9, 2016/21011– 9, 2017/19717-3, 2017/26900–9, 2018/08416–5, 2019/08972-8 and 2019/10662-7; Comisión Nacional de Investigación Científica y Tecnológica.S

Análise de parentesco e variabilidade genética de pacu (Piaractus mesopotamicus) por meio de marcadores SNPs: subsídios para o melhoramento genético

Pacu (Piaractus mesopotamicus) é uma espécie de peixe Neotropical amplamente distribuída nas bacias dos rios Paraná e Paraguai, e uma das espécies de peixe neotropicais de maior valor para a aquicultura. Uma melhor compreensão do genoma do pacu é necessária para o manejo genético na conservação dos estoques naturais e cultivados. O principal objetivo foi identificar SNPs (Single Nucleotide Polymorphisms) gene-associados no transcriptoma de fígado do pacu e, em seguida, aplicar em análises de variabilidade genética e de parentesco visando um manejo adequado desta importante espécie não modelo na aquicultura. O sequenciamento do transcriptoma foi realizado por meio da plataforma Roche/454, que resultou na formação de 4.110 contigs não redundantes. Destes, 2.051 genes foram identificados e funcionalmente anotados a fim de revelar genes relacionados às características econômicas interessantes para a aquicultura. Foram encontrados 464 SNPs localizados em 5’UTR (10.0%), 3’UTR (17.2%) e em regiões CDS (71,1%), e classificados como sinônimos (70,6%) e não sinônimos (29,4%). Foram genotipados 32 SNPs por meio da técnica Sequenom MassARRAY, dos quais alguns estavam relacionados com sistema imune. A variabilidade genética foi estimada em populações de indivíduos selvagens (Rio Paraná) e de indivíduos cultivados em sete pisciculturas do estado de São Paulo (FF1, FF2, FF3, FF4, FF5, FF6 e FF7). Não foram observadas diferenças significativas entre heterozigosidade observada (Hobs) e esperada (Hexp) para cada população. Análises de diferenciação genética mostraram baixo nível de estruturação genética entre as populações (Fst = 0.064, AMOVA = 93,59% da variação dentro de populações, P<0,05). Análises de parentesco mostraram que a maioria das estações de piscicultura possuíam pelo menos 40% de indivíduos aparentados, com risco de endogamia e necessidade de realização de um programa de acasalamentos direcionados. Nossos resultados proporcionaram importantes recursos genéticos para o pacu, com aplicabilidade para a aquicultura.Pacu (Piaractus mesopotamicus) is a Neotropical freshwater fish widely distributed in Parana, Paraguay Basin. Wild populations of pacu are threatened by overfishing and it is one of the fish species of highest commercial value for aquaculture. An understanding of the pacu genome is appropriate to genetic management in the conservation of wild and cultivated stocks. The main objective was identify gene-associated SNPs in liver transcriptome of pacu. We used SNPs (Single Nucleotide Polymorphisms) to perform genetic variability and kinship analysis for suitable management of this important non-model species in aquaculture. Transcriptome sequencing was done with the Roche/454 technology and yielded 4,110 non-redundant contigs. Of these, 2,051 genes were identified and functionally annotated to reveal genes correlated to economical traits in aquaculture. We found 464 SNPs in 5’UTR (10.0%), 3’UTR (17.2%) and CDS (71,1%), classified in synonymous (70,6%) and non-synonymous (29,4%). We genotyped 32 feasible SNPs through Sequenom MassARRAY platform and we obtained some SNPs related to immune system. Genetic diversity was estimated in wild individuals (Parana river) and in seven farm fish populations (FF1, FF2, FF3, FF4, FF5, FF6 and FF7). There were no significant differences between observed heterozygosity (Hobs) and expected (Hexp) for each population; and also between observed heterozygosity, expected heterozygosity and minimum allele frequency (MAF), when the population averages were compared (P <0.05). In addition, genetic differentiation analyzes showed low genetic structure of wild and cultivated populations of pacu (Fst = 0.064; AMOVA = 93.59% of the variation within populations; P<0,05). Kinship analysis showed most hatchery stations had at least 40% of related individuals, at risk of inbreeding and the need to perform a directed mating program. Our results showed unprecedented genomic resources for pacu.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Assessing genetic diversity for a pre-breeding program in Piaractus mesopotamicus by SNPs and SSRs

The pacu (Piaractus mesopotamicus) is a Neotropical fish with remarkable productive performance for aquaculture. Knowledge of genetic resources in Neotropical fish is essential for their applications in breeding programs. The aim of this study was to characterize the genetic diversity of seven farmed populations of pacu which will constitute the basis for a broodstock foundation for coming breeding programs in Brazil. Analysis of one wild population (Paraná River) was used as a reference to compare genetic parameters in the farmed populations. The analyses were performed using 32 single-nucleotide polymorphisms (SNP) and 8 simple sequence repeat (SSR) markers. No significant differences in genetic diversity between populations estimated through the number of alleles and allelic richness, observed heterozygosity, expected heterozygosity, and minimum allele frequency were detected (p > 0.05). Low genetic diversity was observed in all farmed stocks and the wild population. Moreover, we detected low genetic structure when comparing farmed and wild populations for SNPs (FST = 0.07; K = 3) and SSRs (FST = 0.08; K = 2). Analysis of molecular variance (AMOVA) demonstrated that genetic variation was mostly within populations. Kinship analysis showed that most fish farms included related individuals at a proportion of at least 25%. Our results suggest that the basal broodstock for pacu breeding programs should be founded with individuals from different fish farms for higher genetic diversity and to avoid inbreeding risks.Para citar este articulo: Mastrochirico-Filho, V.A.; del Pazo, F.; Hata, M.E.; Villanova, G.V.; Foresti, F.; Vera, M.; Martínez, P.; Porto-Foresti, F.; Hashimoto, D.T. Assessing Genetic Diversity for a Pre-Breeding Program in Piaractus mesopotamicus by SNPs and SSRs. Genes 2019, 10, 668. https://doi.org/10.3390/genes10090668Fil: Mastrochirico-Filho, Vito Antonio. São Paulo State University (Unesp). Unesp's Aquaculture Center (Caunesp); Brazil.Fil: Del Pazo, Felipe. Universidad Nacional de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio Mixto de Biotecnología Acuática (LMBA - UNR - Ministerio de Ciencia, Tecnología e Innovación productiva de Santa Fe); Argentina.Fil: Del Pazo, Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); Argentina.Fil: Hata, Milene Elissa. São Paulo State University (Unesp). Unesp's Aquaculture Center (Caunesp); Brazil.Fil: Villanova, Gabriela Vanina. Universidad Nacional de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio Mixto de Biotecnología Acuática (LMBA - UNR - Ministerio de Ciencia, Tecnología e Innovación productiva de Santa Fe); Argentina.Fil: Villanova, Gabriela Vanina. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); Argentina.Fil: Foresti, Fausto. São Paulo State University (Unesp). Institute of Biosciences; Brazil.Fil: Vera, Manuel. Universidad de Santiago de Compostela (USC). Facultad de Veterinaria. España.Fil: Vera, Manuel. Universidad de Santiago de Compostela (USC). Instituto de Acuicultura; España.Fil: Martínez, Paulino. Universidad de Santiago de Compostela (USC). Facultad de Veterinaria. España.Fil: Martínez, Paulino. Universidad de Santiago de Compostela (USC). Instituto de Acuicultura; España.Fil: Porto-Foresti, Fábio. São Paulo State University (Unesp). School of Sciences; Brazil.Fil: Hashimoto, Diogo Teruo. São Paulo State University (Unesp). Unesp's Aquaculture Center (Caunesp); Brazil

High-quality genome assembly and annotation of the pacu Piaractus mesopotamicus: an aquatic genetic resource of South American aquaculture

Piaractus mesopotamicus, popularly known as pacu, is a freshwater fsh native to the Paraguay-Paraná River basin of South America. This species has been historically exploited by commercial fshermen, and nowadays, it is very important for aquaculture in many countries of South America. Good quality meat and excellent adaptability to culture systems are some of its remarkable farming features. To support studies into the biology, genetics, and genomics of the pacu, we have produced the frst high-quality chromosome-scale genome for P. mesopotamicus. A 1.28 Gbp genome containing 27 linkage groups has been obtained by integrating HiFi PacBio reads, and SNP linkage map data developed in this work. The proportion of BUSCO genes identifed was near 98%, a value that positions this assembly as one of the highest-quality genomes available for Neotropical fsh. Furthermore, genome annotation of genes, repetitions, and non-coding transcripts were performed. Comparison with the genome of the related species Colossoma macropomum showed a high level of synteny, which gives important information regarding the origin of both species and for hybrid management. A broader comparison showed the phylogenetic relationship between pacu and related Characiformes species as well the presence of groups of ortholog proteins. Moreover, specifc pacu proteins were identifed as well as their functional description and their own metabolic pathways, which may be useful for pacu aquaculture. The high-quality genome assembled in this work and its annotation provide a valuable genomic resource for future studies, and they are a helpful resource for pacu aquaculture.Fil: Mascali, Florencia Carla. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Biotecnología Acuática; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Mastrochirico Filho, Vito Antonio. Universidade de Sao Paulo; BrasilFil: Posner, Victoria Maria. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Biotecnología Acuática; ArgentinaFil: Rubiolo, Juan Andrés. Universidad de Santiago de Compostela; España. Universidade de Sao Paulo; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Teruo Hashimoto, Diogo. Universidade de Sao Paulo; BrasilFil: Villanova, Gabriela Vanina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Biotecnología Acuática; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentin

Gene-associated markers as a genomic and transcriptomic resource for a highly migratory and apex predator shark (Isurus oxyrinchus)

The shortfin mako, Isurus oxyrinchus, is an oceanic pelagic shark species found worldwide in tropical and subtropical waters. It is frequently caught by pelagic longline fisheries, but despite its commercial importance and ecological significance, little is still known about its biology and ecology at the molecular level. Therefore, we combined two massive parallel sequencing approaches, double digest restriction site-associated DNA sequencing (ddRAD) and RNA sequencing (RNAseq), for single nucleotide polymorphism (SNP) discovery in the shortfin mako. The ddRAD yielded a total of 82,676 putative SNPs. For RNAseq, a total of 129,663 putative SNPs were found. After the stricter filtering procedure, 405 SNPs from ddRAD and 1165 SNPs from RNAseq were retained and suitable for further analysis. Annotation analysis of SNPs from ddRAD revealed a total of 55 gene associated SNP markers, of which 32 SNPs (58.2%) are associated with diseases and defense responses, 9 SNPs (16.4%) are associated with developmental process, and 3 SNPs (5.4%) are present in genes involved in the reproductive function. For RNAseq, 739 SNPs were annotated and associated to relevant functions amongst which 10 SNPs (0.53%) were related with reproduction, 6 SNPs (0.32%) with growth, and 9 (0.48%) with locomotion. Overall, the genotyping of the SNPs was followed by the validation of 255 SNPs from ddRAD and 646 for RNAseq in 31 individuals from the Atlantic and Indian oceans. Our results provide valuable sequence resources for future population genomics analysis, comparative genomics, phylogenomics, and molecular evolution of the globally endangered shortfin mako shark.info:eu-repo/semantics/publishedVersio

Comparative eye and liver differentially expressed genes reveal monochromatic vision and cancer resistance in the shortfin mako shark (Isurus oxyrinchus)

The shortfin mako, Isurus oxyrinchus is an oceanic pelagic shark found worldwide in tropical and subtropical waters. However, the understanding of its biology at molecular level is still incipient. We sequenced the messenger RNA isolated from eye and liver tissues. De novo transcriptome yielded a total of 705,940 transcripts. A total of 3774 genes were differentially expressed (DEGs), with 1612 in the eye and 2162 in the liver. Most DEGs in the eye were related to structural and signaling functions, including nonocular and ocular opsin genes, whereas nine out of ten most overexpressed genes in the liver were related to tumor suppression, wound healing, and human diseases. Furthermore, DEGs findings provide insights on the monochromatic shark vision and a repertory of cancer-related genes, which may be insightful to elucidate shark resistance to cancer. Therefore, our results provide valuable sequence resources for future functional and population studies.Fundação de Amparo a Pesquisa do Estado de Sao Paulo (Brasil) (FAPESP) [FAPESP 2014/19740-7]Fundacao para a Ciencia e Tecnologia (FCT, Portugal) Portuguese Foundation for Science and TechnologyFundacao de Amparo a Pesquisa do Estado de Sao PauloFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [FAPESP 2017/02420-8, 2018/21319-9]FCT Portuguese Foundation for Science and Technology European Commission [UID/Multi/04423/2019, PTDC/CTA-AMB/31774/2017, POCI-01-0145-FEDER/031774/2017]European Regional Development Fund -program PT2020European Structural and Investment Funds Competitiveness and Internationalization Operational ProgramCOMPETE 2020info:eu-repo/semantics/publishedVersio