A high-density linkage map and sex-determination loci in Pacific white shrimp (Litopenaeus vannamei).

BACKGROUND: Expansion of genomic resources for the Pacific white shrimp (Litopenaeus vannamei), such as the construction of dense genetic linkage maps, is crucial for the application of genomic tools in order to improve economically relevant traits. Sexual dimorphism exists in Pacific white shrimp, and the mapping of the sex-determination region in this species may help in future reproductive applications. We have constructed male, female, and sex-averaged high-density genetic maps using a 50 K single-nucleotide polymorphism (SNP) array, followed by a genome-wide association study (GWAS) to identify genomic regions associated with sex in white shrimp. RESULTS: The genetic map yielded 15,256 SNPs assigned to 44 linkage groups (LG). The lengths of the male, female, and sex-averaged maps were 5,741.36, 5,461.20 and 5,525.26 cM, respectively. LG18 was found to be the largest for both sexes, whereas LG44 was the shortest for males and LG31 for females. A sex-determining region was found in LG31 with 21 statistically significant SNPs. The most important SNP was previously identified as a sex-linked marker and was able to identify 99% of the males and 88% of the females. Although other significant markers had a lower ability to determine sex, putative genes were intercepted or close to them. The oplophorus-luciferin 2-monooxygenase, serine/arginine repetitive matrix protein and spermine oxidase genes were identified as candidates with possible participation in important processes of sexual differentiation in shrimp. CONCLUSIONS: Our results provide novel genomic resources for shrimp, including a high-density linkage map and new insights into the sex-determining region in L. vannamei, which may be usefulfor future genetics and reproduction applications.

Genome-wide association study of host resistance to the ectoparasite Ichthyophthirius multifiliis in the Amazon fish Colossoma macropomum.

BACKGROUND: Tambaqui, Colossoma macropomum, is the most important native fish species farmed in South America, particularly in Brazil, where its production is limited in the southern and southeastern regions due to disease outbreaks caused by the parasite Ichthyophthirius multifiliis. Therefore, genome level analysis to understand the genetic architecture of the host resistance against I. multifiliis is fundamental to improve this trait in tambaqui. The objective of the present study was to map QTL (quantitative trait loci) associated with resistance to I. multifiliis in tambaqui by GWAS (genome-wide association study). METHODS AND RESULTS: Individuals belonging to seven families, which were previously submitted to an experimental challenge to assess the natural resistance to the parasite I. multifiliis, were used for genomic analysis. A total of 7717 SNPs were identified in this population by ddRAD (double digest restriction site associated DNA). GWAS revealed four SNPs significantly associated in the LGs (linkage groups) 2, 9, 11 and 20 for the traits time of death and parasite load. The SNPs explained a low proportion of the variance to I. multifiliis resistance for time of death and parasite load (about 0.622% and 0.375%, respectively). The SNPs were close to 11 genes related to the immune system: abcf3, znf830, ccr9, gli3, ackr4, tbata, ndr2, tgfbr3, nhej1, znf644b, and cldn10a. CONCLUSIONS: In conclusion, the resistance to I. multifiliis is probably under polygenic control in tambaqui, in which different QTLs of low variance can be involved in the immune responses against this ectoparasite.

Computer vision system using deep learning to predict rib and loin yield in the fish Colossoma macropomum.

Computer vision system (CVSs) are effective tools that enable large-scale phenotyping with a low-cost and non-invasive method, which avoids animal stress. Economically important traits, such as rib and loin yield, are difficult to measure; therefore, the use of CVS is crucial to accurately predict several measures to allow their inclusion in breeding goals by indirect predictors. Therefore, this study aimed (1) to validate CVS by a deep learning approach and to automatically predict morphometric measurements in tambaqui and (2) to estimate genetic parameters for growth traits and body yield. Data from 365 individuals belonging to 11 full-sib families were evaluated. Seven growth traits were measured. After biometrics, each fish was processed in the following body regions: head, rib, loin, R + L (rib + loin). For deep learning image segmentation, we adopted a method based on the instance segmentation of the Mask R-CNN (Region-based Convolutional Neural Networks) model. Pearson's correlation values between measurements predicted manually and automatically by the CVS were high and positive. Regarding the classification performance, visible differences were detected in only about 3% of the images. Heritability estimates for growth and body yield traits ranged from low to high. The genetic correlations between the percentage of body parts and morphometric characteristics were favorable and highly correlated, except for percentage head, whose correlations were unfavorable. In conclusion, the CVS validated in this image dataset proved to be resilient and can be used for large-scale phenotyping in tambaqui. The weight of the rib and loin are traits under moderate genetic control and should respond to selection. In addition, standard length and pelvis length can be used as an efficient and indirect selection criterion for body yield in this tambaqui population.

Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish.

BACKGROUND: Eukaryote genomes frequently harbor supernumerary B chromosomes in addition to the "standard" A chromosome set. B chromosomes are thought to arise as byproducts of genome rearrangements and have mostly been considered intraspecific oddities. However, their evolutionary transcendence beyond species level has remained untested. RESULTS: Here we reveal that the large metacentric B chromosomes reported in several fish species of the genus Astyanax arose in a common ancestor at least 4 million years ago. We generated transcriptomes of A. scabripinnis and A. paranae 0B and 1B individuals and used these assemblies as a reference for mapping all gDNA and RNA libraries to quantify coverage differences between B-lacking and B-carrying genomes. We show that the B chromosomes of A. scabripinnis and A. paranae share 19 protein-coding genes, of which 14 and 11 were also present in the B chromosomes of A. bockmanni and A. fasciatus, respectively. Our search for B-specific single-nucleotide polymorphisms (SNPs) identified the presence of B-derived transcripts in B-carrying ovaries, 80% of which belonged to nobox, a gene involved in oogenesis regulation. Importantly, the B chromosome nobox paralog is expressed > 30× more than the A chromosome paralog. This indicates that the normal regulation of this gene is altered in B-carrying females, which could potentially facilitate B inheritance at higher rates than Mendelian law prediction. CONCLUSIONS: Taken together, our results demonstrate the long-term survival of B chromosomes despite their lack of regular pairing and segregation during meiosis and that they can endure episodes of population divergence leading to species formation.

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.

Haplotypes traceability and genetic variability of the breeding population of pacu (Piaractus mesopotamicus) revealed by mitochondrial DNA.

The main objective of this study was to estimate the genetic diversity levels and haplotype traceability in pacu Piaractus mesopotamicus from the breeding program located in Brazil by analyses of the mitochondrial DNA control region (mtDNA). Moreover, broodstocks from eight commercial fish farms were used for comparative evaluation, four from Brazil (Br1-Br4) and four from Argentina (Ar1-Ar4). The descriptive results revealed 47 polymorphic sites and 51 mutations, which evidenced 34 haplotypes. Ten haplotypes were shared among fish farms and 24 were exclusive. The nucleotide diversity (π) ranged from 0.00031 to 0.01462 and haplotype diversity (Hd) from 0.125 to 0.868. The analysis of molecular variance (AMOVA) indicated high structure present in the analyzed stocks (FST = 0.13356 and ФST = 0.52707). The genetic diversity was high in most of the commercial broodstocks, especially those from Brazil. We observed seven haplotypes in the genetic breeding population, of which four were exclusive and three shared among the commercial fish farms. The genetic diversity was moderate (π = 0.00265 and Hd = 0.424) and considered appropriated for this breeding population of pacu. Our results provide support for the genetic diversity maintenance and mtDNA traceability of pacu commercial broodstocks.

Development of a SNP linkage map and genome-wide association study for resistance to Aeromonas hydrophila in pacu (Piaractus mesopotamicus).

BACKGROUND: Pacu (Piaractus mesopotamicus) is one of the most important Neotropical aquaculture species from South America. Disease outbreaks caused by Aeromonas hydrophila infection have been considered significant contributors to the declining levels of pacu production. The current implementation of genomic selection for disease resistance has been adopted as a powerful strategy for improvement in fish species. This study aimed to investigate the genetic architecture of resistance to A. hydrophila in pacu via Genome-Wide Association Study (GWAS), the identification of suggestive Quantitative Trait Loci (QTLs) and putative genes associated with this trait. The genetic data were obtained from 381 juvenile individuals belonging to 14 full-sibling families. An experimental challenge was performed to gain access to the levels of genetic variation for resistance against the bacteria using the following trait definitions: binary test survival (TS) and time of death (TD). RESULTS: The analyses of genetic parameters estimated moderate heritability (h2) for both resistance traits: 0.20 (± 0.09) for TS and 0.35 (± 0.15) for TD. A linkage map for pacu was developed to enable the GWAS, resulting in 27 linkage groups (LGs) with 17,453 mapped Single Nucleotide Polymorphisms (SNPs). The length of the LGs varied from 79.95 (LG14) to 137.01 (LG1) cM, with a total map length of 2755.60 cM. GWAS identified 22 putative QTLs associated to A. hydrophila resistance. They were distributed into 17 LGs, and were considered suggestive genomic regions explaining > 1% of the additive genetic variance (AGV) for the trait. Several candidate genes related to immune response were located close to the suggestive QTLs, such as tbk1, trim16, Il12rb2 and lyz2. CONCLUSION: This study describes the development of the first medium density linkage map for pacu, which will be used as a framework to study relevant traits to the production of this species. In addition, the resistance to A. hydrophila was found to be moderately heritable but with a polygenic architecture suggesting that genomic selection, instead of marker assisted selection, might be useful for efficiently improving resistance to one of the most problematic diseases that affects the South American aquaculture.

Development of microsatellite markers using next-generation sequencing for the fish Colossoma macropomum.

Tambaqui (Colossoma macropomum) is a fish species from the Amazon and Orinoco Rivers, with favorable characteristics to the cultivation system and great market acceptance in South America. However, the construction of a genetic map for the genetic improvement of this species is limited by the low number of molecular markers currently described. Thus, this study aimed to validate gene-associated and anonymous (non-genic) microsatellites obtained by next generation sequencing (RNA-seq and whole genome shotgun-WGS, respectively), for future construction of a genetic map and search for quantitative trait loci (QTL) in this species. In the RNA-seq data, the observed and expected heterozygosity (Ho and He) ranged from 0.09 to 0.73, and 0.09 to 0.85, respectively. In the WGS data, Ho and He ranged from 0.33 to 0.95, and 0.28 to 0.92, respectively. In general, the evaluation of 200 markers resulted in 45 polymorphic loci, of which 14 were gene-associated (RNA-Seq) and 31 were anonymous (WGS). Moreover, some markers were related to genes of the immune system, biological regulation/control and biogenesis. This study contributes to increase the number of molecular markers available for genetic studies in C. macropomum, which will allow the development of breeding programs assisted by molecular markers.

Characterization of lipid metabolism genes and the influence of fatty acid supplementation in the hepatic lipid metabolism of dusky grouper (Epinephelus marginatus).

Dusky grouper is an important commercial fish species in many countries, but some factors such as overfishing has significantly reduced their natural stocks. Aquaculture emerges as a unique way to conserve this species, but very little biological information is available, limiting the production of this endangered species. To understand and generate more knowledge about this species, liver transcriptome sequencing and de novo assembly was performed for E. marginatus by Next Generation Sequencing (NGS). Sequences obtained were used as a tool to validate the presence of key genes relevant to lipid metabolism, and their expression was quantified by qPCR. Moreover, we investigated the influence of supplementing different dietary fatty acids on hepatic lipid metabolism. The results showed that the different fatty acids added to the diet dramatically changed the gene expression of some key enzymes associated with lipid metabolism as well as hepatic fatty acid profiles. Elongase 5 gene expression was shown to influence intermediate hepatic fatty acid elongation in all experimental groups. Hepatic triglycerides reflected the diet composition more than hepatic phospholipids, and were characterized mainly by the high percentage of 18:3n3 in animals fed with a linseed oil rich diet. Results for the saturated and monounsaturated fatty acids suggest a self-regulatory potential for retention and oxidation processes in liver, since in general the tissues did not directly reflect these fatty acid diet compositions. These results indicated that genes involved in lipid metabolism pathways might be potential biomarkers to assess lipid requirements in the formulated diet for this species.

Organization and Chromosomal Distribution of Histone Genes and Transposable Rex Elements in the Genome of Astyanax bockmanni (Teleostei, Characiformes).

An important feature of eukaryotic organisms is the number of different repetitive DNA sequences in their genome, a feature not observed in prokaryotes. These sequences are considered to be important components for understanding evolutionary mechanisms and the karyotypic differentiation processes. Thus, we aimed to physically map the histone genes and transposable elements of the Rex family in 6 fish populations of Astyanax bockmanni. FISH results using a histone H1 gene probe showed fluorescent clusters in 2 chromosome pairs in all 6 samples analyzed. In contrast, FISH with a histone H3 probe showed conspicuous blocks in 4 chromosomes in 5 of the 6 populations analyzed. The sixth population revealed 7 chromosomes marked with this probe. Probes for the transposable elements Rex1 and Rex6 showed small sites dispersed on most chromosomes of the 6 populations, and the Rex3 element is located in a big block concentrated in only 1 acrocentric chromosome of 2 populations. As for the other populations, a Rex3 probe showed large blocks in more than 1 chromosome. Fish from Alambari and Campo Novo Stream have Rex3 elements dispersed along most of the chromosomes. Additionally, the conspicuous signals of Rex1, Rex3, and Rex6 were identified in the acrocentric B microchromosome of A. bockmanni found only in individuals of the Alambari River. Thus, we believe that different mechanisms drive the spread of repetitive sequences among the populations analyzed, which appear to be organized differently in the genome of A. bockmanni. The presence of transposable elements in the B chromosome also suggests that these sequences could play a role in the origin and maintenance of the supernumerary element in the genome of this species.

Development of a multi-species SNP array for serrasalmid fish Colossoma macropomum and Piaractus mesopotamicus.

Scarce genomic resources have limited the development of breeding programs for serrasalmid fish Colossoma macropomum (tambaqui) and Piaractus mesopotamicus (pacu), the key native freshwater fish species produced in South America. The main objectives of this study were to design a dense SNP array for this fish group and to validate its performance on farmed populations from several locations in South America. Using multiple approaches based on different populations of tambaqui and pacu, a final list of 29,575 and 29,612 putative SNPs was selected, respectively, to print an Axiom AFFYMETRIX (THERMOFISHER) SerraSNP array. After validation, 74.17% (n = 21,963) and 71.25% (n = 21,072) of SNPs were classified as polymorphic variants in pacu and tambaqui, respectively. Most of the SNPs segregated within each population ranging from 14,199 to 19,856 in pacu; and from 15,075 to 20,380 in tambaqui. Our results indicate high levels of genetic diversity and clustered samples according to their hatchery origin. The developed SerraSNP array represents a valuable genomic tool approaching in-depth genetic studies for these species.

Genetic Characterization of the Fish Piaractus brachypomus by Microsatellites Derived from Transcriptome Sequencing.

The pirapitinga, Piaractus brachypomus (Characiformes, Serrasalmidae), is a fish from the Amazon basin and is considered to be one of the main native species used in aquaculture production in South America. The objectives of this study were: (1) to perform liver transcriptome sequencing of pirapitinga through NGS and then validate a set of microsatellite markers for this species; and (2) to use polymorphic microsatellites for analysis of genetic variability in farmed stocks. The transcriptome sequencing was carried out through the Roche/454 technology, which resulted in 3,696 non-redundant contigs. Of this total, 2,568 contigs had similarity in the non-redundant (nr) protein database (Genbank) and 2,075 sequences were characterized in the categories of Gene Ontology (GO). After the validation process of 30 microsatellite loci, eight markers showed polymorphism. The analysis of these polymorphic markers in farmed stocks revealed that fish farms from North Brazil had a higher genetic diversity than fish farms from Southeast Brazil. AMOVA demonstrated that the highest proportion of variation was presented within the populations. However, when comparing different groups (1: Wild; 2: North fish farms; 3: Southeast fish farms), a considerable variation between the groups was observed. The FST values showed the occurrence of genetic structure among the broodstocks from different regions of Brazil. The transcriptome sequencing in pirapitinga provided important genetic resources for biological studies in this non-model species, and microsatellite data can be used as the framework for the genetic management of breeding stocks in Brazil, which might provide a basis for a genetic pre-breeding programme.

Microsatellites Associated with Growth Performance and Analysis of Resistance to Aeromonas hydrophila in Tambaqui Colossoma macropomum.

Tambaqui, Colossoma macropomum, is the main native fish species produced in Brazil, and is an important species for genetic improvement in aquaculture. In addition, breeding studies on this species can be optimized with the use of molecular markers associated with productive phenotypes. The objective of the present study was to test the performance of growth traits and resistance to the bacteria, Aeromonas hydrophila, in association with microsatellite markers in C. macropomum. In this study, three full-sib families were subjected to bacterial challenge and morphometric growth assessments. Tambaqui families subjected to the bacterial challenge differed significantly in death time and mortality rate. There was, however, no association between resistance to bacteria and microsatellite markers. In relation to growth traits, we observed a marker/phenotype association in two microsatellites. The marker in the 6b isoform x5 gene (TNCRC6b) was associated with length, whereas an anonymous marker was associated with height. The present study highlighted the evaluation of molecular markers associated with growth traits, and can serve as the basis for future marker-assisted selection (MAS) of tambaqui.

A Glimpse into the Satellite DNA Library in Characidae Fish (Teleostei, Characiformes).

Satellite DNA (satDNA) is an abundant fraction of repetitive DNA in eukaryotic genomes and plays an important role in genome organization and evolution. In general, satDNA sequences follow a concerted evolutionary pattern through the intragenomic homogenization of different repeat units. In addition, the satDNA library hypothesis predicts that related species share a series of satDNA variants descended from a common ancestor species, with differential amplification of different satDNA variants. The finding of a same satDNA family in species belonging to different genera within Characidae fish provided the opportunity to test both concerted evolution and library hypotheses. For this purpose, we analyzed here sequence variation and abundance of this satDNA family in ten species, by a combination of next generation sequencing (NGS), PCR and Sanger sequencing, and fluorescence in situ hybridization (FISH). We found extensive between-species variation for the number and size of pericentromeric FISH signals. At genomic level, the analysis of 1000s of DNA sequences obtained by Illumina sequencing and PCR amplification allowed defining 150 haplotypes which were linked in a common minimum spanning tree, where different patterns of concerted evolution were apparent. This also provided a glimpse into the satDNA library of this group of species. In consistency with the library hypothesis, different variants for this satDNA showed high differences in abundance between species, from highly abundant to simply relictual variants.

Molecular identification of intergenus crosses involving catfish hybrids: risks for aquaculture production

Monitoring of the interspecific hybrid production and trade is essential for the appropriate management of these animals in fish farms. The identification of catfish hybrids by morphological analysis is unreliable, particularly of juveniles and post-F1 individuals. Therefore, in the present study, we used five molecular markers (four nuclear genes and one mitochondrial gene) to detect hybrids in the trade of pimelodid juvenile fish from different stocks purchased of five seed producers in Brazil. Samples commercialized as pintado (pure species Pseudoplatystoma corruscans ) from three fish farms were genetically identified as hybrid cachapinta ( P. reticulatum x P. corruscans ). In the stocks purchased as cachandiá (hybrid between P. reticulatum x Leiarius marmoratus ) and cachapira (hybrid between P. reticulatum x Phractocephalus hemioliopterus ), we suggested the occurrence of intergenus crosses involving the hybrid cachapinta, which was used instead of the pure species P. reticulatum . The problems involving the hybrid cachapinta production were discussed in the present study, especially because these animals have caused genetic contamination and threatened the genetic integrity of natural and cultivated populations. In order to improve the surveillance of the production and provide criteria for the correct management of catfish hybrids, genetic markers has become an excellent alternative to the morphological identification, including juveniles or post-F1 generations.

O monitoramento da produção e comércio de híbridos interespecíficos é essencial para o manejo adequado desses animais em pisciculturas. A identificação de híbridos de bagres por análise morfológica não é confiável, especialmente de juvenis e indivíduos pós-F1. Portanto, no presente estudo, cinco marcadores moleculares (quatro genes nucleares e um gene mitocondrial) foram utilizados para detectar híbridos no comércio de juvenis pimelodídeos de diferentes estoques, comprados de cinco produtores de alevinos no Brasil. As amostras comercializadas como pintado (espécie pura Pseudoplatystoma corruscans ) foram geneticamente identificadas como híbrido cachapinta ( P. reticulatum x P. corruscans ). Nos estoques comprados como cachandiá (híbrido entre P. reticulatum x Leiarius marmoratus ) e cachapira (híbrido entre P. reticulatum x Phractocephalus hemioliopterus ), sugere-se a ocorrência de cruzamentos intergêneros envolvendo o híbrido cachapinta, que foi usado ao invés da espécie pura P. reticulatum . Os problemas envolvendo a produção de cachapinta foram discutidos no presente estudo, especialmente porque estes animais têm causado contaminação genética e ameaçado a integridade genética das populações naturais e cultivadas. Com o intuito de melhorar a fiscalização da produção e fornecer critérios para o manejo correto dos híbridos de bagre, marcadores genéticos têm se tornado uma excelente alternativa para a identificação morfológica, incluindo juvenis ou gerações pós-F1.