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.

Accuracy of genotype imputation of a low-density SNP array for the Amazon fish Colossoma macropomum.

In South America, Tambaqui (Colossoma macropomum) stands as the primary target for aquaculture, yet breeding programs for this Amazon native species are in their early stages. While high-density single nucleotide polymorphism (SNP) arrays are pivotal for aquaculture breeding, their costs can be prohibitive for non- or semi-industrial species. To overcome this, a cost-effective approach involves developing low-density SNP arrays followed by genotype imputation to higher densities. In this study, a 1K SNP array for tambaqui was created and validated, offering a balance between SNP quantity and genome representativity. The imputation accuracy from various SNP densities to a medium-density array was evaluated, with the 1K density demonstrating the best trade-off (accuracy of 0.93). This subset was further utilized to construct a commercial array through Agriseq™ targeted genotyping-by-sequencing, validated in 192 DNA samples, affirming its high quality for genotyping tambaqui. The low-density SNP array, with genome-wide coverage and high polymorphism, emerges as an effective tool for exploring genetic variation within diverse populations. Population analyses using the 1K panel proved to be an efficient tool for genetic characterization of sampled broodstocks, making it a valuable resource for genetic improvement programs targeting this Amazon native species.

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.

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 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.

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.

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.