Chromosome-level genome sequence of the Genetically Improved Farmed Tilapia (GIFT, Oreochromis niloticus) highlights regions of introgression with O. mossambicus.

BACKGROUND: The Nile tilapia (Oreochromis niloticus) is the third most important freshwater fish for aquaculture. Its success is directly linked to continuous breeding efforts focusing on production traits such as growth rate and weight. Among those elite strains, the Genetically Improved Farmed Tilapia (GIFT) programme initiated by WorldFish is now distributed worldwide. To accelerate the development of the GIFT strain through genomic selection, a high-quality reference genome is necessary. RESULTS: Using a combination of short (10X Genomics) and long read (PacBio HiFi, PacBio CLR) sequencing and a genetic map for the GIFT strain, we generated a chromosome level genome assembly for the GIFT. Using genomes of two closely related species (O. mossambicus, O. aureus), we characterised the extent of introgression between these species and O. niloticus that has occurred during the breeding process. Over 11 Mb of O. mossambicus genomic material could be identified within the GIFT genome, including genes associated with immunity but also with traits of interest such as growth rate. CONCLUSION: Because of the breeding history of elite strains, current reference genomes might not be the most suitable to support further studies into the GIFT strain. We generated a chromosome level assembly of the GIFT strain, characterising its mixed origins, and the potential contributions of introgressed regions to selected traits.

The tilapias' chromosomes influencing sex determination.

The sex chromosomes of tilapias (family Cichlidae; genera Oreochromis, Sarotherodon and Tilapia) have been studied for over 50 years, which has gained interest from both agricultural and basic scientific perspectives. Several closely related tilapia species which can interbreed have been studied, and it has been repeatedly demonstrated that there is variation within and between species in the chromosomal sex-determination mechanism. Both male and female heterogametic sex-determination systems have been characterized, as well as epistatic and environmental influences on sex determination. Three different linkage groups (LG1, LG3 and LG23) have been identified as sex-associated chromosomes and have been subjected to further cytogenetic research and analyses of the genes located around the sex-determining region. Variation in the genetic and physical characteristics of the sex chromosomes makes tilapias an excellent model system for studying the evolution of vertebrate sex chromosomes. This review summarizes the progress made along 5 decades of research and the current knowledge of the tilapias' sex chromosomes.

Fine-mapping of a locus on linkage group 23 for sex determination in Nile tilapia (Oreochromis niloticus).

Genetic markers in tilapia species associated with loci affecting sex determination (SD), sex-specific mortality or both were mapped to linkage groups (LG) 1, 2, 3, 6 and 23. The objective of this study was to use these markers to fine-map the locus with the greatest effect on SD in Oreochromis niloticus. Our parental stock, full-sibs of Nile tilapia (Swansea origin), were divided into three groups: (i) untreated, (ii) feminized by diethylstilbestrol and (iii) masculinized by 17α-methyltestosterone. We analysed the first group for association of microsatellite markers representing these five LGs. The strongest association with gender was found on LG23 for marker UNH898 (χ(2) ; P=8.6×10(-5) ). Allele 276 was found almost exclusively in males, and we hypothesized that this allele is a male-associated allele (MAA). Sex-reversed individuals were used for mating experiments with and without the segregating MAA. Mating of individuals lacking the MAA resulted in all-female progeny. Mating of two heterozygotes for MAA gave rise to 81 males and 30 females. Analysis of association between gender and genotypes identified the MAA in 98.6% of males as opposed to 8.0% of females (χ(2) ; P=2.5×10(-18) ). Eight markers that flank UNH898 were genotyped to map the locus on LG23 within a confidence interval of 16-21 cM. Mating of homozygous individuals for MAA is underway for production of all-male populations.

Sexual development in fish, practical applications for aquaculture.

Aquaculture is one of the fastest rising sectors of world food production. Hundreds of fish species are cultured, providing an affordable, high quality food source. Two aspects of sexual development are critically important for the continued improvement of cultured fish stocks: sexual dimorphism and control of reproduction. In this paper, we review the main methods used to control sex determination in fish and their application in some of the most widely cultured species. Specifically, we review the techniques available for the production of all-male, all-female, and sterile populations. Techniques for endocrinological control of reproduction are also discussed.

Genetics of sex determination in tilapiine species.

We identified DNA markers linked to sex determining genes in six closely related species of tilapiine fishes. The mode of sex determination differed among species. In Oreochromis karongae and Tilapia mariae the sex-determining locus is on linkage group (LG) 3 and the female is heterogametic (WZ-ZZ system). In O. niloticus and T. zillii the sex-determining locus is on LG1 and the male is heterogametic (XX-XY system). A more complex pattern was observed in O. aureus and O. mossambicus, in which markers on both LG1 and LG3 were associated with sex. We found evidence for sex-linked lethal effects on LG1, as well as interactions between loci in the two linkage groups. Comparison of genetic and physical maps demonstrated a broad region of recombination suppression harboring the sex-determining locus on LG3. Sex-specific recombination suppression was found in the female heterogametic sex. Sequence analysis showed the accumulation of repetitive elements in this region. Phylogenetic analysis suggests that at least two transitions in the mode of sex determination have occurred in this clade. This variation in sex determination mechanisms among closely related species makes tilapias an excellent model system for studying the evolution of sex chromosomes in vertebrates.

Mapping of sox2 and sox14 in tilapia (Oreochromis spp.).

Sox genes encode transcription factors that are involved in a variety of embryonic developmental pathways. Sox2 and Sox14 are located on the same chromosomal arm in several mammalian and bird species and on the basis of comparative maps were suggested as candidate genes for the major sex-determining locus on tilapia LG3. We have sequenced the sox2 and sox14 genes in four tilapia species and mapped them to different chromosomes, LG17 and LG23 respectively. Although excluded as being one of the major sex-determining genes so far mapped in tilapia, sox14 did fall within a QTL region for growth, stress response, embryonic mortality and a minor effect on sex determination.

Genome-scan analysis for quantitative trait loci in an F2 tilapia hybrid.

We searched for genetic linkage between DNA markers and quantitative trait loci (QTLs) for innate immunity, response to stress, biochemical parameters of blood, and fish size in an F2 population derived from an interspecific tilapia hybrid (Oreochromis mossambicusx O. aureus). A family of 114 fish was scanned for 40 polymorphic microsatellite DNA markers and two polymorphic genes, covering approximately 80% of the tilapia genome. These fish had previously been phenotyped for seven immune-response traits and six blood parameters. Critical values for significance were P <0.05 with the false discovery rate (FDR) controlled at 40%. The genome-scan analysis resulted in 35 significant marker-trait associations, involving 26 markers in 16 linkage groups. In a second experiment, nine markers were re-sampled in a second family of 79 fish of the same species hybrid. Seven markers (GM180, GM553, MHC-I, UNH848, UNH868, UNH898 and UNH925) in five linkage groups (LG 1, 3, 4, 22 and 23) were associated with stress response traits. An additional six markers (GM47, GM552, UNH208, UNH881, UNH952, UNH998) in five linkage groups (LG 4, 16, 19, 20 and 23) were verified for their associations with immune response traits, by linkage to several different traits. The portion of variance explained by each QTL was 11% on average, with a maximum of 29%. The average additive effect of QTLs was 0.2 standard deviation units of stress response traits and fish size, with a maximum of 0.33. In three linkage groups (LG 1, 3 and 23) markers were associated with stress response, body weight and sex determination, confirming the location of QTLs reported by several other studies.

Association between loci with deleterious alleles and distorted sex ratios in an inbred line of tilapia (Oreochromis aureus).

Three microsatellite markers (UNH159, UNH231, and UNH216) were examined for association with both deleterious genes and sex-ratio distortions in a full-sib family of 222 progeny from the fourth generation of a meiogynogenetic tilapia line (Oreochromis aureus). The three markers were mapped previously to different linkage groups and were shown to be associated with genes with deleterious alleles in this line. A restricted maximum likelihood model was used for analysis of major effects and their interactions on sex ratio and viability. This model was based on selective mortality of genders, ignoring effects of possible sex-determining genes. The results showed that deleterious genes linked to UNH216 and UNH231 exert higher lethality in females than in males (P < .0005 and P < .05, respectively). UNH159 was not associated directly with sex ratio distortion, but acts strongly as a modifier of sex ratio in combination with UNH216 and UNH231. Each of the three loci was found to have a significant effect on viability (P < .05) in the maximum likelihood analysis. The deleterious single-locus effects act strongly against females, while most of the epistatic interactions exert higher lethality in males. This contradiction results in a close to 1:1 sex ratio at maturity. The genetic mechanism and significance of such a balance between genders are still unknown. A detailed analysis of sex-specific lethality may be applied by screening in appropriate series of matings and fine mapping with additional markers. Our data suggest that UNH216 and UNH231 are linked to sex ratio distortion genes and that UNH159 may be linked to a modifier of these genes.