Species-Specific Marker Discovery in Tilapia

Tilapias (family Cichlidae) are of importance in aquaculture and fisheries. Hybridisation and introgression are common within tilapia genera but are difficult to analyse due to limited numbers of species-specific genetic markers. We tested the potential of double digested restriction-site associated DNA (ddRAD) sequencing for discovering single nucleotide polymorphism (SNP) markers to distinguish between 10 tilapia species. Analysis of ddRAD data revealed 1,371 shared SNPs in the de novo-based analysis and 1,204 SNPs in the reference-based analysis. Phylogenetic trees based on these two analyses were very similar. A total of 57 species-specific SNP markers were found among the samples analysed of the 10 tilapia species. Another set of 62 species-specific SNP markers was identified from a subset of four species which have often been involved in hybridisation in aquaculture: 13 for Oreochromis niloticus, 23 for O. aureus, 12 for O. mossambicus and 14 for O. u. hornorum. A panel of 24 SNPs was selected to distinguish among these four species and validated using 91 individuals. Larger numbers of SNP markers were found that could distinguish between the pairs of species within this subset. This technique offers potential for the investigation of hybridisation and introgression among tilapia species in aquaculture and in wild populations

A high quality assembly of the Nile Tilapia (Oreochromis niloticus) genome reveals the structure of two sex determination regions

Background  Tilapias are the second most farmed fishes in the world and a sustainable source of food. Like many other fish, tilapias are sexually dimorphic and sex is a commercially important trait in these fish. In this study, we developed a significantly improved assembly of the tilapia genome using the latest genome sequencing methods and show how it improves the characterization of two sex determination regions in two tilapia species.  Results  A homozygous clonal XX female Nile tilapia (Oreochromis niloticus) was sequenced to 44X coverage using Pacific Biosciences (PacBio) SMRT sequencing. Dozens of candidate de novo assemblies were generated and an optimal assembly (contig NG50 of 3.3Mbp) was selected using principal component analysis of likelihood scores calculated from several paired-end sequencing libraries. Comparison of the new assembly to the previous O. niloticus genome assembly reveals that recently duplicated portions of the genome are now well represented. The overall number of genes in the new assembly increased by 27.3%, including a 67% increase in pseudogenes. The new tilapia genome assembly correctly represents two recentvasagene duplication events that have been verified with BAC sequencing. At total of 146Mbp of additional transposable element sequence are now assembled, a large proportion of which are recent insertions. Large centromeric satellite repeats are assembled and annotated in cichlid fish for the first time. Finally, the new assembly identifies the long-range structure of both a ~9Mbp XY sex determination region on LG1 in O. niloticus, and a ~50Mbp WZ sex determination region on LG3 in the related species O. aureus.  Conclusions  This study highlights the use of long read sequencing to correctly assemble recent duplications and to characterize repeat-filled regions of the genome. The study serves as an example of the need for high quality genome assemblies and provides a framework for identifying sex determining genes in tilapia and related fish species

Genetic, abiotic and social influences on sex differentiation in cichlid fishes and the evolution of sequential hermaphroditism

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73799/1/j.1467-2979.2005.00184.x.pd

Impact of Daily Thermocycles on Hatching Rhythms, Larval Performance and Sex Differentiation of Zebrafish

In the wild, water temperature cycles daily: it warms up after sunrise, and cools rapidly after sunset. Surprisingly, the impact of such daily thermocycles during the early development of fish remains neglected. We investigated the influence of constant vs daily thermocycles in zebrafish, from embryo development to sexual differentiation, by applying four temperature regimens: two constant (24°C and 28°C) and two daily thermocycles: 28:24°C, TC (thermophase coinciding with daytime, and cryophase coinciding with night-time) and 24:28°C, CT (opposite to TC) in a 12:12 h light:dark cycle (LD). Embryo development was temperature-dependent but enhanced at 28°C and TC. Hatching rhythms were diurnal (around 4 h after lights on), but temperature- and cycle-sensitive, since hatching occurred sooner at 28°C (48 hours post fertilization; hpf) while it was delayed at 24°C (96 hpf). Under TC, hatching occurred at 72 hpf, while under CT hatching displayed two peaks (at 70 hpf and 94 hpf). In constant light (LL) or darkness (DD), hatching rhythms persisted with tau close to 24 h, suggesting a clock-controlled "gating" mechanism. Under 28°C or TC, larvae showed the best performance (high growth and survival, and low malformations). The sex ratio was strongly influenced by temperature, as the proportion of females was higher in CT and TC (79 and 83% respectively), contrasting with 28°C and 24°C, which led to more males (83 and 76%). Ovarian aromatase (cyp19a) expression in females was highest in TC and CT (6.5 and 4.6 fold higher than at 28°C, respectively); while anti-müllerian hormone (amh) expression in males increased in testis at 24°C (3.6 fold higher compared to TC) and particularly at 28°C (14.3 fold increase). Taken together, these findings highlight the key role of environmental cycles during early development, which shaped the daily rhythms in fish embryo and larvae, and ultimately influenced sex differentiation

Sex Reversal in Zebrafish fancl Mutants Is Caused by Tp53-Mediated Germ Cell Apoptosis

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA–repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination

Sex Determination in the Squalius alburnoides Complex: An Initial Characterization of Sex Cascade Elements in the Context of a Hybrid Polyploid Genome

BACKGROUND:Sex determination processes vary widely among different vertebrate taxa, but no group offers as much diversity for the study of the evolution of sex determination as teleost fish. However, the knowledge about sex determination gene cascades is scarce in this species-rich group and further difficulties arise when considering hybrid fish taxa, in which mechanisms exhibited by parental species are often disrupted. Even though hybridisation is frequent among teleosts, gene based approaches on sex determination have seldom been conducted in hybrid fish. The hybrid polyploid complex of Squalius alburnoides was used as a model to address this question. METHODOLOGY/PRINCIPAL FINDINGS:We have initiated the isolation and characterization of regulatory elements (dmrt1, wt1, dax1 and figla) potentially involved in sex determination in S. alburnoides and in the parental species S. pyrenaicus and analysed their expression patterns by in situ hybridisation. In adults, an overall conservation in the cellular localization of the gene transcripts was observed between the hybrids and parental species. Some novel features emerged, such as dmrt1 expression in adult ovaries, and the non-dimorphic expression of figla, an ovarian marker in other species, in gonads of both sexes in S. alburnoides and S. pyrenaicus. The potential contribution of each gene to the sex determination process was assessed based on the timing and location of expression. Dmrt1 and wt1 transcripts were found at early stages of male development in S. alburnoides and are most likely implicated in the process of gonad development. CONCLUSIONS/SIGNIFICANCE:For the first time in the study of this hybrid complex, it was possible to directly compare the gene expression patterns between the bisexual parental species and the various hybrid forms, for an extended set of genes. The contribution of these genes to gonad integrity maintenance and functionality is apparently unaltered in the hybrids, suggesting that no abrupt shifts in gene expression occurred as a result of hybridisation