Elodie Pepey

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.

A high-resolution map of the Nile tilapia genome: a resource for studying cichlids and other percomorphs

BackgroundThe Nile tilapia (Oreochromis niloticus) is the second most farmed fish species worldwide. It is also an important model for studies of fish physiology, particularly because of its broad tolerance to an array of environments. It is a good model to study evolutionary mechanisms in vertebrates, because of its close relationship to haplochromine cichlids, which have undergone rapid speciation in East Africa. The existing genomic resources for Nile tilapia include a genetic map, BAC end sequences and ESTs, but comparative genome analysis and maps of quantitative trait loci (QTL) are still limited.ResultsWe have constructed a high-resolution radiation hybrid (RH) panel for the Nile tilapia and genotyped 1358 markers consisting of 850 genes, 82 markers corresponding to BAC end sequences, 154 microsatellites and 272 single nucleotide polymorphisms (SNPs). From these, 1296 markers could be associated in 81 RH groups, while 62 were not linked. The total size of the RH map is 34,084 cR3500 and 937,310 kb. It covers 88% of the entire genome with an estimated inter-marker distance of 742 Kb. Mapping of microsatellites enabled integration to the genetic map. We have merged LG8 and LG24 into a single linkage group, and confirmed that LG16-LG21 are also merged. The orientation and association of RH groups to each chromosome and LG was confirmed by chromosomal in situ hybridizations (FISH) of 55 BACs. Fifty RH groups were localized on the 22 chromosomes while 31 remained small orphan groups. Synteny relationships were determined between Nile tilapia, stickleback, medaka and pufferfish.ConclusionThe RH map and associated FISH map provide a valuable gene-ordered resource for gene mapping and QTL studies. All genetic linkage groups with their corresponding RH groups now have a corresponding chromosome which can be identified in the karyotype. Placement of conserved segments indicated that multiple inter-chromosomal rearrangements have occurred between Nile tilapia and the other model fishes. These maps represent a valuable resource for organizing the forthcoming genome sequence of Nile tilapia, and provide a foundation for evolutionary studies of East African cichlid fishes.

An EST resource for tilapia based on 17 normalized libraries and assembly of 116,899 sequence tags

BackgroundLarge collections of expressed sequence tags (ESTs) are a fundamental resource for analysis of gene expression and annotation of genome sequences. We generated 116,899 ESTs from 17 normalized and two non-normalized cDNA libraries representing 16 tissues from tilapia, a cichlid fish widely used in aquaculture and biological research.ResultsThe ESTs were assembled into 20,190 contigs and 36,028 singletons for a total of 56,218 unique sequences and a total assembled length of 35,168,415 bp. Over the whole project, a unique sequence was discovered for every 2.079 sequence reads. 17,722 (31.5%) of these unique sequences had significant BLAST hits (e-value < 10-10) to the UniProt database.ConclusionNormalization of the cDNA pools with double-stranded nuclease allowed us to efficiently sequence a large collection of ESTs. These sequences are an important resource for studies of gene expression, comparative mapping and annotation of the forthcoming tilapia genome sequence.

Temperature induced-masculinisation in the Nile tilapia causes rapid up-regulation of both dmrt1 and amh expressions.

Nile tilapia has primarily a XX/XY sex determining system but minor genetic factors as well as temperature can override the major factors. Female XX progenies can be sex-reversed into functional males by rearing at high temperatures (>34°C) from 10dpf onwards. Temperature effects on the molecular pathways during sex differentiation in teleosts need to be deciphered. The temporal expression profiles of cyp19a1a and foxl2, two ovarian-developmental genes and dmrt1 and amh, two testes-developmental genes were analysed during key stages of the sex differentiation of genetic all-females, all-males and temperature-masculinised XX females (TM) tilapia. Overall QPCR analysis was similar between gonads and trunks. Both amh and dmrt1 expressions were up-regulated simultaneously in TM already at 13-15dpf. Dmrt1 expression became markedly elevated ∼3-fold higher than XY male levels at 20-26dpf whereas amh had similar levels to XY males. Foxl2 and cyp19a1a expression profiles were similar. Both were up-regulated at early stages in TM but repressed after 17-19dpf, whilest levels continued to increase in XX-females. Our results show that temperature action on tilapia testis development induces the rapid increase of both dmrt1 and amh expressions followed by the down-regulation of foxl2 and cyp19a1a. This suggests that dmrt1 and/or amh may be the modulator(s) of the down-regulation of foxl2 and/or cyp19a1a.

Elevated amh Gene Expression in the Brain of Male Tilapia (Oreochromis niloticus) during Testis Differentiation

Anti-müllerian hormone (AMH) is expressed in male embryos and represses development of müllerian ducts during testis differentiation in mammals, birds and reptiles. Amh orthologues have been identified in teleosts despite them lacking müllerian ducts. Previously we found sexually dimorphic aromatase activity in tilapia brains before ovarian differentiation. This prompted us to search for further dimorphisms in tilapia brains during sex differentiation and see whether amh is expressed. We cloned the tilapia amh gene and found that it contains 7 exons but no spliced forms. The putative protein presents highest homologies with Amh proteins of pejerrey and medaka as compared to other Perciformes. We analysed amh expression in adult tissues and found elevated levels in testes, ovary and brain. Amh expression was dimorphic with higher levels in XY male brains at 10–15 dpf, when the gonads were still undifferentiated and gonadal amh was not dimorphic. Male brains had 2.7-fold higher amh expression than gonads. Thereafter, amh levels decreased in the brain while they were up-regulated in differentiating testes. Our study indicates that amh is transcribed in male brains already at 10 dpf, suggesting that sexual differentiation may be occurring earlier in tilapia brain than in gonads.

Chromosomal localisation of sex-specific BACs and of selected cDNAs identified by microdissection in two tilapia species, #Oreochromis niloticus# and "Oreochromis aureus#

1. Chromosome Architecture 1033 2. Clinical Cytogenetics 1035 3. New Technologies and Microarray 1041 4. Karyotype Evolution and Comparative Genomics 1042 5. Nuclear Organization, Gene Expression and Gene Mapping 1048 6. Avian Cytogenetics 1053 7. Cytogenetics of Reproduction 1054 8. Fish Cytogenetics 1059 9. Cytogenetics and Ecotoxicology 1064Cichlid fish of the Tilapia group are an ideal model for studying sex determination. Sequencing of the Tilapia (Oreochromis niloticus) genome has started in USA and France and genetic and physical linkage maps based on high resolution fingerprints of BAC clones are produced. We have in charge the chromosomal anchoring of some sex linked BACs from the genetic mapping. Some BAC libraries constructed from a male O. niloticus have been assembled into a physical map. Generally, species with a major sex determination locus on linkage group 1 (LG1) are male heterogametic and species with a major sex determination locus on linkage group 3 (LG3) are female heterogametic. In order to confirm that LG1 and LG3 are on two different, unlinked chromosomes, we physically mapped the sex-linked markers by chromosomal FISH on the chromosomes of two closely related Tilapia species, O. niloticus and O. aureus. These experiments demonstrated that LG3 corresponds to the largest chromosome in the Tilapia karyotype, and that LG1 corresponds to a different, smaller chromosome. The chromosome pair corresponding to LG3 in O. niloticus has been manually microdissected and a series of cDNAs isolated from this microdissected product have been cloned, sequenced and selected for chromosomal FISH. Their hybridization helps to validate the results. (Texte integral)