Progress 01/15/04 to 01/14/07
Outputs
We identified DNA markers linked to sex determining genes in six species of tilapia. The mode of sex determination differed among species. Oreochromis karongae and Tilapia mariae have a sex-determining locus on linkage group 3 and the female is heterogametic (WZ-ZZ system). O. niloticus and T. zillii have a sex-determining locus on linkage group 1 and the male is heterogametic (XX-XY system). A more complex pattern observed in O. aureus and O. mossambicus, with sex associated loci on both linkage group 1 and linkage group 3, as well as deleterious effects from interactions between these loci. Linkage group 3 has several characteristics of a derived sex chromosome. Comparison of genetic and physical maps demonstrated a suppression of recombination over a broad region around the sex-determining locus, and 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 over the past 10 million years. Our previous work showed that sex-determining region in Nile tilapia lies in LG1 between UNH995 and GM201.5 tCYT19a is located at the end of LG1, 22.1cM away from this region. WT1_2 maps in the sex determining region 2.4cM from GM201, and 2.5cM from SEX. Breakpoint analysis using additional markers in the region found 2 recombinants that excluded WT1_2 as a candidate for sex determination. We have continued to fine-map the region surrounding this sex locus, and expect to have a complete contig of BACs spanning a 1Mb region by the end of the year. Several studies have reported QTL In various species and strains of tilapia, regions contributing to sex determination have been identified on linkage groups 1, 3, and 23. Genes contributing to sex-specific mortality have been detected on linkage groups 2, 6 and 23. To test whether the same genes might control sex determination in mammals and fishes, we mapped 11 genes
that are considered as putative master key regulators of sex determination: Amh, Cyp19, Dax1, Dmrt2, Dmrta2, Fhl3l, Foxl2, Ixl, Lhx9, Sf1 and Sox8. We identified polymorphisms in non-coding regions of these genes and genotyped these sites for 90 individuals of an F2 mapping family. Mapping of Dax1 joined LG16 and LG21 into a single linkage group. The Amh and Dmrta2 genes were mapped to two distinct regions of LG23. The Amh gene was mapped 5 cM from UNH879 within a QTL region for sex determination, and 2 cM from UNH216 within a QTL region for sex-specific mortality. Dmrta2 was mapped 4 cM from UNH848 within another QTL region for sex determination. Cyp19 was mapped to LG1 far from a previously reported QTL region for sex determination on this chromosome. Seven other candidate genes mapped to LG 4, 11, 12, 14 and 17.
Impacts
Tilapia imports to the US continue to grow at 25% per year, and tilapia are one of a handful of key species that will allow aquaculture to meet the growing demand for seafood. Control of sexual differentiation is key to profitability of this industry. Stocking of all-male populations is preferred, because females begin to reproduce at a small size, diverting energy from growth, and producing a population of small unmarketable fish. By understanding the genetic mechanisms by which sex is determined early in development, we hope to design hybrid crosses that will reliably produce all-male offspring for commercial growout.
Publications
- Shirak A, Seroussi E, Cnaani A, Howe AE, Domokhovsky R, Zilberman N, Kocher TD, Hulata G, Ron M. 2006. Amh and Dmrta2 genes map to tilapia (Oreochromis spp.) linkage group 23 within QTL regions for sex determination. Genetics 174(3): 1573-1581.
- Lee BY, Kocher TD. 2007. Exclusion of Wilms Tumor (WT1_2) and ovarian aromatase (CYP19A1) as candidates for sex determining genes in Nile tilapia (Oreochromis niloticus). Animal Genetics (in press).
- Cnaani A, Lee BY, Zilberman N, Ozouf-Costaz C, Hulata G, Ron M, D'Hont A, Baroiller JF, D'Cotta H, Penman DJ, Tomasino E, Coutanceau JP, Pepey E, Shirak A, Kocher TD. 2007. Rapid evolution of the sex determination mechanism among closely related cichlid fish. (submitted).
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Progress 10/01/03 to 09/30/04
Outputs
We are breeding variety of tilapia species and strains in order to determine the genetic mechanism of sex determination in each population. By screening pedigreed families with DNA markers we can map the sex determination loci to particular chromosomes, and by following the inheritance of parental alleles we can determine the sex determination mechanism (XY or WZ). At least two different chromosomes, LG1 and LG3, are involved in sex determination in tilapia. In Oreochromis niloticus, the sex-determining region has been mapped to LG1 (Lee et al. 2004). The sex-determining locus lies between markers GM201 and UNH 995 (11cM). Using our resource of fingerprinted BAC clones (Katagiri et al. 2005), we identified a BAC contig containing UNH995. Several BACs in the contig were end- or shotgun sequenced. These sequences were blasted against the Tetraodon genome sequence databases for comparative mapping. Based on the BLAST analysis, we identified that Tetraodon chromosome 5 is
the homolog of tilapia LG1. The sex-determining region of LG1 seems to be syntenic with 2.7Mb4.8Mb region of Tetraodon chr5. Other microsatellite markers (UNH868, UNH213, and GM041) from LG1 also hit tetraodon chr5 and the order of these markers is consistent between tilapia LG1 and tetraodon chr5. Published cichlid ESTs corresponding to genes in the syntenic region of tetraodon chr5 were identified by BLAST and nine additional EST-derived markers were successfully mapped in the sex-determining region. This work further narrowed the sex-determining region to a 2.6 cM and excluded two genes on LG1 (WT1 and ovarian aromatase) as candidates for the sex determining locus. Although we have developed four additional EST markers within this region, so far we have not identified any recombinants between these markers and sex. So, we have genotype about 1000 additional animals to find recombinants. We are also developing more markers from BAC clones corresponding to this interval. In
Oreochromis aureus (Mehadrin strain, originated from Israel) seven markers on LG3 are highly associated with sex. An attempt to construct a linkage map from the marker genotypes, using the Joinmap software, was unsuccessful. A manual analysis of the data showed that in a region of four adjacent markers the maternal alleles were inherited mostly in the parental type, with a strong reduction in recombination rate (only double recombinants were identified). Among other markers, and in the paternal alleles, recombination rates were similar to what was previously observed in tilapia (Lee et al. 2005). This pattern can be explained by the existence of an inversion in one of the female's chromosomes. We are testing the inversion hypothesis by physically mapping of the markers along this chromosome using FISH. These experiments were conducted with a collaborator, Dr. Catherine Ozouf, in Paris.
Impacts
The sex-linked markers we have identified are already useful for genotyping broodstock in commercial breeding programs to identify sex-reversed and YY male broodstock. They also provide tools for further investigations of the genetic and environmental basis of sex determination in cultured tilapia.
Publications
- Katagiri T, Kidd C. Tomasino E, Davis JT, Wishon C, Stern JE, Carleton KL, Howe AE, Kocher TD. 2005. A BAC-based physical map of the Nile tilapia genome. BMC Genomics 6:89.
- Lee BY, Lee WJ, Streelman JT, Carleton KL, Howe AE, Hulata G, Slettan A, Stern JE, Terai Y, Kocher TD. 2005. A second generation genetic linkage map of tilapia (Oreochromis spp.) Genetics 170: 237-244.
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