Genetic and functional characterization of the rainbow trout "mal" masculinizing mutation. – SEXYTROUT

In contrast to the situation observed in mammals and birds, in which a major sex-determining system has been maintained over at least 100 million years of evolution, a very large diversity of sex determination mechanisms has been reported in fish. In salmonids, sex determination is generally regarded as being strictly genetic, with male heterogamety (males XY and females XX). However, unexpected males have been observed in homozygote XX rainbow trout (Oncorhynchus mykiss) lines resulting from gynogenesis. Genetic analysis of this maleness phenotype pointed to the existence of a recessive mutation in a minor sex-determining gene called mal. The present project combines genetics, genomics and transcriptomics approaches to identify mal. Three convergent strategies will be developed. Firstly, in a positional cloning approach, the mal locus will be localized on rainbow trout linkage map. Linked bacterial artificial chromosome (BAC) clones will be subsequently sequenced to identify genes potentially related to mal. Secondly, in a candidate gene approach, genes involved in sex determination and differentiation will be identified in silico in fish sequence databases. These genes will be then genetically mapped to test their possible co-localization with the mal locus. Thirdly, after characterization of the gonadal phenotype of mal-carrying populations and analysis of the effect of temperature on its occurrence, microarray-based expression profiling followed by quantitative RT-PCR and in situ hybridization analyses will be performed to identify deregulations of genes and pathways associated with the maleness phenotype. Deregulated genes and their putative master regulators, which are candidates for the mal locus, will be then localized on the linkage map of O. mykiss. Gene candidates uncovered through these three complementary strategies, chosen on the basis of their genetic co-localization with the mal locus and their expression pattern compatible with a role in sex determination and differentiation, will be then sequenced in XX females and mal males in order to identify the mutation responsible for the maleness phenotype. Finally, the functional effect of the mal mutation on the gene products will be investigated both in vitro and in vivo. This project involves three research teams with recognized expertise in the domain of sex determination and sex differentiation of the trout and of other fish species, with complementary competences in the fields of genetics, genomics, transcriptomics, molecular physiology, evolution and developmental biology. The project will provide unprecedented new information on the genetics and molecular physiology of sexual development in rainbow trout and will therefore significantly contribute to a better understanding of the molecular and evolutionary basis of sex determination and differentiation in fish. It will also provide new insights into how interactions between genome and environmental factors can modulate phenotype in living organisms. In an aquaculture context, a better knowledge of the genetic and environmental bases of masculinization of XX fish will help breeders to manage similar problems reported to occur in fish farms. Finally, deciphering how environment can modulate gonadal differentiation in trout might provide new opportunities to implement original temperature-based method of sex control in fish production as an alternative of the steroid hormone treatments currently used to produced the XX neomales, that are needed to obtain genetic all-females’ populations.