This project aims to identify the master sex-determining gene of the platyfish Xiphophorus maculatus as well as other sex-chromosomal genetic loci involved in melanoma formation, pigmentation and puberty using a positional cloning strategy. New insights into both the early steps driving the molecular differentiation between sex chromosomes and the evolution of sex-linked gene functions are expected from the comparison between the X and Y chromosomes. This study should significantly contribute to our understanding of the molecular mechanisms and adaptive constraints driving the variability of sex determination observed in fish and other organisms.

As an essential prerequisite to achieve these objectives, the first bacterial artificial chromosome (BAC) library for a Xiphophorus species has been constructed (Froschauer et al. 2002, Gene 295:247). Using BAC clones containing Xmrk, egfrb and other sex-linked molecular markers as starting points for a chromosome walking, BAC contigs covering several megabases of the sex-determining region on the X and Y chromosomes have been assembled. BAC clones from these contigs are now being sequenced (Genoscope). The different candidate genes identified during the sequencing of the sex-determining region will be characterized at the functional level in different fish species (expression analysis, RNA/DNA microinjection in early embryos, morpholino antisense nucleotide technology, transgenesis), with transfer of knowledge to aquaculture-relevant species (University of Würzburg). The evolutionary dynamics of the sex chromosomes will be analyzed in related and more divergent fish species by fluorescent in situ hybridization (Muséum National d'Histoire Naturelle, Paris).

Genoscope is undertaking the sequencing of 50 BAC clones from the sex-determining region of the X and Y chromosomes of the platyfish Xiphophorus maculatus. For each BAC clone, a plasmid library (5 kb inserts) will be created in pcdna2.1 and clones from each library will be sequenced at both ends, with an expected sequencing volume of about 200,000 reads. BAC clones will be assembled in phase 2, with oriented contigs.