We characterized the male-specific region on the Y chromosome of rainbow trout, which contains both sdY (the sex-determining gene) and the male-specific genetic marker, OmyY1. Several clones containing the OmyY1 marker were screened from a BAC library from a YY clonal line and found to be part of an 800？kb BAC contig. Using fluorescence in situ hybridization (FISH), these clones were localized to the end of the short arm of the Y chromosome in rainbow trout, with an additional signal on the end of the X chromosome in many cells. We sequenced a minimum tiling path of these clones using Illumina and 454 pyrosequencing. The region is rich in transposons and rDNA, but also appears to contain several single-copy protein-coding genes. Most of these genes are also found on the X chromosome; and in several cases sex-specific SNPs in these genes were identified between the male (YY) and female (XX) homozygous clonal lines. Additional genes were identified by hybridization of the BACs to the cGRASP salmonid 4x44K oligo microarray. By BLASTn evaluations using hypothetical transcripts of OmyY1-linked candidate genes as query against several EST databases, we conclude at least 12 of these candidate genes are likely functional, and expressed. 1. Introduction Salmonid fishes have the XX/XY system of sex determination [1]. Most rainbow trout have morphologically distinguishable sex chromosomes [2], with the short arm being longer in the X chromosome than the Y, primarily due to presence of 5S rDNA sequences on the X chromosome (reviewed in [3]). Some wild fish and several male clonal lines, including the Swanson YY and Arlee YY clones have a Y chromosome that resembles the X chromosome in having a longer short arm including the 5S rDNA sequences [4]. This fact and the viability seen in rainbow trout and Chinook salmon YY individuals [5, 6] suggest that the X and Y chromosomes share considerable genetic content. The SEX locus is not found on a common linkage group in Pacific salmon and trout, but rather each species has the SEX locus on a different linkage group as shown by genetic mapping and localization of male-specific markers using in situ hybridization with clones to GH-Y [7–9]. (Rainbow and cutthroat trout are an exception to this [10], and hybrids between these two species are interfertile.) Although it is possible that each species has a different master sex determining gene, this is unlikely because there are several male-specific markers shared by the Oncorhynchus species. These include OmyY1, found in all Pacific trout and salmon [11], OtY2 [12] which is
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