Teleosts comprise more than half of all vertebrate species and have adapted to a variety of marine and freshwater habitats 1 . Their genome evolution and diversification are important subjects for the understanding of vertebrate evolution. Although draft genome sequences of two pufferfishes have been published 2,3 , analysis of more fish genomes is desirable. Here we report a high-quality draft genome sequence of a small egg-laying freshwater teleost, medaka (Oryzias latipes). Medaka is native to East Asia and an excellent model system for a wide range of biology, including ecotoxicology, carcinogenesis, sex determination 4-6 and developmental genetics 7 . In the assembled medaka genome (700 megabases), which is less than half of the zebrafish genome, we predicted 20,141 genes, including 2,900 new genes, using 59-end serial analysis of gene expression tag information. We found single nucleotide polymorphisms (SNPs) at an average rate of 3.42% between the two inbred strains derived from two regional populations; this is the highest SNP rate seen in any vertebrate species. Analyses based on the dense SNP information show a strict genetic separation of 4 million years (Myr) between the two populations, and suggest that differential selective pressures acted on specific gene categories. Four-way comparisons with the human, pufferfish (Tetraodon), zebrafish and medaka genomes revealed that eight major interchromosomal rearrangements took place in a remarkably short period of 50 Myr after the whole-genome duplication event in the teleost ancestor and afterwards, intriguingly, the medaka genome preserved its ancestral karyotype for more than 300 Myr.We applied the whole-genome shotgun approach to an inbred strain, , derived from the southern Japanese population, as the main target. A total of 13.8 million reads amounting to approximately 10.6-fold genome coverage were obtained from the shotgun plasmid, fosmid and bacterial artificial chromosome (BAC) libraries. A newly developed RAMEN assembler was used to process the shotgun reads to generate contigs and scaffolds. The N50 values (50% of nucleotides in an assembly are in scaffolds-or contigs-longer than or equal to the N50 value) are ,1.41 megabases (Mb) for scaffolds and ,9.8 kilobases (Kb) for contigs. The total length of the contigs reached 700.4 Mb, which, from now on, we refer to as the medaka genome size.To construct ultracontigs, the scaffolds were integrated with the medaka genetic map by using SNP markers. For this purpose, we further obtained about 2.8-fold coverage of shotgun reads from another inbred strain HNI (refs 9, 10), which is derived from the northern Japanese population. The reads were assembled by RAMEN to scaffolds covering 648 Mb. Aligning the HNI contigs with the HdrR genome using BLASTZ 11 , we identified 16.4 million SNPs as well as 1.40 million insertions and 1.45 million deletions in non-repetitive regions (Supplementary Table 2). We selected 2,401 SNPs and genetically mapped them onto medaka chromosomes using a backcross panel between the...

Three sex-determining (SD) genes, SRY (mammals), Dmy (medaka), and DM-W (Xenopus laevis), have been identified to date in vertebrates. However, how and why a new sex-determining gene appears remains unknown, as do the switching mechanisms of the master sex-determining gene. Here, we used positional cloning to search for the sex-determining gene in Oryzias luzonensis and found that Gsdf Y (gonadal soma derived growth factor on the Y chromosome) has replaced Dmy as the master sex-determining gene in this species. We found that Gsdf Y showed high expression specifically in males during sex differentiation. Furthermore, the presence of a genomic fragment that included Gsdf Y converts XX individuals into fertile XX males. Luciferase assays demonstrated that the upstream sequence of Gsdf Y contributes to the male-specific high expression. Gsdf is downstream of Dmy in the sex-determining cascade of O. latipes, suggesting that emergence of the Dmy-independent Gsdf allele led to the appearance of this novel sexdetermining gene in O. luzonensis. IN most vertebrates, sex is determined genetically. Mammals and birds with cytogenetically well-differentiated sex chromosomes have sex determination systems that differ between the taxonomic classes but not within them (Solari 1994). In mammals, for example, the sex-determining (SD) gene SRY/Sry on the Y chromosome has a universal role in sex determination (Gubbay et al. 1990;Sinclair et al. 1990;Koopman et al. 1991;Foster et al. 1992). By contrast, some fish groups, such as salmonids, sticklebacks, and Oryzias fishes, have sex chromosomes that differ among closely related species (Devlin and Nagahama 2002;Woram et al. 2003;Takehana et al. 2007a;Ross et al. 2009).A DM-domain gene, Dmy, was the first SD gene identified in a nonmammalian vertebrate, the fish medaka Oryzias latipes (Matsuda et al. 2002(Matsuda et al. , 2007. In this species, the term Y chromosome is employed to refer to a recombining chromosome that carries the male-determining gene Dmy, and X is used for the homologous chromosome; these are not a heteromorphic pair. This gene is conserved among all wild populations of O. latipes examined to date . The closely related species O. curvinotus also has Dmy on its Y chromosome, which is orthologous to the O. latipes Y chromosome (Matsuda et al. 2003). However, Dmy has not been detected in any other type of fish, including other Oryzias fishes (Kondo et al. 2003). Analysis of the Y-specific region of the O. latipes sex chromosome has demonstrated that Dmy arose from duplication of the autosomal Dmrt1 gene (Nanda et al. 2002;Kondo et al. 2006). This Dmrt1 duplication is estimated to have occurred within the last 10 million years in a common ancestor of O. latipes, O. curvinotus, and O. luzonensis. In O. luzonensis, however, no functional duplicated copy of Dmrt1 has been detected (Kondo et al. 2003) (Figure 5A).O. luzonensis possesses an XX-XY system, which is homologous to an autosomal linkage group (LG 12) and Uwa 1985). In the d-rR strain, the wild-type alle...

The mapping of Hox clusters and many duplicated genes in zebrafish indicated an extra whole-genome duplication in ray-fined fish. However, to reconstruct the preduplication chromosomes (proto-chromosomes), the comparative genomic studies of more distantly related teleosts are essential. Medaka and zebrafish are ideal for this purpose, because their lineages separated from their last common ancestor ∼140 million years ago. To reconstruct ancient vertebrate chromosomes, including the chromosomes of the vertebrate ancestor of humans from 450 million years ago, we mapped 818 genes and expressed sequence tags (ESTs) on a single meiotic backcross panel obtained from inbred strains of the medaka, Oryzias latipes. Comparisons of linkage relationships of orthologous genes among three species of vertebrates (medaka, zebrafish, and human) indicate the number and content of the chromosomes of the last common ancestor of ray-fined fish and lobe-fined fish (including humans), and the extra whole genome duplication event in the ray-fin lineage occurred in the common ancestor of perhaps all teleosts.

Sex chromosomes harbour a primary sex-determining signal that triggers sexual development of the organism. However, diverse sex chromosome systems have been evolved in vertebrates. Here we use positional cloning to identify the sex-determining locus of a medaka-related fish, Oryzias dancena, and find that the locus on the Y chromosome contains a cis-regulatory element that upregulates neighbouring Sox3 expression in developing gonad. Sex-reversed phenotypes in Sox3 Y transgenic fish, and Sox3 Y loss-of-function mutants all point to its critical role in sex determination. Furthermore, we demonstrate that Sox3 initiates testicular differentiation by upregulating expression of downstream Gsdf, which is highly conserved in fish sex differentiation pathways. Our results not only provide strong evidence for the independent recruitment of Sox3 to male determination in distantly related vertebrates, but also provide direct evidence that a novel sex determination pathway has evolved through co-option of a transcriptional regulator potentially interacted with a conserved downstream component.