Evidence for different origins of sex chromosomes in closely related Oryzias fishes: substitution of the master sex-determining gene

Abstract

The medaka Oryzias latipes and its two sister species, O. curvinotus and O. luzonensis, possess an XX-XY sex-determination system. The medaka sex-determining gene DMY has been identified on the orthologous Y chromosome [O. latipes linkage group 1 (LG1)] of O. curvinotus. However, DMY has not been discovered in other Oryzias species. These results and molecular phylogeny suggest that DMY was generated recently [approximately 10 million years ago (MYA)] by gene duplication of DMRT1 in a common ancestor of O. latipes and O. curvinotus. We identified seven sex-linked markers from O. luzonensis (sister species of O. curvinotus) and constructed a sex-linkage map. Surprisingly, all seven sex-linked markers were located on an autosomal linkage group (LG12) of O. latipes. As suggested by the phylogenetic tree, the sex chromosomes of O. luzonensis should be "younger" than those of O. latipes. In the lineage leading to O. luzonensis after separation from O. curvinotus approximately 5 MYA, a novel sex-determining gene may have arisen and substituted for DMY. Oryzias species should provide a useful model for evolution of the master sex-determining gene and differentiation of sex chromosomes from autosomes.

Figure 1.—

Phylogenetic relationship of Oryzias species based on mitochondrial DNA sequences. Using cytochrome b gene sequences from Takehana et al. (2003), we redrew a linearized neighbor-joining tree. The separation of O. luzonensis from O. curvinotus was estimated to have occurred ∼5 MYA according to a divergence rate of 2.8%/MY. DMY has been identified only in O. latipes and O. curvinotus.

Figure 2.—

Search for the DMY gene and mapping of DMY-related genes. (A) DMY structure of the O. latipes and positions of primers used in this study. Open boxes, shaded boxes, and horizontal lines indicate exons, the DM domain, and introns, respectively. (B) Agarose gel electrophoresis (1%) of PCR products using the 49S and 48U primers. Only O. latipes and O. curvinotus gave a male-specific band (DMY). (C) Polyacrylamide gel electrophoresis (9%) of PCR products using the ex4.1 and 48U primers. MboI-digested and undigested samples were loaded on the gel. The lower band of digested samples was judged as Oludmrt1, which has an Sau3AI (isochizomer of MboI) restriction site (Kondo et al. 2004). (D) Linkage analysis using CL1 cross. Map distances between markers are shown in centimorgans.

Figure 3.—

Sex-linked polymorphisms of O. luzonensis. (A) Polyacrylamide gel electrophoresis (9%) of PCR products for AU167284, MF01SSA025F03, and OLb24.08a. Arrowheads mark male-specific bands. (B) Sequence analysis of polymorphic PCR products. For EST AU167284, the female PCR products show seven T repeats, whereas males display both seven and eight T repeats. In EST MF01SSA025F03, males show a (C/T) SNP. In OLb24.08a, the 39-bp deletion links to maleness.

Figure 4.—

FISH analysis of male metaphase chromosomes in O. luzonensis using O. latipes BAC clones. (A) Chromosomal location of the O. luzonensis sex-determining region (BAC Md0171M23, red) and of the O. latipes sex chromosomal marker SL1 (BAC Md0173J11, green). (B) Chromosomal location of the O. luzonensis sex-determining region (BAC Md0171M23, red) and the O. latipes DMRT1 gene (BAC Md0172B19, green).

Figure 5.—

Sex-determination mechanisms and sex linkage groups in Oryzias species. The phylogenetic information was taken from Takehana et al. (2005).

Figure 6.—

Comparative recombination map between sex chromosomes of O. luzonensis and LG12 (autosome) of O. latipes. The sex-determining gene (Sex, arrowhead) of O. luzonensis is located adjacent to the b gene. Map distances between markers are shown in centimorgans and total map lengths are shown below each map.