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Genetics. 2007 Apr; 175(4): 2039–2042.
PMCID: PMC1855111

The Sex-Determining Locus in the Tiger Pufferfish, Takifugu rubripes


The tiger pufferfish (fugu), Takifugu rubripes, is a model fish that has had its genome entirely sequenced. By performing genomewide linkage analyses, we show that the sex of fugu is determined by a single chromosomal region on linkage group 19 in an XX–XY system.

ALTHOUGH the existence of female and male sexes in vertebrates is a conserved phenomenon, there is considerable diversity in the mechanisms that control this choice, either by environmental or genetic factors. Even in the case of animals where sex is determined by the genome, the primary sex-determining gene(s) is not conserved in divergent species (Capel 2000). DMY/dmrt1bY and SRY have been identified as the primary sex-determining genes in the medaka (Oryzias latipes) and in most mammals, respectively (Matsuda et al. 2002). Apart from these cases, the molecular nature of the primary sex-determining gene(s) in the remainder of the vertebrates remains unknown (Schartl 2004).

Teleost fish are an attractive group of organisms with which to study the evolution and mechanisms of sex determination systems. This is because this group of organisms includes different types of sexuality—from hermaphroidism to gonochorism and from environmental to genetic sex determination (Schartl 2004). The tiger pufferfish (fugu), Takifugu rubripes, is a model species of fish that has had its genome entirely sequenced (Aparicio et al. 2002). Since extensive searches in the fugu genome sequence suggested the absence of the previously known sex-determining genes mammalian SRY and medaka DMY (Koopman et al. 2004; Schartl 2004), it follows that the fugu may provide an ideal resource to use in the search for a novel sex-determining gene. However, even the mode of sex determination is poorly understood in this fish and other closely related species. In this article, we characterize the genetic basis of sex determination in fugu.

To investigate whether the choice of developing testicular or ovarian tissues in the fugu is determined by genetic or environmental factors, we analyzed the progeny produced from two independent genetic crosses by taking advantage of a linkage map previously constructed by our laboratory (Kai et al. 2005). To accelerate the search for sex-linked markers, we chose several markers from 22 linkage groups (LGs) and genotyped 15 progeny of each sex from each family. We found that the marker f120 on LG19 cosegregated with phenotypic sex (Figure 1).

Figure 1.
Male and female meiotic maps of fugu LG19. The markers f2006, f542-2, and SDY (the locus determining male sex phenotype) showed no recombination. Genetic maps were generated using 75 males and 55 females from family 1. Allelic bridges are indicated by ...

The fugu linkage map reported previously contained only five markers anchoring four scaffolds on LG19 (Kai et al. 2005). When analyzing model organisms in which almost the complete genome sequence is available, the candidate region for markers on a particular chromosome can be selected by referring to the genome sequence data. However, this approach currently remains limited for fugu because of the fragmented nature of the draft genome sequence (Aparicio et al. 2002). In contrast, the draft genome sequence of Tetraodon nigroviridis, another model fish, is less fragmented than that of fugu (Ensembl version 36.1d). We therefore assessed whether it might be practical to use the syntenic conservation between the genome of fugu and that of Tetroadon to selectively enrich markers covering the whole range of the putative sex chromosome for fugu. We first compared the whole catalog of genes from these two species and selected fugu genes orthologous to Tetraodon genes on chromosome 11. Of 614 fugu genes identified in this search, 531 genes were contained in 10 major fugu scaffolds (Table 1). We then obtained microsatellite loci from these scaffolds and examined their linkage relationships. These analyses enabled us to anchor 11 fugu scaffolds spanning 10.9 Mb along LG19 and, thus, to establish a genomic tool for analyzing the sex chromosome of fugu (Table 1).

Fugu scaffolds anchored to LG19

By using these genetic and physical maps, we were able to add new markers and successfully locate the sex-determining locus at the region where the markers f542-2 and f2006, on scaffolds 87 and 33, respectively, exhibit perfect segregation with phenotypic sex among the 130 progeny from family 1 and the 30 progeny from family 2 (Figure 1; supplemental data 2 at http://www.genetics.org/supplemental/). The sex-linked alleles were inherited from male parents in both families (Figure 1), suggesting that the fugu adopts an XX–XY sex-determining system. Comparison of male and female maps revealed that recombination between markers around the sex-determining locus was reduced during meiosis in males relative to meiosis in females (Figure 1). This could reflect either a general reduction of recombination in male fish (Kai et al. 2005) or region-specific reductions due to the sex-specific differences in DNA sequence close to the sex-determining locus (Peichel et al. 2004).

At present, the draft genome sequences of five species of fish have been fully sequenced or are in the process of being sequenced. Species involved are fugu, medaka, Tetraodon, stickleback (Gasterosteus aculeatus), and zebrafish (Danio rerio). Of these, the sex determination system and sex chromosome has been determined only in medaka and stickleback (Matsuda et al. 2002; Peichel et al. 2004). To compare the sex chromosome between these fish and fugu, we identified medaka and stickleback genes orthologous to the genes surrounding the sex-determining locus in fugu (genes on scaffolds 33, 56, 77, and 87) and ascertained their genomic positions via Ensemble databases. These analyses revealed that none of the genes tested mapped to the sex chromosome of either medaka or stickleback (Table 2). Therefore, it is likely that the sex chromosome in fugu has evolved independently from that of medaka or stickleback.

The number and genomic position of medaka and stickleback genes orthologous to genes located around the sex-determining locus of fugu (scaffolds 33, 56, 77, and 87)

DMRT1 and SOX9 are regarded as conserved key players in the downstream cascade of the sex-determining/differentiation pathway in divergent species including mammals, reptiles, and fish (Capel 2000; Koopman 2005). Existing literature highlights the possibility that these genes may serve as the primary sex-determining genes in some nonmammalian vertebrates (Nanda et al. 1999; Capel 2000; Volff and Schartl 2002). By surveying the fugu genome database, previous studies have identified two sox9 genes (sox9a and sox9b) and one dmrt1 gene in fugu (Koopman et al. 2004; Yamaguchi et al. 2006). To determine whether any of these genes resides in the primary sex-determining locus, we mapped microsatellite loci physically linked to these genes on the meiotic panel. This linkage analysis revealed that none of these genes mapped to the fugu sex chromosome (Table 3). To identify candidates for the sex-determining gene in fugu, we searched the scaffolds around the sex-determining locus (scaffolds 33, 56, 77, and 87) for genes belonging to the sox or dmrt gene families or for genes related to the hormonal pathways involved in sex determination/differentiation. These searches successfully identified amhrII (NEWSINFRUG00000143070) and inhbb (NEWSINFRUG00000130534) on scaffold 33. Since these genes play key roles in the sex differentiation of mammals (Vigier et al. 1989; Brown et al. 2000), it is reasonable to consider that these genes may be strong candidates for the primary sex-determining gene in fugu. Given the freely available nature of the draft fugu genome sequence, a positional cloning strategy should make it possible to accurately identify the sex-determining gene of fugu. Successful identification of the fugu sex-determining gene should provide significant insight into our understanding of the evolution of the XX–XY system.

Mapping position of the fugu sox9a, sox9b, and dmrt1 genes


We thank Byrappa Venkatesh for critical comments on the manuscript and Akiyuki Ozaki for technical advice. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


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