A trans-species missense SNP in Amhr2 is associated with sex determination in the tiger pufferfish, Takifugu rubripes (fugu)

Abstract

Heterogametic sex chromosomes have evolved independently in various lineages of vertebrates. Such sex chromosome pairs often contain nonrecombining regions, with one of the chromosomes harboring a master sex-determining (SD) gene. It is hypothesized that these sex chromosomes evolved from a pair of autosomes that diverged after acquiring the SD gene. By linkage and association mapping of the SD locus in fugu (Takifugu rubripes), we show that a SNP (C/G) in the anti-Müllerian hormone receptor type II (Amhr2) gene is the only polymorphism associated with phenotypic sex. This SNP changes an amino acid (His/Asp384) in the kinase domain. While females are homozygous (His/His384), males are heterozygous. Sex in fugu is most likely determined by a combination of the two alleles of Amhr2. Consistent with this model, the medaka hotei mutant carrying a substitution in the kinase domain of Amhr2 causes a female phenotype. The association of the Amhr2 SNP with phenotypic sex is conserved in two other species of Takifugu but not in Tetraodon. The fugu SD locus shows no sign of recombination suppression between X and Y chromosomes. Thus, fugu sex chromosomes represent an unusual example of proto-sex chromosomes. Such undifferentiated X-Y chromosomes may be more common in vertebrates than previously thought.

Figure 1. Fine mapping of the sex-determining locus in the pedigreed families of fugu.

(A) Genetic map of the sex-determining (SD) region. The frequency of recombinant markers with the sexual phenotype observed in the initial genetic mapping is shown at the top of the linkage map. In the box below, each row is a recombinant individual found in the subsequent screening of 1034 additional siblings and those found in the initial mapping. IDs of males (green boxed) and females (red boxed) are shown in the first column, followed by allele type at markers along the sex chromosome. Alleles shown in red and green are transmitted to nonrecombinant females and males, respectively. Empty blocks indicate that genotypes are non-informative or not assigned. Marker names are indicated at the top of column and their genomic positions are shown above. A combination of markers sca77 and f540 or sca77 and 1189 k is used for screening recombinants from 1034 siblings. The boxed 17.5 kb region shows perfect correlation between the haplotype and phenotypic sex. (B) Schematic diagram of the SD region and gene structures. The positions of SNPs defining the 17.5 kb SD region and two predicted genes in this region are shown. Orange arrows indicate the transcriptional orientation of the genes. Green and red lines indicate the position of genomic DNA clones that were amplified by PCR, cloned into plasmids and sequenced. Two clones each for the genomic regions SD2–14k and SD3–14k were sequenced from X (red line) and Y (green line) chromosomes.

Figure 2. Association mapping of the sex-determining locus in a natural population of fugu.

(A) Schematic diagram of the SD region. The 17.5 kb SD region and both of the only two predicted genes are shown. (B) Plot of −log₁₀ (P value) versus chromosome position for association test of the SD region. Position starts from the 5′ end of the genomic clone covering the entire Amhr2 gene shown in Figure 1B (SD3–14k region). SNP 7271 in Amhr2 showed perfect correlation with phenotypic sex (green dotted vertical line). Bonferroni correction gives a significance threshold of −log₁₀(P) = 3.2. (C) Linkage disequilibrium (LD) around the SD region. The square of the correlation coefficient (r²) is estimated for each pairwise comparison of SNPs, with darker grey indicating stronger LD (white, r² = 0; shades of grey, 0<r²<1; black, r² = 1).

Figure 3. A trans-specific SNP in Amhr2 is correlated with phenotypic sex in Takifugu.

(A) Sequence traces of Amhr2 from a male (left) and a female (right) fugu. The male is heterozygous at the non-synonymous SNP site that converts His384 codon into Asp384 codon. (B) Sexual phenotypes, genotypes and statistical significance of trans-species SNPs. Proportion of heterozygotes among females and males in three species of Takifugu are shown. P-values are from 2×2 Fisher's exact test of the genotype count data. A horizontal arrow indicates the transcriptional orientation of the genes. (C) Comparison of a part of kinase domain sequences of Amhr2 from various vertebrates. The sequences from Takifugu species are boxed in black. The amino acid specific to the male chromosome of Takifugu is boxed in red. His/Asp384 of fugu Amhr2 corresponds to Asp397 of human AMHR2. The position of the amino acid mutation in the medaka (hotei) that results in a female phenotype when homozygous is boxed in green. The amino acid position of a natural mutation leading to the loss-of-function of human AMHR2 is boxed in cyan.

Figure 4. Expression pattern of Amhr2 in fugu.

(A) RT-PCR analysis of the tissue distribution of Amhr2 mRNA in juvenile fugu at 147 days after fertilization (dpf). EF1a was used as an internal control. PCR analysis was performed for three males and females, and only representative results for a male and a female are shown. (B) In situ hybridization on undifferentiated and differentiating gonads. Genotypic sexes are shown as XY and XX. In situ hybridization with antisense probe (a–d, and i–l) and sense probe (e–h) for Amhr2 are shown. (a, e) Undifferentiated gonad in XY fish at 62 dpf. (b, f) Differentiating gonad in XY fish at 90 dpf. (c, g) Undifferentiated gonad in XX fish at 62 dpf. (d, h, l) Differentiating gonad in XX fish at 90 dpf. The ovarian cavity (OC) is apparent only in differentiating XX gonad. The large and round germ cells (arrows in l) were evident under DIC optics. (i–k) Differentiating gonad in XY fish at 126 dpf. The germ cells containing large and round nucleus (arrows) were apparent in DAPI stained (j) and overlay (k) images. Scale bar, 20 µm (a, e, c, g), 50 µm (b, f, d, h) and 10 µm (i–l).

Figure 5. Mediation of AMH signaling by wild-type and mutated human AMHR2 (His397) constructs.

Each assay was done in triplicate, and the results were expressed as mean ± SEM. Human AMHR2^H397 mediated significantly less signaling compared to wild-type human AMHR2 following the addition of human rAMH. Double asterisks indicate significant difference (P<0.01) between groups.