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Fig. 1. Genotypic and phenotypic sex of X. tropicalis samples. (A) Genotypic sex of estrogen-treated animals (T356 and T357) was determined using several sex-linked SSLP markers with informative polymorphisms in their progenitors. The father of clutches T356 and T357 (T260) is homozygous for the SSLP marker shown in A; the mother (T290.3) is heterozygous, with the longer allele (red dot) located on the W chromosome and the shorter allele (blue dot) located on the Z chromosome (data obtained from grandparents). When this marker is used, ZW individuals have two bands, and ZZ individuals have only one. Sex-reversed males are identified as phenotypic females with a ZZ genotypic constitution for several informative sex-linked markers. (B and C) X. tropicalis gonads (g) attached to mesonephroi (m) from male (B) and female (C) tadpoles at stage NF 60. When the gonad has differentiated morphologically, the sex of tadpoles can be established by direct observation of the gonad under the stereoscope after dissection.
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Fig. S1. Pedigree chart of the golden animals used in sex-reversal experiments (estradiol and fadrozole treatments). SR indicates sex-reversed individuals.
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Fig. S2. Pedigree chart of the N/IC family used in estradiol sex-reversal and gynogenesis experiments. SR indicates sex-reversed individuals. Color coding refers to the strain the sex chromosomes come from: red, Nigerian; blue, Ivory Coast; Green, PacBio; orange, Cam4/N.
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Fig. 2. Gynogenetic animals: production, confirmation of absence of paternal DNA, and verification of double haploids and gynogenetic diploids obtained by LCS and ECS, respectively. (A) To obtain gynogenetic offspring, the paternal contribution is blocked by UV-irradiation of sperm. Haploid (nonviable) embryos are rescued to viable diploidy either by suppressing second polar body formation with ECS to produce gynogenetic diploid embryos (heterozygous at many distal loci) or by blocking first cytokinesis using LCS and producing completely homozygous double haploids. (B) Chromosomal position of the two markers shown in C (red) on X. tropicalis chromosome 7 according to Wells et al. (36). (C) Genotypes for markers 010E04 (centromeric) and 095F08 (distal) in nongynogenetic diploid controls, double haploids, and gynogenetic diploids. A three-allele system for marker 010E04 allows the identification of the paternal contribution because the father is homozygous for allele 2, not present in the mothers (T290.1 and T290.2), heterozygous for alleles 1 and 3. Both triploid and nongynogenetic leaker individuals (labeled as 3n and *, respectively) were observed among true double haploids and gynogenetic diploids. The polymorphism for marker 095F08 (together with all markers included in Table 2) distinguishes true isogenic double haploids from gynogenetic diploids (GD) arising from spontaneous polar body failure. Double-haploid individuals are homozygous for all markers analyzed, whereas gynogenetic diploids are homozygous for centromeric markers, with a high probability of heterozygosity at distal loci.
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Fig. 3. Genotyping of triploid offspring. Triploid offspring are genotyped using polymorphic markers when both progenitors are heterozygous for different pairs of alleles (results for marker 033F11 are shown; see Table S1 for details). This type of marker allows identification of paternal (T139, heterozygous for alleles 1 and 3) and maternal (T290.5, heterozygous for alleles 2 and 4) contributions. Most triploid individuals (T354) will have three different alleles (one paternal and both maternal alleles), although triploids with two copies of one of the maternal alleles are also possible (*). The use of several markers differentiates diploids from triploids with two copies of the same allele.
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Fig. S3. Pedigrees and sex ratios of triploid and diploid siblings. (A) Pedigree and sex ratios of triploid and diploid siblings obtained from hybrid progenitors. The progenitors used in A were produced from interstrain crosses to obtain hybrids with high heterozygosity. The strain/stock of origin and proposed sex chromosome constitution are indicated (color-coded according to the strain/stock they come from; red: Nigerian; blue: Ivory Coast; green: PacBio; orange: Cam4/N hybrids). (B) Pedigree and sex ratios for triploids obtained from IC progenitors. The proposed sex chromosome constitutions are stated.
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Fig. 4. Sex ratios and sex-linked marker inheritance in interstrain pairings prove the existence of Y, W, and Z sex chromosomes. The pedigree chart of the interstrain pairings performed shows the sex of the offspring obtained in different spawns (P0 to P91). All-female offspring were observed in two types of crosses (labeled in red), both involving golden ZZ males (obtained from breeding sex-reversed ZZ females with ZZ males). These offspring are possible only if the mothers are WW. Without sex reversal or gynogenesis, WW females can be produced only if males with a W chromosome (YW) are bred with ZW or WW females. Because all hybrid N/TGA females analyzed were WW, the sex chromosome constitution of the progenitors used to produce these hybrids was YW (TGA male) and WW (NNB1 female). Other TGA and NNB1 males used in this pedigree have a Y chromosome, although the data do not distinguish YZ from YW. The proposed sex chromosome constitution for all the animals in the pedigree chart is depicted together with a color code that indicates the strain or provenance (green: TGA; red: Nigerian; blue: golden). SR indicates a sex-reversed individual obtained by ethynylestradiol exposure (see pedigree chart in Fig. S1).
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Fig. S4. Sex-linked marker in the offspring of cross X29. The genotype for a sex-linked marker is shown for the parents and offspring of cross X29. The father (M.Po1) is heterozygous for the alleles labeled as Z and Y (sample number 16 in upper row). The mother (T290.7) is heterozygous for alleles Z and W (sample number 15 in upper row). Phenotypic males are YZ (A: 1, 2, 4, 14, 15; B: 1, 3, 11), YW (A: 7, 8, 9, 10; B: 7, 8), or ZZ (A: 3, 5, 6, 11; B: 6, 12, 14). Phenotypic females can be only ZW (A: 12, 13, 15; B: 2, 4, 5, 10 and 13). A: upper row; B: lower row.
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