Fig 1.
The karyotype of Strigamia maritima.
(A) Inverted image of a DAPI-stained mitotic metaphase prepared from embryonic material, of which the sex was unknown (left panel). The constructed karyotype (right panel) is a representative example of karyotypes prepared from at least 10 other embryos. It shows a large pair of metacentric chromosomes and seven middle to small acrocentric pairs, gradually decreasing in size. All karyotypes give qualitatively the same result and none of them show evidence for a heteromorphic sex chromosome pair. Scale bar = 10 μm. (B) Comparative genomic hybridization (CGH) on a spermatogonial mitotic metaphase prepared from the testes of a sub-adult male (left panel). Chromosomes were counterstained with DAPI (blue). Female-derived genomic probe was labelled with fluorescein-12-dUTP (green) and male-derived genomic probe with Cy3-dUTP (red). Both probes highlighted one arm of the two large metacentric chromosomes (arrows) and the centromeric heterochromatin of all chromosomes (asterisks), but did not differentiate a sex chromosome pair as demonstrated in the constructed karyotype (right panel). Scale bar = 10 μm.
Fig 2.
Identification and validation of X- and Y-linked genomic scaffolds.
(A) Plot of the fold difference in coverage of each genomic scaffold between two female individuals (blue) or a male and a female individual (red), in genome re-sequencing data. Fold difference for each scaffold is shown as a logarithm to base 2. The plot reveals a number of scaffolds with different patterns of abundance in the two sexes. The regions of the plot containing scaffolds over- or underrepresented in males are indicated with brackets on the right. Only scaffolds 10 kb or more in length are shown. The scaffolds are ordered by length along the horizontal axis, with largest scaffolds on the left. Scaffolds independently validated as being located on the X or Y chromosome are indicated with arrowheads or arrows, respectively. (B) Quantitative PCR confirms that three tested genomic scaffolds are present at approximately twice the copy number in females as in males. The Y-axis is the mean ratio of CT value of the candidate scaffold to the CT value of the autosomal reference scaffold (for scaffolds 1–3: males n = 12, females n = 9; for scaffold 4: males n = 9, females n = 8). Error bars represent the standard error of the mean (SEM). Student’s t test * P<0.05; ** P<0.001; n.s. = not significant. Scaffolds 1–3 are scf7180001248200, scf7180001248049 and scf7180001247190, respectively; the control scaffold is scf7180001247533. (C) Identification of six male-specific genomic scaffolds. Primers designed against these 6 scaffolds only amplify a PCR product from male genomic DNA samples. In contrast, a PCR against an autosomal scaffold amplifies a product in both sexes (control). None of the primer sets amplified a product with water as the template (NTC = no template control). Scaffolds 1–6 are scf7180001247258, scf7180001245067, scf7180001243011, scf7180001247286, scf7180001247324 and scf7180001247297, respectively; the control scaffold is scf7180001247533. Original, uncropped gels are provided in S2 Fig. The raw data for this figure are provided in S2 Table.
Fig 3.
Identification of the X chromosome in the Strigamia karyotype by FISH with a set of X-chromosome derived DNA probes.
(A) The relative positions and sizes of the five PCR fragments, distributed on two different X-linked scaffolds, which were used to produce the X-probes. Two of the fragments are located on scf7180001248200 and three on scf7180001248049. The genomic distance between these scaffolds is not known. The black line depicts the scaffold. The numbers above the line indicate the number of bases, starting at 1 on the left hand side. Green boxes represent the length and relative position of the PCR products, numbered arbitrarily from 1 to 5. (B) A mitotic metaphase chromosome spread prepared from a single embryo. Hybridization signals of the X-probes identify a middle-sized element in the Strigamia karyotype as the X chromosome. As there are two chromosomes with the X-probe signals, we infer that this chromosome spread is derived from a female embryo (XX). (C) Two Strigamia karyotypes constructed from the mitotic metaphases of embryonic cells. They are derived from different embryos. Upper panel: karyotype derived from the female metaphase shown in (B). Lower panel: karyotype derived from an inverted image of a DAPI-stained metaphase of unknown sex. It is the same as that shown in Fig 1A. We infer that the pair of sex chromosomes represents the 4th pair of chromosomes by size (asterisks). (D, E, F) Meiotic chromosome spreads, prepared from sub-adult male testes. (D) Late zygotene complement showing a clump of incompletely paired bivalents. The X-probes label the longer chromosome of a partially paired bivalent, as schematically illustrated in (D’). We thus infer that this is the X chromosome, and that the other shorter chromosome, without hybridization signals, is the Y chromosome. The X and Y chromosomes are only paired at the distal part of the X chromosome, with a large proximal part unpaired. (E) A particularly clear and well-spread XY bivalent at a similar stage to (D). It shows hybridization signals of X-probes on the unpaired proximal part of the X chromosome, while the Y chromosome is completely paired except for the DAPI-highlighted centromere (see schematic drawing below the XY bivalent). (F) Pachytene complement showing 8 bivalents, each with DAPI-highlighted centromeric chromatin. X-probe hybridization signals are visible on the unpaired segment of the longer chromosome, near the centromere (see schematic drawing on the right-hand side). The X and Y chromosomes now appear almost equal in length in the bivalent. Scale bar is equal to 5 μm in (B) and 10 μm in (D, E, F). Chromosomes were counterstained with DAPI (blue). Arrowheads indicate hybridization signals of the digoxigenin-labelled X-probes (green); arrows indicate a pair of the largest chromosomes (B) or the largest bivalent (F).