Chromosomal locations of <i>S. triporcatus</i> homologs of 16 chicken Z-linked genes in male <i>S. triporcatus</i>.

<p>(A, B) FISH pattern of <i>NFIB</i> on PI-stained metaphase spread (A) and Hoechst-stained pattern of the same metaphase spread (B). (C–Z, A’–F’) FISH signals of <i>TMOD</i> (C, D), <i>ACO1</i> (E, F), <i>RNF20</i> (G, H), <i>DMRT1</i> (I, J), <i>RPS6</i> (K, L), <i>NTRK2</i> (M, N), <i>SPIN</i> (O, P), <i>FER</i> (Q, R), <i>CHD1</i> (S, T), <i>HMGCR</i> (U, V), <i>KIF2A</i> (W, X), <i>GHR</i> (Y, Z), <i>ATP5A1</i> (A’, B’), <i>NARS</i> (C’, D’), and <i>VCP</i> (E’, F’) on PI-stained X and Y chromosomes. Arrows indicate the hybridization signals of the genes. Scale bars represent 10 µm (A, B) and 2.5 µm (C–Z, A’–F’).</p

The <i>Staurotypus</i> Turtles and Aves Share the Same Origin of Sex Chromosomes but Evolved Different Types of Heterogametic Sex Determination

<div><p>Reptiles have a wide diversity of sex-determining mechanisms and types of sex chromosomes. Turtles exhibit temperature-dependent sex determination and genotypic sex determination, with male heterogametic (XX/XY) and female heterogametic (ZZ/ZW) sex chromosomes. Identification of sex chromosomes in many turtle species and their comparative genomic analysis are of great significance to understand the evolutionary processes of sex determination and sex chromosome differentiation in Testudines. The Mexican giant musk turtle (<i>Staurotypus triporcatus</i>, Kinosternidae, Testudines) and the giant musk turtle (<i>Staurotypus salvinii</i>) have heteromorphic XY sex chromosomes with a low degree of morphological differentiation; however, their origin and linkage group are still unknown. Cross-species chromosome painting with chromosome-specific DNA from Chinese soft-shelled turtle (<i>Pelodiscus sinensis</i>) revealed that the X and Y chromosomes of <i>S</i>. <i>triporcatus</i> have homology with <i>P. sinensis</i> chromosome 6, which corresponds to the chicken Z chromosome. We cloned cDNA fragments of <i>S</i>. <i>triporcatus</i> homologs of 16 chicken Z-linked genes and mapped them to <i>S</i>. <i>triporcatus</i> and <i>S</i>. <i>salvinii</i> chromosomes using fluorescence in situ hybridization. Sixteen genes were localized to the X and Y long arms in the same order in both species. The orders were also almost the same as those of the ostrich (<i>Struthio camelus</i>) Z chromosome, which retains the primitive state of the avian ancestral Z chromosome. These results strongly suggest that the X and Y chromosomes of <i>Staurotypus</i> turtles are at a very early stage of sex chromosome differentiation, and that these chromosomes and the avian ZW chromosomes share the same origin. Nonetheless, the turtles and birds acquired different systems of heterogametic sex determination during their evolution.</p></div

The cDNA fragments of ostrich (<i>S. camelus</i>, SCA) homologs of chicken Z-linked genes and nucleotide sequence identities among <i>S. triporcatus</i> (STR), ostich and chicken (<i>Gallus gallus</i>, GGA) cDNA fragments.

a<p><i>ACO1</i>, aconitase 1, soluble; <i>ATP5A1</i>, ATP synthase, H⁺ transporting, mitochondrial F1 complex, alpha subunit, isoform 1, cardiac muscle; <i>CHD1</i>, chromodomain helicase DNA binding protein 1; <i>DMRT1</i>, <i>doublesex</i> and <i>mab-3</i> related transcription factor 1; <i>FER</i>, (fps/fes related) tyrosine kinase; <i>GHR</i>, growth hormone receptor; <i>HMGCR</i>, 3-hydroxy-3-methylglutaryl-CoA reductase; <i>KIF2A</i>, kinesin heavy chain member 2A; <i>NARS</i>, asparaginyl-tRNA synthetase; <i>NFIB</i>, nuclear factor I/B; <i>NTRK2</i>, neurotrophic tyrosine kinase receptor, type 2; <i>RNF20</i>, ring finger protein 20, E3 ubiquitin protein ligase; <i>RPS6</i>, ribosomal protein S6; <i>SPIN</i>, spindlin; <i>TMOD</i>, tropomodulin 1; <i>VCP</i>, valosin containing protein.</p>b<p>The number in parenthesis indicates the number of identical bases/the number of bases in the overlapped region between cDNA fragments of two species.</p>c<p>The nucleotide sequences were obtained from Tsuda et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105315#pone.0105315-Tsuda1" target="_blank">[27]</a>.</p>d<p>The nucleotide sequence was obtained from Ishijima et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105315#pone.0105315-Ishijima1" target="_blank">[18]</a>.</p

The cDNA fragments of <i>S. triporcatus</i> (STR) homologs of chicken Z-linked genes and nucleotide sequence identities between <i>S. triporcatus</i> and chicken (<i>Gallus gallus</i>, GGA) cDNA fragments.

a<p><i>ACO1</i>, aconitase 1, soluble; <i>ATP5A1</i>, ATP synthase, H⁺ transporting, mitochondrial F1 complex, alpha subunit, isoform 1, cardiac muscle; <i>CHD1</i>, chromodomain helicase DNA binding protein 1; <i>DMRT1</i>, <i>doublesex</i> and <i>mab-3</i> related transcription factor 1; <i>FER</i>, (fps/fes related) tyrosine kinase; <i>GHR</i>, growth hormone receptor; <i>HMGCR</i>, 3-hydroxy-3-methylglutaryl-CoA reductase; <i>KIF2A</i>, kinesin heavy chain member 2A; <i>NARS</i>, asparaginyl-tRNA synthetase; <i>NFIB</i>, nuclear factor I/B; <i>NTRK2</i>, neurotrophic tyrosine kinase receptor, type 2; <i>RNF20</i>, ring finger protein 20, E3 ubiquitin protein ligase; <i>RPS6</i>, ribosomal protein S6; <i>SPIN</i>, spindlin; <i>TMOD</i>, tropomodulin 1; <i>VCP</i>, valosin containing protein.</p>b<p>The number in parenthesis indicates the number of identical bases/the number of bases in the overlapped region between cDNA fragments of two species.</p

Giemsa-stained karyotypes of male <i>S. triporcatus</i> and <i>S. salvinii</i>.

<p>(A) <i>S. triporcatus.</i> (B) <i>S. salvinii.</i> The X and Y chromosomes have large and small secondary constrictions, respectively. Scale bars = 10 µm.</p

Chromosome painting with chromosome 6-specific DNA probe of <i>P. sinensis</i> to metaphase spread of male <i>S. triporcatus</i>.

<p>(A) The probe painted the X and Y chromosomes on PI-stained metaphase spread of <i>S. triporcatus</i> (indicated by arrows). (B) Hoechst-stained pattern of the same metaphase spread as in (A). Scale bar = 10 µm.</p

C-banded metaphase spreads of male <i>S. triporcatus</i> and <i>S</i>. <i>salvinii</i>.

<p>(A) <i>S. triporcatus.</i> (B) <i>S. salvinii.</i> (C, D) Enlarged photographs of the X and Y chromosomes of <i>S. triporcatus</i> (C) and <i>S</i>. <i>salvinii</i> (D). Scale bars = 10 µm.</p

Chromosomal distribution of the 18S-28S rRNA genes and NORs on metaphase spreads of male <i>S</i>. <i>triporcatu</i>s and <i>S</i>. <i>salvinii</i>.

<p>(A–C) <i>S</i>. <i>triporcatu</i>s. (D–F) <i>S</i>. <i>salvinii</i>. FISH signals of the 18S–28S rRNA genes were localized to the secondary constrictions of the X and Y chromosomes (indicated by arrows), one of the copies of chromosome 2 (an arrowhead), and a pair of microchromosomes (a circle) in <i>S</i>. <i>triporcatus</i> (A), and the secondary constrictions of the X and Y chromosomes in <i>S</i>. <i>salvinii</i> (D). Ag-stained NORs were also distributed in the secondary constrictions of the X and Y chromosomes in <i>S</i>. <i>triporcatus</i> (C) and <i>S</i>. <i>salvinii</i> (F). However, no NORs were detected on chromosome 2 and a pair of microchromosomes in <i>S</i>. <i>triporcatus</i>, where the FISH signals of the rRNA genes were detected. (B, E) Hoechst-stained patterns of the same PI-stained metaphase spreads (A) and (D), respectively. Scale bars = 10 µm.</p

List of 86 cDNA clones mapped to the Hokou gecko (<i>Gekko hokouensis</i>) chromosomes and their chromosomal locations in the sand lizard (<i>Lacerta agilis</i>), the water monitor lizard (<i>Varanus salvator macromaculatus</i>), the butterfly lizard (<i>Leiolepis reevesii rubritaeniata</i>), the Japanese four-striped rat snake (<i>Elaphe quadrivirgata</i>), the green anole (<i>Anolis carolinensis</i>), and the chicken (<i>Gallus gallus</i>).

<p>^aNucleotide sequences of two accession numbers were determined separately by forward and reverse primers in one clone.</p><p>^bThe cDNA fragment were obtain from <i>G</i>. <i>hokouensis</i>, which were mapped in our previous study (Kawai et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134829#pone.0134829.ref039" target="_blank">39</a>]). For mapping of <i>ATP5A1</i>, <i>ACO1/IREBP</i> and <i>CHD1</i>, total length of cDNA fragment concatenated with multiple</p><p>–: No data</p><p>List of 86 cDNA clones mapped to the Hokou gecko (<i>Gekko hokouensis</i>) chromosomes and their chromosomal locations in the sand lizard (<i>Lacerta agilis</i>), the water monitor lizard (<i>Varanus salvator macromaculatus</i>), the butterfly lizard (<i>Leiolepis reevesii rubritaeniata</i>), the Japanese four-striped rat snake (<i>Elaphe quadrivirgata</i>), the green anole (<i>Anolis carolinensis</i>), and the chicken (<i>Gallus gallus</i>).</p

Chromosomal locations of the 18S–28S and 5S rRNA genes and (TTAGGG)n sequences in female <i>Gekko hokouensis</i>.

<p>(<b>a</b>) Hybridization pattern of FITC-labeled 18S–28S rRNA genes (green) and rhodamine-labeled 5S rRNA genes (red). Arrows indicate FISH signals of the 18S–28S rRNA genes, and arrowheads indicate signals of the 5S rRNA genes. (<b>b</b>) Hybridization pattern of FITC-labeled TTAGGG repeats (green) and rhodamine-labeled 5S rRNA genes (red). Arrows indicate signals of interstitial telomeric sites, and arrowheads indicate signals of the 5S rRNA genes. Scale bars represent 10 μm.</p