Mitotic metaphase chromosome spreads and karyotypes of maternal backcross.

<p>(a) BC-M1 ((<i>Hoc*</i>/<i>Hag</i>) × <i>Hoc</i>) type 1, (b) large metacentric chromosomes of BC-M1 ((<i>Hoc*</i>/<i>Hag</i>) × <i>Hoc</i>) type 2, (c)) large metacentric chromosomes and microchromosome of BC-M1 ((<i>Hoc*</i>/<i>Hag</i>) × <i>Hoc</i>) type 3; (d) BC-M2 ((<i>Hoc*</i>/<i>Hot</i>) × <i>Hoc</i>) type 1, (e)) large metacentric chromosomes and microchromosome of BC-M2 ((<i>Hoc*</i>/<i>Hot</i>) × <i>Hoc</i>) type 2. Arrow heads showed large metacentric chromosomes. The remaining spreads of BC-M1 ((<i>Hoc*</i>/<i>Hag</i>) × <i>Hoc</i>) and BC-M2 ((<i>Hoc*</i>/<i>Hot</i>) × <i>Hoc</i>) were showed in S4 and S5, respectively.</p

Mitotic metaphase chromosome spread and karyotypes.

<p>(a) <i>H</i>. <i>octogrammus</i>, (b) F₁-hybrid (<i>Hoc</i> × <i>Hag</i>), (c) F₁-hybrid (<i>Hoc</i> × <i>Hot</i>). Spreads of F₁-hybrids were showed in S1.</p

The lineages of specimens used in this study.

<p>(a) artifial F₁-hybrids, (b) paternal backcross lineage, (c) maternal backcross lineage. All abbreviated species signs were referred to text. Yellow, light blue and purple of species signs indicate the <i>H</i>.<i>otakii</i>, <i>H</i>. <i>octogrammus</i> and <i>H</i>. <i>aggrammus</i> genome, respectively. Uncolored signs indicate species that not observed in this study.</p

Reproductive mode of maternal backcross (BC-M) inferred from genotyping offspring of (<i>Hoc</i> x BC-M; Cross 1–3) and (BC-M x <i>Hoc</i>; Cross 4 and 5) using microsatellite DNA.

<p>In BC-M retaining two <i>Hoc</i> genome sets, alleles (orange) from hemiclonal hybrids (grandmother: <i>Hoc/Hag</i>) were inherited by offspring after recombination. Alleles (blue) from fathers (<i>Hoc</i>) are colored to facilitate discrimination.</p

Mitotic metaphase chromosome spreads and karyotypes.

<p>(a) BC-M1 × <i>Hoc</i>; (b) <i>Hoc</i> × BC-M1.</p

Karyological observations of the three <i>Hexagrammos</i> species, and the natural hybrids and the artificial hybrids.

<p>Yellow cells indicate the most frequent chromosome numbers of each species and hybrid.</p

Mitotic metaphase chromosome spreads and karyotypes of maternal backcross.

<p>(a) BC-M1 ((<i>Hoc*</i>/<i>Hag</i>) × <i>Hoc</i>) type 1, (b) large metacentric chromosomes of BC-M1 ((<i>Hoc*</i>/<i>Hag</i>) × <i>Hoc</i>) type 2, (c)) large metacentric chromosomes and microchromosome of BC-M1 ((<i>Hoc*</i>/<i>Hag</i>) × <i>Hoc</i>) type 3; (d) BC-M2 ((<i>Hoc*</i>/<i>Hot</i>) × <i>Hoc</i>) type 1, (e)) large metacentric chromosomes and microchromosome of BC-M2 ((<i>Hoc*</i>/<i>Hot</i>) × <i>Hoc</i>) type 2. Arrow heads showed large metacentric chromosomes. The remaining spreads of BC-M1 ((<i>Hoc*</i>/<i>Hag</i>) × <i>Hoc</i>) and BC-M2 ((<i>Hoc*</i>/<i>Hot</i>) × <i>Hoc</i>) were showed in S4 and S5, respectively.</p

Schema of meiosis of natural hybrids and BC-M; (<i>Hoc*/Hag</i>) × <i>Hoc</i>.

<p>Figures of natural hybrids were cited from a putative description of chromosome elimination proposed by Ogielska (2009) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180626#pone.0180626.ref020" target="_blank">20</a>]. Interphase to Anaphase I is included in the first meiotic division and Anaphase II in the second meiotic division. Large chromosomes are composed of a set of two chromosomes. In natural hybrids, the large chromosome appears to be transmitted intact without recombination to the gametes. However, in BC-M, the large chromosome becomes fissured into two chromosomes after segregation and recombination of genomes from the female and the male.</p

A schematic illustration of development of sturgeon PGCs during embryonic development.

<p>(A) 1-cell stage. (B) Early cleavage stage. (C) Gastrula stage: a cross-section cut along animal-vegetal axis. (D) Neurula stage: a cross-section cut along animal-vegetal axis. (E) Closed neural tube stage: posterior lateral view. (F) 4-dpf embryo: lateral view. (G) 5-dpf embryo: lateral view. The regions colored gray show the position labeled by GFP or FITC-dextran injected at the vegetal pole of an early-stage embryo. The red regions in (A) and (B) show the embryonic area where germplasm was observed by TEM. Red dots in (C) and (D) are presumptive PGCs. PGCs were difficult to observe as a single cell, since the PGCs still remained in a group of GFP/FITC positive vegetal cells at this stage. Red dots in (E–G) are PGCs in migration. At stage (E), PGCs showed the crescent-like localization on the yolky cells around the posterior part of the embryonic body. Around stage (E) to (F), PGCs exhibited active movements with the cellular protrusions. After stage (G), however, most PGCs transformed into a round shape and were passively moved toward the gonadal ridge via the mesenchyme.</p

Expression of GFP-Buc protein in an embryo and observation of germplasm with transmission electron microscopy.

<p>(A) Expression of GFP-Buc protein at the animal pole. Insert indicates the diagram of this view. (B) The magnified image of the box in (A). (C) Expression of GFP-Buc protein at the vegetal pole. Insert indicates the diagram of this view. Yolky vegetal blastomeres are bigger than those of the animal hemisphere. (D) The magnified image of the box in (C). (E) Vegetal view of a blastula stage embryo injected with GFP-<i>buc</i> mRNA at the vegetal pole. Blastomeres were semi-dissociated in order to observe the expression of GFP-Buc protein in the embryo. (F) The magnified and fluorescent image of the boxed area in (E). Arrowheads indicate accumulations of GFP-Buc protein. Note that one accumulation is inherited by a small cell (red arrowhead). (G) Ultrastructure of embryo at the vegetal pole region at 8-cell stage. Mitochondrial cloud and “nuage” were observed near the cleavage furrow (arrows). (H) The magnified image of the box in (G). The “nuage” of the sturgeon appears as distinct amorphous inclusions without the surrounding membrane. This structure is very similar to that is seen in other species <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086861#pone.0086861-Herpin1" target="_blank">[20]</a>. C, Chorion; CF, cleavage furrow; YG, yolk granule. Scale bars in (C) and (E) indicate 500 µm, and 100 µm in (D) and (F); the scale bar in (G) indicates 5 µm, and 1 µm in (H).</p