Pollen meiosis in synthetic <i>T. mirus.</i>

<p>(A,B) Feulgen staining showing in (A) 12 regular bivalents and (B) one quadrivalent (arrow) and bivalents in close association with each other and a nucleolus. (C–H). Fluorochrome colours and GISH probes are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003353#pone-0003353-g002" target="_blank">Figure 2</a>. (C–D) Note chromosome A^du in association with the nucleolus. Two bivalents, one from the <i>T. dubius</i> genome (yellow) and one from the <i>T. porrifolius</i> genome (orange), overlapped (arrow), perhaps occurring as a multivalent. (E–F). Note the rDNA and the bivalents carrying these genes associated with the nucleolus. A quadrivalent with two chromosomes of <i>T. dubius</i> origin and two of <i>T. porrifolius</i> origin occurring in a ring (arrow). (G–H) Note two bivalents (A^du and D^po) with rDNA associated with the nucleolus and the A^po bivalent with condensed rDNA unassociated with the nucleolus. N = nucleolus. Scale bar (C–H) is 10 µm.</p

Karyotype organisation of individuals of <i>T. miscellus</i> from two populations (localities of each population are indicated in column 1).

<p>The parental origins of the chromosomes were determined by GISH. Chromosome nomenclature of homeologous groups (A–F) followed Ownbey and McCollum <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003353#pone.0003353-Ownbey2" target="_blank">[25]</a> and Pires <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003353#pone.0003353-Pires1" target="_blank">[24]</a>. Superscript letters indicate the genome origins of the chromosomes, du = <i>T. dubius</i>, pr = <i>T. pratensis</i>. Chromosomes carrying translocations are indicated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003353#pone-0003353-t001" target="_blank">Table 1</a>. E-indicates parental chromosome set as expected from the diploid parent.</p

Karyotype organisation of individuals of <i>T. mirus</i> from three populations (localities of each population are indicated in column 1).

<p>The parental origins of the chromosomes were determined by GISH. Chromosome nomenclature of homeologous groups (A–F) followed Ownbey and McCollum <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003353#pone.0003353-Ownbey2" target="_blank">[25]</a> and Pires <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003353#pone.0003353-Pires1" target="_blank">[24]</a>. Superscript letters indicate the genome origins of the chromosomes, du = <i>T. dubius</i>, po = <i>T. porrifolius</i>. Chromosomes carrying translocations are indicated by naming the chromosome according to the genomic origins of the centromeres, followed by the genome origins of the translocated segments. E-indicates chromosome set as expected from the diploid parents.</p

Genomic origin of decondensed rDNA compared with previously published data from Kovarik <i>et al.</i>[22] and Matyasek <i>et al.</i>[23] using genomic–cleaved amplified polymorphic sequence (g-CAP) and reverse transcription-cleaved amplified polymorphic sequence (RT-CAP) to determine the genomic origin of rDNA sequences in the genome and the cDNA, respectively.

<p>Ns–FISH image not shown</p

(A-C) Karyotype analyses of <i>T. mirus</i> from individuals in three populations (A) 2601, (B) 2603 and (C) 2602.

<p>Homeologous chromosome group nomenclature (A-F) follows Ownbey and McCollum <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003353#pone.0003353-Ownbey2" target="_blank">[25]</a> and Pires <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003353#pone.0003353-Pires1" target="_blank">[24]</a>. Fluorochrome colours: yellow/green (FITC, digoxigenin-labelled probes), orange/red (Cy3, biotin-labelled probes), blue (DAPI staining). Each karyotype is shown with DAPI staining, sometimes also simultaneously labelled for 45S rDNA (yellow fluorescence) or 5S rDNA (red fluorescence), and after GISH with <i>T. dubius</i> (green fluorescence) and <i>T. porrifolius</i> (red fluorescence) total genomic DNA probes. Monosomic or trisomic chromosomes are underlined. Note: (1) intergenomic translocations to chromosome C^po/du (arrows) in individuals 2601-7 and 2601-8 and D^du/po in individual 2602-0-3-4 (A). (2) The 45S rDNA locus on the two homologues of chromosome A^du in individuals 2602-4 and 2602-0-3-4 show different levels of decondensation, one being condensed and the other with a secondary constriction (C). (3) A large reduction in size of the 45S rDNA locus on chromosome A^du of individual 2603-33A (#) compared with other individuals (D). (4) No secondary constrictions of 45S rDNA loci on <i>T. porrifolius</i> origin chromosomes (A^po, D^po) in individual 2603-33B. (D-E) Root-tip interphase nuclei of <i>T. mirus</i> after FISH with digoxigenin-labelled pTa71 for 45S rDNA. (D) Individual 2601-4, showing four large condensed 45S rDNA loci (arrows) and (E) Individual 2602-0-3-10 with two large condensed 45S rDNA loci (arrows). Scale bar (top right) is 10 µm.</p

Analyses of root tip metaphases of synthetic <i>T. mirus</i> (A, B is plant 73-14; C-E is plant 134-16-3).

<p>(A, C) Metaphase (merged images) after FISH for 45S rDNA (red) and 5S rDNA (green) and counterstained with DAPI (blue). (B) and (D) Metaphases (merged images) from (A) and (C), respectively, after reprobing using GISH with labeled genomic DNA from <i>T. porrifolius</i> (pink) and <i>T. dubius</i> (green) and counterstained with DAPI. In (B) note the particularly there are only four 45S rDNA sites, the sites on A^du. Chromosomes are particularly large (arrows) and the site expected on chromosome D^po is missing. In (D) note the <i>T. porrifolius</i> translocation to a <i>T. dubius</i> chromosome (red arrow) and the satellites to both A^du homologues are distant from the rest of the chromosome (see connecting white lines). (E) Karyotype of (D) revealing that the <i>T. porrifolius</i> origin translocation is to chromosome C^du and that no <i>T. porrifolius</i> chromosomes lack chromatin. Scale bar is 10 µm.</p

Genomic analysis of 5S rDNA repeats.

<p>(A) Southern blot hybridisation of 5S rDNA probe to <i>Taq</i>I-digested genomic DNA of the progeny of 2602-0-3 individual and diploid parental accessions. (B) Quantitative representation of the parental 5S gene families determined by a phosphorimager scanning of radioactivity signals on blots (three independent experiments).</p

(A–C) Karyotype analyses of <i>T. miscellus</i> from two populations (A) 2604 and (B) 2605.

<p>Homeologous chromosome group nomenclature (A–F) follows Ownbey and McCollum <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003353#pone.0003353-Ownbey2" target="_blank">[25]</a> and Pires <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003353#pone.0003353-Pires1" target="_blank">[24]</a>. Fluorochrome colours and probes are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003353#pone-0003353-g002" target="_blank">Figure 2</a> except that biotinylated <i>T. pratensis</i> genomic DNA was used in GISH experiments. Monosomic and trisomic chromosomes are underlined. Note in (A) that there is a large intergenomic translocation, chromosome A^du/pr (arrow). Scale bar (top right) is 10 µm.</p