Figure 2. Chromosome preparations of the F₁ hybrid Trifolium nigrescens ssp. nigrescens x T. occidentale. (a) Giemsa-stained somatic metaphase cell (2n = 16). Eight larger chromosomes can be distinguished. Arrows indicate nucleolus organizer region (NOR)-bearing chromosomes. (b–f) Examples of fluorescence in situ hybridization (FISH) results. DNA probes are indicated with respective colors. Chromosomes are counterstained with DAPI. (b) A mitotic metaphase cell. Chromosomes with a secondary constriction (NOR) are indicated by arrows. (c) The same cell as in (b) after FISH with 5S and 18S rDNA sequences; FISH signals of the two probes on two NOR-bearing chromosomes are very prominent. A minute 5S rDNA signal on the short arm of a T. occidentale–derived chromosome is indicated by an arrowhead. (d) A mitotic prometaphase cell after FISH with TrR350. Five large centromeric FISH signals on T. occidentale-derived chromosomes are very evident. Some of the small signals are not clear. Dotted lines on two NOR-bearing chromosomes represent stretched secondary constrictions. (e) A meiotic diplotene cell shows eight bivalents and homeologous pairing of NOR-bearing chromosomes derived from the two parents. A T. occidentale–derived chromosome with a 5S signal (arrowhead) forms a bivalent, probably with a T. nigrescens-derived chromosome. (f) A meiotic pachytene cell with a single and prominent 18S rDNA signal denotes homeologous pairing of NOR-bearing chromosomes from the two parents. A minute 5S signal on a T. occidentale–derived chromosome is indicated by an arrowhead. Bar represents 10 µm.