Fluorescence in situ Hybridization Analysis of Oligonucleotide 5S Ribosomal DNA, 45S Ribosomal DNA, and (TTTAGGG)3 Locations in Gloriosa superba L

Abstract

Introduction: Gloriosa superba L. is a horticulturally and medicinally important plant native to Africa. However, the few cytogenetic studies of the species are mainly focused on chromosome counting and chromosome morphology-based karyotyping. Fluorescence in situ hybridization (FISH) is a powerful tool for the detection of DNA repetitive elements in a specific region of a chromosome.

Methods: Here, detailed karyotypes of G. superba were constructed by FISH using 5S and 45S rDNAs, and telomeric repeat (TTTAGGG)3 oligonucleotides.

Results and conclusion: Twenty-two chromosomes were observed. Two 5S rDNA hybridization signals were detected in the proximal regions of the short arms of one pair of chromosomes, which were adjacent to the (TTTAGGG)3 terminal signals. Four 45S rDNA signals were detected near the centromere region of the short arm of the four chromosomes, but one of these was very weak and almost undetectable compared to the others. Telomeric repeat hybridization signals were distributed at the terminal region of each chromosome. The chromosomes displayed were intact, and the chromosome counts were accurate. Chromosome length ranged from 3.46 to 9.31 μm. These results will facilitate the cytogenetic mapping of other major repeats, thus contributing to an improved understanding of the G. superba genome structure and evolutionary history.

Introduction: Gloriosa superba L. is a horticulturally and medicinally important plant native to Africa. However, the few cytogenetic studies of the species are mainly focused on chromosome counting and chromosome morphology-based karyotyping. Fluorescence in situ hybridization (FISH) is a powerful tool for the detection of DNA repetitive elements in a specific region of a chromosome.

Methods: Here, detailed karyotypes of G. superba were constructed by FISH using 5S and 45S rDNAs, and telomeric repeat (TTTAGGG)3 oligonucleotides.

Results and conclusion: Twenty-two chromosomes were observed. Two 5S rDNA hybridization signals were detected in the proximal regions of the short arms of one pair of chromosomes, which were adjacent to the (TTTAGGG)3 terminal signals. Four 45S rDNA signals were detected near the centromere region of the short arm of the four chromosomes, but one of these was very weak and almost undetectable compared to the others. Telomeric repeat hybridization signals were distributed at the terminal region of each chromosome. The chromosomes displayed were intact, and the chromosome counts were accurate. Chromosome length ranged from 3.46 to 9.31 μm. These results will facilitate the cytogenetic mapping of other major repeats, thus contributing to an improved understanding of the G. superba genome structure and evolutionary history.

Keywords: Fluorescence in situ hybridization; Gloriosa; Oligonucleotide probes; Ribosomal DNA; Telomere.

Fig. 1.

a–c Somatic metaphase chromosomes of G. superba after FISH using 5S rDNA (red), 45S rDNA (green), and Arabidopsis-type telomere ((TTTAGGG)₃) (red) oligonucleotide probes. a, b and c DAPI stained metaphase chromosome spreads (blue) (white arrows indicate the NOR sites); a1, b1, c1, a2, b2 and c2 45S rDNA signals (green), the chromosomes with a weak 45s rDNA signal are marked as green arrows; a2, b2 and c2 5S rDNA signals (red); a3, b3 and c3 (TTTAGGG)₃ signals (red). All scale bars, 10 μm. NOR, nucleolus organizer region.

Fig. 2.

FISH karyotyping of G. superba through hybridization with 5S rDNA (red signals, a, b and c), 45S rDNA (green signals, a, b and c), and Arabidopsis-type telomere ((TTTAGGG)₃) (red signals, a′, b′ and c′) oligonucleotides. All chromosomes are captured from Fig. 1. For example, the chromosomes in a and a′ are from Figure 1a1, a2, and a3, respectively. Because weak 45S rDNA signals were easily obscured after image composition, chromosomes (orange) with the hybridization signal are included as show in a, b and c. The chromosomes were aligned by length (from the longest to shortest) and signal pattern (a–c′). The left/right and bottom numbers indicate chromosome lengths and chromosome counts (a, b and c), respectively.