Roco ÁS et al. (2021), Comparative Distribution of Repetitive Sequence...

XB-ART-58052

Genes (Basel) 2021 Apr 21;125:. doi: 10.3390/genes12050617.

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Comparative Distribution of Repetitive Sequences in the Karyotypes of Xenopus tropicalis and Xenopus laevis (Anura, Pipidae).

Roco ÁS , Liehr T , Ruiz-García A , Guzmán K , Bullejos M .

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Xenopus laevis and its diploid relative, Xenopus tropicalis, are the most used amphibian models. Their genomes have been sequenced, and they are emerging as model organisms for research into disease mechanisms. Despite the growing knowledge on their genomes based on data obtained from massive genome sequencing, basic research on repetitive sequences in these species is lacking. This study conducted a comparative analysis of repetitive sequences in X. laevis and X. tropicalis. Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) with Cot DNA of both species revealed a conserved enrichment of repetitive sequences at the ends of the chromosomes in these Xenopus species. The repeated sequences located on the short arm of chromosome 3 from X. tropicalis were not related to the sequences on the short arm of chromosomes 3L and 3S from X. laevis, although these chromosomes were homoeologous, indicating that these regions evolved independently in these species. Furthermore, all the other repetitive sequences in X. tropicalis and X. laevis may be species-specific, as they were not revealed in cross-species hybridizations. Painting experiments in X. laevis with chromosome 7 from X. tropicalis revealed shared sequences with the short arm of chromosome 3L. These regions could be related by the presence of the nucleolus organizer region (NOR) in both chromosomes, although the region revealed by chromosome painting in the short arm of chromosome 3L in X. laevis did not correspond to 18S + 28S rDNA sequences, as they did not colocalize. The identification of these repeated sequences is of interest as they provide an explanation to some problems already described in the genome assemblies of these species. Furthermore, the distribution of repetitive DNA in the genomes of X. laevis and X. tropicalis might be a valuable marker to assist us in understanding the genome evolution in a group characterized by numerous polyploidization events coupled with hybridizations.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: crot

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	Figure 1. GISH and FISH with Cot DNA. Metaphase chromosomes from X. tropicalis (A,C,E,G) and X. laevis (B,D,F,H) hybridized using, as a probe: (A,B) genomic DNA from X. tropicalis (female), (C,D) genomic DNA from X. laevis (male), (E,F) Cot DNA from X. tropicalis (female) and (G,H) Cot DNA from X. laevis (male). All metaphases were derived from female individuals. All probes were labeled with biotin 11–dUTP, and three rounds of amplification were used during immunological detection. The arrowheads point to the NOR. Chromosome XTR8 is mounted in C. Scale: 5 µm.
	Figure 2. Analysis of X. tropicalis chromosomes in female samples. (A) DAPI-stained metaphase from X. tropicalis. (B) Ag-NOR staining revealing active NOR located on the secondary constriction of chromosome 7. (C,D) Triple staining using CMA3/DA/DAPI. (C) Metaphase spread from X. tropicalis using DAPI filter; (D) the same metaphase using a FITC filter. The arrow points to the secondary constrictions on pair 7 (NOR). The asterisk (*) signals the short arm of XTR3. (E) In situ hybridization on X. tropicalis metaphase chromosomes using X. tropicalis SpectrumGreen-labeled Cot-20 DNA as a probe (direct detection). (F) In situ hybridization using a telomeric probe, labeled with biotin 11–dUTP (after three rounds of amplification). Scale: 5 µm.
	Figure 3. Chromosome painting with XTR-7 probes on X. tropicalis (A,B) and X. laevis (C,D) metaphase chromosomes from female samples, using two painting probes from X. tropicalis labeled with Texas Red–dUTP: XTR-7w (A,C) or XTR-7p (B,D). The insert in A and B shows chromosome 7 from X. tropicalis at a higher magnification after hybridization with XTR-7w or XTR-7p, respectively. Note the absence of hybridization signal on the secondary constriction (NOR) of chromosome 7. The arrowhead points to the centromere; the arrow points to the NOR. The signals observed are from direct fluorescence. Scale: 5 µm.
	Figure 4. FISH with rDNA probe vs chromosome painting with XTR-7. Metaphase chromosomes from female X. tropicalis (A) and male X. laevis (B) samples, hybridized with an rDNA probe (18S + 28S) labeled with Texas Red–dUTP. (C–F) Detail of chromosome 3L from X. laevis hybridized with the rDNA probe (C,D) and with the painting probe XTR-7w (E,F). In all cases, the stained chromosome was compared with the image of the same chromosome stained only with DAPI. The image comparison revealed that the hybridization signal with the ribosomal DNA probe, which was located in the region of the secondary constriction, did not coincide with the hybridization signal when XTR-7w was used as a probe, which was located at the end of the short arm of the chromosome. All signals correspond to direct fluorescence. Complete metaphases corresponding to Figure 4C–F are depicted in Figure S5. Scale: 5 µm.
	Figure 5. Summary of the results obtained by GISH, FISH and chromosome painting comparing the signals observed in the karyotypes of X. tropicalis and X. laevis. The probes used and the color codes are indicated in the box. (*) Alternative possibilities for a positive signal identified by GISH/Cot with chromosomes and probes from X. laevis (represented only in XLA3S; according to chromosome morphology could be on XLA4L instead). The size and shape of the chromosomes of both species are based on the ideograms by [28], but the nomenclature follows [9] for X. tropicalis and [32] for X. laevis.

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