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FISH mapping in Xenopus pygmaeus refines understanding of genomic rearrangements and reveals jumping NORs in African clawed frogs.
Bergelová B
,
Gvoždík V
,
Knytl M
.
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Chromosomal rearrangements are fundamental evolutionary drivers leading to genomic diversification. African clawed frogs (genus Xenopus, subgenera Silurana and Xenopus) represent an allopolyploid model system with conserved chromosome numbers in species with the same ploidy within each subgenus. Two significant interchromosomal rearrangements have been identified: a translocation between chromosomes 9 and 2, found in subgenus Silurana, and a fusion between chromosomes 9 and 10, probably widespread in subgenus Xenopus. Here, we study the allotetraploid Xenopus pygmaeus (subgenus Xenopus) based on in-depth karyotype analysis using chromosome measurements and fluorescent in situ hybridization (FISH). We designed FISH probes for genes associated with translocation and fusion to test for the presence of the two main types of rearrangements. We also examined the locations of 5S and 28S ribosomal tandem repeats, with the former often associated with telomeric regions and the latter with nucleolus organizer regions (NORs). The translocation-associated gene mapping did not detect the translocation in X. pygmaeus, supporting the hypothesis that the translocation is restricted to Silurana, but instead identified a pericentromeric inversion on chromosome 2S. The fusion-associated gene mapping confirmed the fusion of chromosomes 9 and 10, supporting this fusion as an ancestral state in subgenus Xenopus. As expected, the 5S repeats were found predominantly in telomere regions on almost all chromosomes. The nucleolar 28S repeats were localized on chromosome 6S, a position previously found only in the closely related species X. parafraseri, whereas other, phylogenetically more distant species have NORs located on different chromosomes. We therefore hypothesize that a jumping mechanism could explain the relatively frequent changes in the location of NORs during Xenopus evolution.
186024 Grantová Agentura, Univerzita Karlova (Charles University Grant Agency), 54123 Grantová Agentura, Univerzita Karlova (Charles University Grant Agency), DKRVO 2024-2028/6.I.a, National Museum of the Czech Republic 00023272 Ministerstvo Kultury (Ministry of Culture, Czech Republic)
Fig. 1. Analysis of measurements and statistics of the length of short and long arms of X. pygmaeus chromosomes.The L-subgenome is shown in blue and the S-subgenome in red. A Relationship between centromeric index (i), x-axis, and chromosome length (l), y-axis. Black dashed vertical lines delineate the intervals 0–12.5, 12.5–25, 25–37.5, and 37.5–50, corresponding to acrocentric, subtelocentric, submetacentric, and metacentric chromosomes, respectively. The plotted values of i and l are medians for each chromosome. Panel (B) shows intrachromosomal variation of l value (y axis) for the haploid complement of 18 X. pygmaeus chromosomes (x axis). C Intrachromosomal variability of i value (y axis) for the haploid complement of 18 X. pygmaeus chromosomes (x axis). B, C Black dashed vertical lines define pairs of homeologous chromosomes. Upper and lower whiskers show minimum and maximum values, respectively; boxes involve the lower (Q1) and upper (Q3) quartiles; horizontal lines inside the boxes indicate the median values (Q2); outliers are indicated by blue (for L) and red (for S) points above and below the whiskers. Significance codes for l and i values at the top of (B, C) define whether pairs of homeologous chromosomes are significantly different based on ANOVA and Tukey’s tests. Significantly different homeologs are depicted by significance codes ***, **, and * showing p values of p < 0.001, p < 0.01, and p < 0.05, respectively.
Fig. 2. Mapping of ribosomal genes using FISH on metaphase spread in X. pygmaeus, sample XPYTaF1-1.A DAPI (black and white, B&W) counter-stained metaphase spread shows all 36 chromosomes. B FISH with the 28S rDNA probe (B&W), labeled with digoxigenin, reveals a signal on the long arm of chromosome 6S, indicating a nucleolar secondary constriction. C FISH with the 5S rDNA probe (B&W), labeled with biotin, highlights nearly all chromosomes in X. pygmaeus except chromosome 8S. D Merged metaphase (RGB) with DAPI (blue-purple) stain, 28S (red), and 5S (green) signals. Scale bars represent 10 μm. E Karyotype (RGB) arranged according to the l and i values. White lines indicate the position of the centromere.
Fig. 3. Mapping of single-copy genes by FISH-TSA with positive red signals on X. pygmaeus chromosomes (XPY), sample XPYTaF1-1.Each of the cept1 and gyg2 probes map to both X. pygmaeus chromosomes 2L and 2S. The fn1.L, ndufs1.L, sf3b1.S and nomo3.L loci are localized on the q arm of X. pygmaeus chromosomes 9_10L, 9_10L, 9_10S and 9_10L, respectively. The bmp7.L and sox9.L are mapped on the p arm of X. pygmaeus chromosomes 9_10L. White lines indicate the position of the centromere.
Fig. 4. Locations of selected single-copy genes in four Xenopus species and phylogenetic relationships of these species with respect to NOR locations.The gene positions in X. tropicalis and X. laevis were taken from genome databases (Bredeson et al. 2024; Session et al. 2016). Gene localizations in X. mellotropicalis and X. pygmaeus were adopted from Knytl et al. (2018) and this study, respectively. Translocation-associated genes (cept1, gyg2, fn1, ndufs1, and sf3b1) are shown in subgenera Silurana (A) and Xenopus (B). A In X. tropicalis (Silurana), genes are considered to have ancestral positions. In X. mellotropicalis, the sf3b1 gene was translocated from chromosome 9b to pericentromeric region of chromosome 2b (indicated by dashed arrow and described in red font). B In X. laevis, translocation-associated genes cept1 and gyg2 were annotated on one homeologous chromosome each, specifically on chromosome 2S and 2L, respectively. The remaining translocation-associated genes fn1, ndufs1, and fn1 were annotated on both homeologous chromosomes 9_10L and 9_10S. In contrast, we mapped all translocation-associated genes on both homeologous chromosomes in X. pygmaeus. In addition, positions of the cept1 and gyg2 genes were inverted on chromosome 2S (indicated by dashed arrows and described in red font). C Fusion-associated genes (fn1, ndufs1, sf3b1, nomo3, bmp7, and sox9) depicted on X. tropicalis chromosomes 9 and 10, and X. laevis and X. pygmaeus chromosomes 9_10L and 9_10S. The order of the fusion-associated genes in these three species revealed that the fusion between chromosomes 9 and 10 is shared in X. laevis and X. pygmaeus. D A schematic phylogenetic tree showing variation in the NOR positioning. Two species from subgenus Silurana (X. tropicalis, X. mellotropicalis) and two species from subgenus Xenopus (X. pygmaeus, X. laevis) are shown for clear understanding of evolution by rearrangements depicted in A, B, and C. Chromosomes in parentheses associated with each species are NOR-carrying chromosomes (this study; Gvoždík et al. 2024; Knytl et al. 2017; Tymowska 1991). Hymenochirus sp. was used as an outgroup. A–C Created with BioRender.com, (D): created with Geneious Prime and modified in Adobe Photoshop.
