Consequences of polyploidy and divergence as revealed by cytogenetic mapping of repetitive elements in African clawed frogs (Xenopus, Pipidae)

doi:10.21203/rs.3.rs-2487507/v1

Download PDF

Research Article

Consequences of polyploidy and divergence as revealed by cytogenetic mapping of repetitive elements in African clawed frogs (Xenopus, Pipidae)

https://doi.org/10.21203/rs.3.rs-2487507/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Repetitive elements have multiple copies in a genome and have been identified in several amphibian genomes using whole genome sequencing, but few studies have used cytogenetic mapping to visualize these elements in this vertebrate group. Here we compared the numbers and positions of repetitive elements in diploid and tetraploid species of the genus Xenopus, specifically between the diploid Xenopus tropicalis and its close allotetraploid relative X. calcaratus (both from Silurana subgenus), and also in more phylogenetically distant allotetraploids from the amieti, muelleri, and laevis species groups (all from subgenus Xenopus). Results allowed us to qualitatively evaluate the relative roles of polyploidization and divergence in the evolution of repetitive elements because our focal species include allotetraploid species derived from two independent polyploidization events – one closely related to the diploid species and one that is more distant. We used fluorescence in situ hybridization (FISH) to map the U1 and U2 small nuclear RNA (snRNA), histone H3, and retroelement Rex3 repeat loci. Our results demonstrated that U1 and U2 snRNA loci were conserved in copy number and position in the Silurana genomes, with one copy of U1 and U2 snRNA in diploid genome and one copy in each subgenome of allotetraploid species in subgenus Silurana and the muelleri species group. However, snRNA content varied among allotetraploid species in the amieti and laevis species groups, with only one U1 and U2 snRNA copy present in the entire allotetraploid genome of three species. Probes interrogating repetitive H3 and Rex3 loci were present on all chromosomes in diploid and allotetraploid species in subgenus Silurana, and in the muelleri species group. In the amieti and laevis species groups, H3 and Rex3 repeats were present on about half of all chromosomes. Overall, the results indicated that variation in the position and copy number of repetitive elements increases with phylogenetic distance, with high conservation in diploid and tetraploid Silurana species but more variation between Xenopus subgenera and among species groups in the subgenus Xenopus. In general and as expected, the effect of allotetraploidization was to increase the copy number of repetitive elements relative to diploid species, with the exception of the amieti and laevis species groups.

Amphibia

Anura

snRNA

Rex3

chromosome

Repetitive elements are genomic sequences that are present in multiple copies, and are found in all eukaryotic genomes, but with varying abundances and genomic distributions. Content of repetitive DNA can range from less than 10% in some fishes (Tetraodon nigroviridis and Cynoglossus semilaevis) and birds, e.g. Phoenicopterus ruber, Struthio camelus, Haliaeetus albicilla [Chalopin et al., 2015; Chen et al., 2014; Zhang et al., 2014; Canapa et al., 2016] to > 90% in some plants (such as the onion, Allium cepa [Fu et al., 2019]).

Repetitive elements in eukaryotes are categorized into transposable elements (TEs) and tandem repeats (TRs). TEs, which are scattered throughout the genome, include retroelements and DNA transposons. Retroelements (Rex1, Rex3, and Rex6) are reversely transcribed to DNA, and replicated using a copy and paste mechanism. Therefore, retroelements may increase genome size [Yampolsky 2016; Clark et al., 2019]. TRs, in contrast, are found as tandem arrays or head-to-tail motifs from several to more than a hundred copies [Myers, 2007]. TRs include microsatellite, minisatellite, satellite DNA, and also ribosomal genes (minor 5S and major 45S), histone genes, and small nuclear RNA (snRNA), which are found as clusters on single chromosomal locus or multiple loci [Valente et al., 2011; Knytl et al., 2018; Sember et al., 2020; Gazoni et al., 2021; Schott et al., 2022]. These loci often undergo concerted evolution [Elder and Turner, 1995; Liao, 1999] which causes paralogous repetitive elements within a species to be more similar to each other than to orthologous repetitive elements, even when the origin of repetitive elements precedes speciation [Liao, 1999]. Repetitive loci on chromosomes show dynamic evolution in terms of where in the genome and how frequently they are found, and they often have a high rate of rearrangement [Bruschi et al., 2014; Liu et al., 2019].

Small nuclear RNA are TR components of the spliceosome that perform pre-mRNA splicing. The spliceosome is composed of five RNA tandem-repeat units (U1, U2, U4, U5 and U6 snRNAs) [Valadkhan, 2005]. Histones are components of the nucleosome that are present in tandem repeat families of five major genes: H1/H5, H2A, H2B, H3, and H4. Retroelements Rex1, Rex3, and Rex6 are widely distributed on teleost chromosomes [Carducci et al., 2018] and first were extracted from the Xiphophorus genome [Volff et al., 1999]. Repetitive elements such as snRNA, histone genes, and Rex elements have been cytogenetically mapped on vertebrate chromosomes but neither has been mapped on Xenopus species.

Due to their repetitive nature, short read sequences typically pose challenges to mapping and repeat quantification, including using genomic data [James-Zorn et al., 2018]. However, repetitive DNA can be precisely mapped by fluorescent in situ hybridization (FISH) techniques. Cytogenetic mapping of repetitive sequences has been studied in vertebrates in teleost fishes [Bishani et al., 2021; Knytl et al., 2022], reptiles [Oliveira et al., 2021; Altmanová et al., 2022], birds [De Oliveira Furo et al., 2017; Kretschmer et al., 2021], and mammals [Milioto et al., 2019; Gerbault-Seureau et al., 2018].

In Amphibia, cytogenetic mapping of repetitive sequences is a subject of study of inter- and intraspecific rearrangements, karyotype evolution, and/or cytotaxonomy [da Silva et al., 2021; Phimphan et al., 2021; Guzmán et al., 2022]. African clawed frogs of the genus Xenopus involve almost 30 species and include two subgenera (Xenopus and Silurana). Subgenus Xenopus includes three species groups: the amieti, laevis, and muelleri [Evans et al., 2015]. Ploidy levels include diploid, tetraploid, octoploid, and dodecaploid [Tymowska, 1991] and at least eight independent polyploidization events (two allotetraploidization events) occurred in this genus [Evans et al., 2015] (Fig. 1). This remarkable variation in independently evolved ploidy level and genome sizes offers a compelling opportunity to explore how TEs and TRs evolve in the wake of genome duplication.

Figure 1. Evolutionary scenario within genus Xenopus. At least two independent allotetraploidization events occurred with respect to our study species – one in subgenus Silurana (square at the top left of the branch) during which two diploid ancestors gave rise to allotetraploid X. calcaratus (square at the top right of the branch). One diploid parental ancestor was X. tropicalis (or their common ancestor, respectively) and the second parental ancestor has not yet been described (dagger). The other allotetraploidization event occurred in subgenus Xenopus (square at the bottom left of the branch). Two diploid species (daggers) fused together, and common allotetraploid ancestor of subgenus Xenopus was formed. Evolutionary affinities of four allotetraploid species of subgenus Xenopus and used in this study (squares) are shown: X. allofraseri, X. pygmaeus, X. laevis, and X. muelleri. Number of chromosomes in all studied and ancestral species are shown in parentheses. The Silurana, amieti, laevis, and muelleri species groups are shown within branch.

Repetitive elements (TEs) are represented by 34.5% and 40% in the diploid Xenopus tropicalis and tetraploid X. laevis genomes compared to a total genomic DNA, respectively [Hellsten et al., 2010; Session et al., 2016]. Ribosomal DNAs (rDNAs) have been widely used for cytogenetic mapping in Xenopus [Schmid and Steinlein, 2015; Knytl et al., 2017; Roco et al., 2021; Knytl et al., 2023]. In allotetraploid species of subgenus Silurana, rDNA FISH analysis identified nucleolar organizer region (NOR) on chromosome 7a [Knytl et al., 2017, 2023]. The fact that only one pair of homologous chromosomes contains the NOR locus in Xenopus allotetraploids suggests deletion of NOR on chromosome 7b after allotetraploidization [Knytl et al., 2023]. There is limited cytogenetic information from other non-rDNA repetitive sequences in Xenopus and thus it is also not known if the numbers of non-rDNA repeats vary among Xenopus species and if so, why.

Here, we used the FISH approach for mapping U1 and U2 snRNA, histone H3, and retroelement Rex3 loci on chromosomes in a diploid-tetraploid model of African clawed frogs (Xenopus, Pipidae) representing two divergent subgenera Silurana and Xenopus (divergence approximately 45 Mya, [Feng et al., 2017]) and two different allotetraploidization events (one occurred in each subgenus). Specifically, from the subgenus Silurana diploid X. tropicalis and its tetraploid close relative X. calcaratus, and from the subgenus Xenopus tetraploid X. pygmaeus, X. allofraseri (both amieti species group), X. laevis (laevis species group), and X. muelleri (muelleri species group). In the subgenus Xenopus subgenomes have evolutionary distance about 30 Mya, while in the tetraploid Silurana about 10 Mya (Evans et al., 2015). With an overarching goal of exploring the effect of polyploidization and divergence on the evolution of repetitive elements, we tested the hypotheses that (i) the proportion of repetitive loci in diploid X. tropicalis is similar to that in the tetraploid species and (ii) the repetitive loci in these species are localized on homologous positions.

Primary cell cultures and metaphase spread preparations

Primary cell cultures were derived from X. tropicalis laboratory strain ‘Ivory Coast’ (diploid Silurana), wild-derived X. calcaratus (tetraploid Silurana) from Cameroon (Bakingili, where it occurs in syntopy with X. allofraseri), and in the tetraploid subgenus Xenopus from wild-derived X. pygmaeus originating from the Democratic Republic of the Congo (Kokolopori, Yalokole, near Luo River, 0.2056°N, 22.8884°E), X. allofraseri originating from Cameroon (Bakingili, 4.0684°N 9.0682°E), the latter two from the amieti species group, X. laevis laboratory strain (laevis species group), and X. muelleri laboratory strain (muelleri species group). Xenopus muelleri animals were originally obtained from the Institute of Zoology at the University of Geneva (Switzerland). All species were bred at Charles University, Faculty of Science, Prague, Czech Republic. Briefly, tadpoles were anesthetized, hind limbs removed and homogenized [Sinzelle et al., 2012] in a cultivation medium [Knytl et al., 2017] and modified according to the study Knytl et al. [2023]. The explants were then cultivated at 29.5°C with 5.5% CO₂ for five days without disturbance. The medium was then changed every day for one week. The first and next passages were performed with trypsin-ethylenediaminetetraacetic acid [Knytl et al., 2017]. After two weeks, Gibco™ Antibiotic-Antimycotic (100X) solution was removed from the cultivation medium, as usually recommended [Knytl et al., 2017], for the reason of faster cell proliferation.

Chromosomal suspensions were prepared according to previous works [Krylov et al., 2010] and stored in fixative solution (methanol: acetic acid, 3:1, v/v) at -20°C. For cytogenetic analysis, a chromosome suspension was dropped onto a slide [Courtet et al., 2001]. Chromosome preparations were aged at -20°C for at least one week. For each experiment, mitotic metaphase spreads were counterstained with ProLong™ Diamond Antifade Mountant with the fluorescent 4’,6-diamidino-2-phenylindole, DAPI stain (Invitrogen by Thermo Fisher Scientific). From ten to twenty metaphase spreads were analyzed per each probe. Microscopy and processing of metaphase images using Leica Microsystem (Wetzlar, Germany) [Seroussi et al., 2019].

FISH with repetitive DNA probes

In order to generate probes for FISH, Xenopus tropicalis was used as a template for amplification of repetitive DNAs (small nuclear DNAs regions (snDNA) U1 and U2, histone H3, and retroelements Rex3). Total gDNA was extracted from the tail tissue of a tadpole using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Conditions for PCR amplification were used according to PPP Master Mix (Top-Bio, Prague, Czech Republic) supplier recommendations. Primers used for amplification are listed in Table 1. The annealing temperature was 54°C and the elongation step 30 sec for all PCR reactions. Labelling PCR was performed as described in previous studies [Knytl and Fornaini, 2021]. Digoxigenin-11-dUTP (Jena Bioscience, Jena, Germany) was used for U2 and H3 labelling. Biotin-16-dUTP (Jena Bioscience) was used for U1 and Rex3 labelling. Xenopus tropicalis U1 and U2 snDNA, H3, and Rex3 probes were hybridized with chromosome spreads of X. tropicalis, X. calcaratus, X. pygmaeus, X. allofraseri, X. laevis, and X. muelleri. The contents of the hybridization mixture, denaturation, and the subsequent overnight hybridization were conducted as it described in rDNA FISH protocol [Knytl et al., 2023]. Post-hybridization washing and blocking reactions were performed as described for painting FISH in Krylov et al. [2010]. Probe visualization was performed as protocol [Knytl et al., 2017]. Slides were then de-stained according following protocol: nail polish was released in xylene (2 min) and benzoin (2 min). Slides with cover slip were incubated in 4x SSC/ 0.1% Tween for 10 min with agitation and then cover slips were manually removed. Slides without cover slip were then incubated in 4x SSC/ 0.1% Tween for 30 min with agitation followed by dehydration with methanol series (70, 90, 100% for 3 min each) and air drying. After slide incubation in fixative solution for 30 min, rinsing with distilled water, and air drying, slides were aged for 90 min at 60°C.

Table 1

Genes used for FISH analysis, their GenBank accession numbers, lengths, sequences of primers, and studies in which primers were designed.
Gene Symbol	Gene Name	Accession No.	Length (bp)	Primer Sequence	Citation
U1	Small nuclear RNA U1	XXX	119	U1F: 5′ -GCAGTCGAGATTCCCACATT-3′	Silva et al. [2015]
U1	Small nuclear RNA U1	XXX	119	U1R: 5′ -CTTACCTGGCAGGGGAGATA-3′	Silva et al. [2015]
U2	Small nuclear RNA U2	XXX	177	U2F: 5′-ATCGCTTCTCGGCCTTATG-3′	Bueno et al. [2013]
U2	Small nuclear RNA U2	XXX	177	U2R: 5′-TCCCGGCGGTACTGCAATA-3′	Bueno et al. [2013]
H3	Histone H3	XXX	364	H3F: 5′-ATGGCTCGTACCAAGCAGAC(ACG)GC-3′	Colgan et al. [1998]
H3	Histone H3	XXX	364	H3R: 5′-ATATCCTT(AG)GGCAT(AG)AT(AG)GTGAC-3′	Colgan et al. [1998]
Rex3	Retroelement of Xiphophorus 3	XXX	224	RTX3-F2: 5′-AACACCTTGGCTGCGCCTAG-3′	Volff et al. [1999]
Rex3	Retroelement of Xiphophorus 3	XXX	224	RTX3-R3: 5′-TGGCAGACNGGGGTGGTGGT-3′	Volff et al. [1999]

Painting FISH

We used whole chromosome painting probes generated by laser microdissection of X. tropicalis chromosomes from previous study Knytl et al. [2023]. Probes were newly labelled either with digoxigenin-11-dUTP (Jena Bioscience) [Krylov et al., 2010] or biotin-16-dUTP (Jena Bioscience) [Knytl et al., 2023]. De-stained slides (after FISH with the U1 and U2 probes) of X. tropicalis, X. calcaratus, and X. laevis were used for cross-species painting FISH (Zoo-FISH) with digoxigenin-labelled probe. Subsequently, slides were de-stained again and used with biotin-labelled probe. The X. tropicalis FISH experiments were performed in the reverse order from the other species (i.e., painting FISH first followed by snDNA FISH after de-staining).

Sequences and genomic locations of probes in available genome assemblies

The U1 and U2 snDNA, H3, and Rex3 nuclear genes resulted in approximately 120 and 180, 360, and 220 bp long amplicons. Based on BLASTn results, the U1 amplicon had 98.3% identity (query cover 100%) with the sequence of X. tropicalis laboratory strain ‘Nigeria’ U1 spliceosomal RNA, LOC116408489 (accession number XR_004220992.1), and U2 amplicon had 98.3% identity (query cover 100%) with X. tropicalis (‘Nigeria’) U2 spliceosomal RNA, LOC116407440 (accession number XR_004220346.1). The H3 amplicon showed 97.2% identity (query cover 98%) with X. tropicalis (‘Nigeria’) histone H3, LOC100497127, mRNA (accession number XM_012953339.3). The Rex3 amplicon had 90.0% identity with X. tropicalis large-insert genomic clones. The U1 sequence on the Xenbase database matched about 20 copies of U1 snDNA on X. tropicalis chromosome 1 and one copy on X. tropicalis chromosome 2. This sequence matched approximately 40 copies of U1 snDNA on X. laevis chromosome 1S and 11 copies on X. laevis chromosome 1L. The U2 sequence matched about 40 copies on X. tropicalis chromosome 8 and 40 copies on X. laevis chromosomes 8L and 12 copies on 8S with the high score but the shorter regions of U2 locus were also mapped on other X. tropicalis and X. laevis chromosomes. The H3 and Rex3 sequences on the Xenbase database showed more variable results. The H3 sequence matched regions of X. tropicalis chromosomes 3, 5, 6, 8, 9, and 10 and X. laevis chromosomes 1L, 3S, 5L, 5S, 6L, 6S, 8L, 9_10L, and 9_10S. The Rex3 sequence had matches on all X. tropicalis and X. laevis chromosomes and some unidentified scaffolds. Our sequences were deposited to the NCBI GenBank database (U1 snDNA accession number XXX, U2 snDNA accession number XXX, H3 accession number XXX, Rex3 accession number XXX).

FISH with U1 and U2 snDNA probes

When hybridized on the diploid species X. tropicalis, the U1 and U2 snDNA probes had intense signals on the long (q) arm of the X. tropicalis chromosome 1 (XTR 1q) and XTR 8q (Fig. 2a), respectively. In the allotetraploid species X. calcaratus, the U1 and U2 snDNA probes hybridized to the q arms of the X. calcaratus chromosomes 1 (XCA 1aq), XCA 1bq and XCA 8aq, XCA 8bq (Fig. 2b), respectively. These U1 and U2 snRNA signals thus hybridized to two pairs of homoeologous chromosomes in X. calcaratus. In the allotetraploid species X. pygmaeus and X. allofraseri, the U1 snDNA probes hybridized most conspicuously to the q arms of X. pygmaeus chromosomes 1L (XPY 1Lq) and X. allofraseri chromosomes 1L (XAL 1Lq), respectively. The U2 snDNA probes showed loci on the XPY 8Sq and XAL 8Sq (Fig. 2c, 2d). In X. laevis, U1 and U2 snDNA probes mapped to XLA 1Sq and XLA 8Lq, respectively (Fig. 2e). In X. muelleri, U1 and U2 snDNA probes mapped to the homoeologous chromosomal quartets (U1 on XMU 1Lq and 1Sq; U2 on XMU 8Lq and 8Sq) (Fig. 2f). Both U1 and U2 loci mapped to a portion of the q arm near a telomere in all investigated species, and the number of loci did not support the expectation that all tetraploid species should have twice as many snRNA loci as the diploid X. tropicalis.

Figure 2. Double-colour fluorescent in situ hybridization (FISH) with U1 and U2 probes. The U1 rDNA probe (red) reveals one clear signal in (a) X. tropicalis, (c) X. pygmaeus, (d) X. allofraseri, and (e) X. laevis, respectively, while the same FISH shows two homoeologous signals in (b) X. calcaratus and (f) X. muelleri. The U2 (green) probe shows one signal in (a) X. tropicalis, (c) X. pygmaeus, (d) X. allofraseri, and (e) X. laevis, respectively, while the U2 probe shows two homoeologous signals in (b) X. calcaratus and (f) X. muelleri. Green and red arrows correspond to U2 and U1 repeat loci, respectively. Painting probes were used for identification of chromosomes 1 (green) and 8 (red) in (a) X. tropicalis. Chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) in blue. Scale bars represent 10 µM.

Painting FISH

We then used FISH to identification of chromosomes that bear the U1 and U2 loci. We used X. tropicalis whole chromosome painting (WCP) probes from XTR 1 and 8 from previous study [Knytl et al., 2023]. Using these painting probes, we successfully identified chromosomes 1 and 8 in X. tropicalis (Fig. 2a), chromosomes 1a, 1b, 8a, and 8b in X. calcaratus (Fig. 3), and chromosomes 1L, 1S, 8L, and 8S in X. laevis (Fig. 4). Homoeologous chromosomes were identified based on the intensity of fluorescence signal [Knytl et al., 2023] and in other species in which the FISH approach was not conducted, homoeologous chromosomes were identified based on visual estimation of differences in chromosome length. The L homoeologous chromosomes are longer than the S chromosomes due to deletions in the S subgenome [Session et al., 2016].

Figure 3. Sequential FISH experiment illustrating that the U1 locus (in red) is localized on X. calcaratus chromosomes 1 (XCA 1a and 1b) and the U2 locus (in green) on XCA 8a and 8b. (a) and (b) are shown in the red channel, (c) is merged in the RGB channels. (c) chromosomes were counterstained with DAPI in gray. Scale bars represent 10 µM.

Figure 4. Sequential FISH experiment illustrating that (b) the U1 locus (in red) is localized on (a) X. laevis chromosomes 1S (XLA 1S), in red, and (b) the U2 locus (in green) on (a) XLA 8L, in green. Both (a) and (b) are merged in the RGB channels. Chromosomes were counterstained with DAPI in blue. Scale bars represent 10 µM.

FISH with H3 and Rex3 probes

We then hybridized H3 and Rex3 probes to chromosome spreads of X. tropicalis, X. calcaratus, X. pygmaeus, X. allofraseri, X. laevis, and X. muelleri. In all of these species, signals from both probes were present in small patches on many chromosomes (Fig. 5). We found H3 and Rex3 signals in all chromosomes in X. tropicalis, X. calcaratus, and X. muelleri. Xenopus pygmaeus, X. allofraseri, and X. laevis had signals on about half of chromosomes. The H3 and Rex3 loci mapped to telomeres, centromeres, pericentromeric regions, and other interstitial chromosomal regions.

Figure 5. Double-colour FISH with H3 and Rex3 probes. The H3 (green) probe shows multiple signals in (a) X. tropicalis, (b) X. calcaratus, (c) X. pygmaeus, (d) X. allofraseri, (e) X. laevis, and (f) X. muelleri, respectively. The Rex3 rDNA probe (red) shows multiple signals in (a) X. tropicalis, (b) X. calcaratus, specially in telomeric positions, respectively, while it shows multiple signals in (c) X. pygmaeus and (d) X. allofraseri, without showing a specific pattern. Green and red arrows correspond to H3 and Rex3 ribosomal loci, respectively. Chromosomes were counterstained with DAPI in blue/gray. Scale bars represent 10 µM.

Quantification and localization of repetitive sequences using high quality genome sequencing and assembly is an expensive and challenging undertaking as compared to using multiple cytogenetic approaches for gene mapping [Knytl et al., 2018, 2023]. As an alternative, here we cytogenetically mapped repetitive elements to diploid Silurana and tetraploid Silurana and Xenopus species with the aim of testing whether repetitive elements were or were not influenced by genome duplication and divergence among species. At least two allotetraploidization events occurred in genus Xenopus with respect to our focal species and both events, including studied species and their diploid extinct or yet undescribed predecessors are depicted in Fig. 1.

We expected allotetraploid species to have twice as many repetitive elements as the diploid X. tropicalis and that the locations of these elements would be unchanged (i.e. on both homoeologous chromosomes). For U1 and U2 snRNA repetitive elements in tetraploid X. calcaratus, and X. muelleri this expectation was supported but for other tetraploids (X. pygmaeus, X. allofraseri, and X. laevis) this expectation was not supported because, although in homologous locations, we found same number of repetitive elements as in diploids (Table 2). This suggests a stronger influence of divergence than polyploidization on U1 and U2 snRNA content. The allotetraploid ancestor of X. muelleri diverged from the common ancestor of X. pygmaeus, X. allofraseri, and X. laevis soon after allotetraploidization in subgenus Xenopus (Fig. 5) [Evans et al., 2015] and therefore X. muelleri can be the case of polyploidization hypothesis unlike the amieti and laevis groups.

Table 2

Number and chromosomal position of U1 and U2 snRNA, H3, and Rex3 signals per haploid genome in diploid *X. tropicalis*, and reduced genome (half of the somatic set) in allotetraploid *X. calcaratus* (both *Silurana* subgenus), *X. pygmaeus*, *X. allofraseri* (both *amieti* species group), *X. laevis* (*laevis* species group), and *X. muelleri* (*muelleri* species group). The XTR, XCA, XPY, XAL, XLA, and XMU abbreviations show chromosome of *X. tropicalis*, *X. calcaratus*, *X. pygmaeus*, *X. allofraseri*, *X. laevis*, and *X. muelleri*, respectively.
Species	Haploid chromosome number	U1 snRNA	U2 snRNA	H3	Rex3
X. tropicalis	10	1 (XTR 1)	1 (XTR 8)	10	10
X. calcaratus	20	2 (XCA 1a and 1b)	2 (XCA 8a and 8b)	20	20
X. pygmaeus	18	1 (XPY 1L)	1 (XPY 8S)	8	9
X. allofraseri	18	1 (XAL 1L)	1 (XAL 8S)	8	9
X. laevis	18	1 (XLA 1S)	1 (XLA 8L)	8	11
X. muelleri	18	2 (XMU 1L and 1S)	2 (XMU 8L and 8S)	18	18

In amphibians, U2 snRNA tandem repeats also have been localized in the cycloramphid genus Thoropa and the leptodactylid genus Leptodactylus [Gazoni et al., 2021], the H3 gene have been localized in the pipid genus Pipa [Zattera et al., 2020], and the Rex elements have been localized in the leptodactylid genus Physalaemus [Nascimento et al., 2015]. To our knowledge, there are no other cytogenetic studies that localize the U1, U2 snRNA, H3, or Rex loci in this vertebrate group.

U1 and U2 snRNA repeat loci

We mapped the U1 snRNA gene on the distal part of the q arm of X. tropicalis chromosome 1 (Fig. 2a). Because chromosome 1 in X. tropicalis is the longest chromosome, it is easily distinguished from others in a metaphase spread [Tymowska, 1991]. In the allotetraploid species X. calcaratus, the U1 snRNA locus mapped to the distal portion of the q arm of chromosomes 1a and 1b (Fig. 2b). Chromosomes 1a and 1b are homoeologous to each other, and each of them is orthologous to X. tropicalis chromosome 1, and the mapped U1 region in X. tropicalis is homologous to the U1 region of X. calcaratus (both species have U1 gene on the distal part of chromosome). The U2 snRNA gene was found on the distal part of the q arm of X. tropicalis chromosome 8. In X. calcaratus, the U2 snRNA locus also was found in a homologous location on the distal region of the q arms of chromosomes 8a and 8b. These results suggest that both diploid Silurana ancestors of X. calcaratus bore U1 and U2 snRNAs on chromosome 1 and 8, respectively.

Compared to mapping of the U1 and U2 snRNA loci recovered different results in the species we studied. Xenopus pygmaeus and X. allofraseri (both amieti species group) had U1 and U2 signals on chromosomes 1Lq and 8Sq, respectively (Fig. 2c, 2d). Xenopus laevis had U1 and U2 signals on chromosomes 1Sq and 8Lq (Fig. 2e). In contrast to these three species which contain only one U1 and U2 signal in allotetraploid genome, X. muelleri had detectable signals on both homoeologous chromosomes, i.e. U1 on chromosomes 1Lq and 1Sq, U2 on chromosomes 8Lq and 8Sq. When we mapped our U1 and U2 snDNA X. tropicalis sequences to X. laevis genome assembly (v10.1), the query sequence matched both X. laevis homoeologs, i.e. the X. tropicalis U1 sequence matched X. laevis 1L and 1S, X. tropicalis U2 sequence matched X. laevis 8L and 8S. The number of FISH signals on X. laevis do not match the number of U1 and U2 snRNA as revealed by BLAST. The inconsistency may be due to among individual variation in tandem repeats in each X. laevis subgenome and possibly by insufficient sensitivity of FISH to detect a low number of repeats, which is less than 20 copies. A snRNA FISH signal was detected on those X. laevis homoeologous chromosomes that bear a higher portion of U1 and U2 repeats. We did not find variability in mapping locations of U1 and U2 snRNA in Xenopus. Owing to the conserved genomic locations of their targets, the U1 and U2 FISH probes permitted identification of chromosomes 1 and 8 in all species of the genus Xenopus inspected here, though we detected differences among species in copy number within these chromosomes. This also suggests that the Xenopus ancestor had the U1 and U2 snRNA on chromosomes 1 and 8, respectively, each with different copy numbers of U1 and U2 repeats. We found twice as many U1 and U2 snRNA signals in tetraploid Silurana as compared to diploid X. tropicalis, which is consistent with expectations based on polyploidization. In subgenus Xenopus, we also found twice as many U1 and U1 snRNA signals in the allotetraploid X. muelleri, but in allotetraploids from the amieti and laevis species groups the number of U1 and U2 repeats was the same as in the diploid species. This highlights an effect of phylogenetic divergence on U1 and U2 repeat copy number.

In another frog genus, Thoropa, U2 snRNA was detected on chromosomal pairs 6 and 7 but some Thoropa species had the U2 loci only on chromosome 6 and some U2 loci on chromosome 7 [Cholak et al., 2020]. One explanation for this finding in different Thoropa species is that U2 loci are universally situated on both pairs 6 and 7 but with different and species-specific copy numbers on each chromosome pair. In Leptodactylus, the U2 loci were identified on chromosome pair 6 in eight species but one of them had additional signals on chromosomes 4, 9, and 10 [Gazoni et al., 2021]. Thus, results of the U2 snRNA mapping on those frogs (both genera have an approximate evolutionary distance from Xenopus about 180 Mya [Hime et al., 2021]) have partial homology in the number and location, but some species also have additional signals on other chromosomes [Gazoni et al., 2021]. The FISH technology deployed here cannot detect snRNAs when they have low copy number (in X. laevis, genome mapping of our U1 and U2 snRNA sequences matched two chromosome pairs, but FISH detected these loci on one chromosome pair) and is an alternative tool for visualization of snRNAs on specific chromosomes such as chromosome 1 (U1) and chromosome 8 (U2). We can conclude that snRNAs can be dynamically accumulated in some chromosomal loci but relatively stable in others, at least in Xenopus.

Histone H3 and Retroelement Rex3 loci

We also tested effects of allotetraploidization and divergence on evolution of H3 and Rex3 repeats. Allotetraploid X. calcaratus and X. muelleri possessed twice as many H3 and Rex3 repeats as the diploid X. tropicalis that indicates polyploidization to duplicate number of these repeats. In allotetraploid X. pygmaeus, X. allofraseri, and X. laevis we found signals on 8–11 homologous pairs and therefore these repeat number was reduced on about half of the genome due to phylogenetic divergence. The H3/Rex3 results are similar with the U1/U2 results in the term of repeat number variability. An exception in the subgenus Xenopus among our study species is X. muelleri (H3 and Rex3 signals on all chromosomes) giving to rise from same allotetraploidization event as X. pygmaeus, X. allofraseri, and X. laevis (H3 and Rex3 signals on about half of chromosome set). The exception herein might be given by X. muelleri divergence which occurred soon after allotetraploidization in subgenus Xenopus and thus the allotetraploidization multiplicated the number of H3 and Rex3 repeats (as well as U1 and U2 snRNAs), while the repeats were reduced in one subgenome in the common ancestor of the amieti and laevis species groups.

Until recently, there was only one study in which the H3 gene was mapped on amphibian chromosomes [Zattera et al., 2020]. In Pipa carvalhoi, a representative of the same family as Xenopus but deeply phylogenetically divergent by about 115–120 Mya [Feng et al., 2017; Hime et al., 2021], H3 signals were detected on chromosomes 1, 5, 6, 8, and 9, and less intense signals were visible on other chromosomes. BLAST searches of the Xenopus tropicalis genome sequence identified H3 genes on chromosomes 3, 5, 6, 8, 9, and 10. Sequence mapping results of our X. tropicalis H3 probe against X. laevis genome sequence database showed chromosomes 1L, 3S, 5L, 5S, 6L, 6S, 8L, 9_10L, and 9_10S and our FISH mapping showed that about half of X. laevis chromosomes contains signals. Localization of H3 and Rex3 genes showed dispersed signals on small regions of all or about half of chromosomes in all investigated species (Table 2) which is consistent with previous findings [Bueno et al., 2013; Mourao et al., 2017]. The dispersed Rex3 signals in Xenopus karyotypes correspond to the BLAST results and to the TEs content which is 35–40% in the Xenopus genome [Hellsten et al., 2010; Session et al., 2016]. We did not find the co-localization of Rex3 with H3 loci, an observation that fails to support the hitchhiking hypothesis in which mobile elements are tightly linked with transposition events of mobile elements and co-occur on the same chromosomal locus [Pucci et al., 2018].

Co-localization of repetitive elements

An interesting finding that emerged from this study is a spatial association of various repetitive elements with ribosomal RNA (rRNA) that can provide evolutionary insights into genome organization. The spatial synteny was found between As51 satellite DNA (originating from TEs) and NORs [Vicari et al., 2008] or between U1 snRNA and 5S rRNA in the characid fish genus Astyanax [Silva et al., 2015]. One of possible roles of a co-localization can be a silencing of ribosomal genes by TEs [Vicari et al., 2008]. All Xenopus species have NOR on a single homologous pair [Tymowska, 1991]. Xenopus species have NORs on chromosomes 7 (X. tropicalis), 7a (Silurana tetraploids), and 3L (Xenopus tetraploids) [Tymowska, 1991; Session et al., 2016; Roco et al., 2021; Knytl et al., 2023]. We have not found any snRNA positive loci on chromosomes 7, 7a, or 3L and this finding excludes spatial association between distinct NOR and snRNA repeats. The 5S ribosomal genes have been found at the distal region of the X. mellotropicalis (tetraploid Silurana) and X. calcaratus chromosomes 8bq [Knytl et al., 2017, 2023] which is the identical locus as the U2 snRNA locus in X. calcaratus (this study) supporting spatial association between rRNA and snRNA. One NOR locus was deleted on chromosome 7b (in Silurana tetraploids) and 3S (Xenopus tetraploids) [Session et al., 2016; Knytl et al., 2023]. However, U1 and U2 loci have been mapped on two chromosome pairs in some tetraploids and thus deletion of snRNA in Xenopus did not take place. There is the possibility that all our investigated Xenopus tetraploids have twice as many snRNA repeats as diploid X. tropicalis but they have unequal content within individual subgenomes which is the case of X. laevis (this study).

Repetitive elements are integral components of a genome, and their evolution can be influenced by mechanism of polyploidization or divergence among species. We studied unique Xenopus frogs with different ploidy levels (diploid-tetraploid relationships) with the aim to localize and quantify repetitive elements in the genome of diploid and subgenomes of allotetraploid Xenopus species. For U1 and U2 snRNA, H3, and Rex3 repeats, we identified variation in copy numbers that was generally in homologous locations. These results demonstrate an effect of allotetraploidization in duplicating repeat number (Silurana subgenus and muelleri species group) as well as by divergence among species that led to loss of repeats (amieti and laevis species groups). Because the studied Silurana species underwent different allotetraploidization than the studied allotetraploids from subgenus Xenopus, the amieti species group and X. laevis are exceptions that revealed unexpected variability in the number of FISH signals showing dynamic evolution of repetitive elements.

Statements

All papers must contain the following statements after the main body of the text and before the reference list:

Acknowledgement

Statement of Ethics

Charles University has registered experimental breeding facilities for pipid frogs (16OZ12891/2018-17214, 37428/2019-MZE-18134). All experimental procedures involving frogs were approved by the Institutional Animal Care and Use Committee of Charles University, according to the directives from the State Veterinary Administration of the Czech Republic, reference number MSMT-20585/2022-4 issued by the Ministry of Education, Youth and Sport of the Czech Republic (MK is a manager of the experimental project on living Xenopus animals). MK is a holder of the Certificate of professional competence to design experiments according to §15d(3) of the Czech Republic Act No. 246/1992 coll. on the Protection of Animals against Cruelty (Registration number CZ 03973), provided by the Ministry of Agriculture of the Czech Republic.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

The research was supported by the institutional support from the IVB CAS (RVO: 68081766); the Ministry of Culture of the Czech Republic (DKRVO 2019–2023/6.VII.e, National Museum, 00023272) (both VG); institutional support from the Ministry of Agriculture of the Czech Republic (MZE-RO0518) (HČ, SK); the Natural Science and Engineering Research Council of Canada (RGPIN-2017-05770) (BJE).

Author Contributions

NRF: Laboratory experiments, Graphic design, Manuscript editing. BB: Laboratory experiments, Manuscript editing. VG: Laboratory experiments, Fieldwork, Funding acquisition, Manuscript editing. HČ: Laboratory experiments, Funding acquisition, Manuscript editing. VK: Manuscript editing. SK: Funding acquisition, Manuscript editing. EBF: Fieldwork. GB: Fieldwork. BJE: Fieldwork, Funding acquisition, Manuscript editing, Supervision. MK: Conceptualization of the study, Visualization, Data curation and validation, Project administration, Original draft preparation, Manuscript editing, supervision. All authors approved the final version of the manuscript.

Data Availability Statement

The Sanger sequences that support the findings of this study are openly available in the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/genbank/).

Altmanová M, Doležálková-Kaštánková M, Jablonski D, Strachinis I, Vergilov V, Vacheva E, et al. Karyotype stasis but species-specific repetitive DNA patterns in Anguis lizards (Anguidae), in the evolutionary framework of Anguiformes, 29 December 2022, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-2413537/v1]

Bishani A, Prokopov DY, Romanenko SA, Molodtseva AS, Perelman PL, Interesova EA, et al. Evolution of Tandemly Arranged Repetitive DNAs in Three Species of Cyprinoidei with Different Ploidy Levels. Cytogenet Genome Res. 2021;161(1–2):32–42.

Bueno D, Palacios-Gimenez OM, Cabral-de-Mello DC. Chromosomal Mapping of Repetitive DNAs in the Grasshopper Abracris flavolineata Reveal Possible Ancestry of the B Chromosome and H3 Histone Spreading. PLoS One. 2013;8(6). DOI: 10.1371/journal.pone.0066532

Bruschi DP, Rivera M, Lima AP, Zúñiga AB, Recco-Pimentel SM. Interstitial Telomeric Sequences (ITS) and major rDNA mapping reveal insights into the karyotypical evolution of Neotropical leaf frogs species (Phyllomedusa, Hylidae, Anura). Mol Cytogenet. 2014;7(1):1–12.

Canapa A, Barucca M, Biscotti MA, Forconi M, Olmo E. Transposons, genome size, and evolutionary insights in animals. Cytogenet Genome Res. 2016;147(4):217–39.

Carducci F, Barucca M, Canapa A, Biscotti MA. Rex retroelements and teleost genomes: An overview. Int J Mol Sci. 2018;19(11). DOI: 10.3390/ijms19113653

Chalopin D, Naville M, Plard F, Galiana D, Volff JN. Comparative analysis of transposable elements highlights mobilome diversity and evolution in vertebrates. Genome Biol Evol. 2015;7(2):567–80.

Chen S, Zhang G, Shao C, Huang Q, Liu G, Zhang P, et al. Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle. Nat Genet. 2014;4

Cholak LR, Haddad CFB, Parise-Maltempi PP. Cytogenetic analysis of the genus thoropa cope, 1865 (Anura-cycloramphidae) with evolutionary inferences based on repetitive sequences. Genet Mol Biol. 2020;43(3):1–9. 6(3):253–60.

Clark DP, Pazdernik NJ, McGehee MR. Mobile DNA. Molecular Biology. Elsevier; 2019; pp 793–829.

Courtet M, Flajnik M, Du Pasquier L. Major histocompatibility complex and immunoglobulin loci visualized by in situ hybridization on Xenopus chromosomes. Dev Comp Immunol. 2001;25(2):149–57.

Da Silva DS, Da Silva Filho HF, Cioffi MB, De Oliveira EHC, Gomes AJB. Comparative Cytogenetics in Four Leptodactylus Species (Amphibia, Anura, Leptodactylidae): Evidence of Inner Chromosomal Diversification in Highly Conserved Karyotypes. Cytogenet Genome Res. 2021;161(1–2):52–62.

De Freitas Mourão AA, Daniel SN, Hashimoto DT, Ferreira DC, Porto-Foresti F. Organization and distribution of repetitive DNA classes in the Cichla kelberi and Cichla piquiti genome. Cytologia (Tokyo). 2017;82(2):193–7.

De Oliveira Furo I, Kretschmer R, Dos Santos MS, De Lima Carvalho CA, Gunski RJ, O’Brien PCM, et al. Chromosomal Mapping of Repetitive DNAs in Myiopsitta monachus and Amazona aestiva (Psittaciformes, Psittacidae) with Emphasis on the Sex Chromosomes. Cytogenet Genome Res. 2017;151(3):151–60.

Elder, JF, Turner BJ. Concerted Evolution of Repetitive DNA Sequences in Eukaryotes. Q Rev Biol. 1995 Sep;70(3):297–320.

Evans BJ, Carter TF, Greenbaum E, Gvoždík V, Kelley DB, McLaughlin PJ, et al. Genetics, morphology, advertisement calls, and historical records distinguish six new polyploid species of African clawed frog (Xenopus, Pipidae) from West and Central Africa. PLoS One. 2015 Dec;10(12):e0142823.

Feng YJ, Blackburn DC, Liang D, Hillis DM, Wake DB, Cannatella DC, et al. Phylogenomics reveals rapid, simultaneous diversification of three major clades of Gondwanan frogs at the Cretaceous–Paleogene boundary. Proc Natl Acad Sci U S A. 2017;114(29):E5864–70.

Fu J, Zhang H, Guo F, Ma L, Wu J, Yue M, et al. Identification and characterization of abundant repetitive sequences in Allium cepa. Sci Rep. 2019;9(1):1–7.

Gazoni T, Dorigon NS, Da Silva MJ, Cholak LR, Haddad CFB, Parise-Maltempi PP. Chromosome Mapping of U2 snDNA in Species of Leptodactylus (Anura, Leptodactylidae). Cytogenet Genome Res. 2021;161(1–2):63–9.

Gerbault-Seureau M, Cacheux L, Dutrillaux B. The Relationship between the (In-)Stability of NORs and Their Chromosomal Location: The Example of Cercopithecidae and a Short Review of Other Primates. Cytogenet Genome Res. 2018;153(3):138–46.

Guzmán K, Roco ÁS, Stöck M, Ruiz-García A, García-Muñoz E, Bullejos M. Identification and characterization of a new family of long satellite DNA, specific of true toads (Anura, Amphibia, Bufonidae). Sci Rep. 2022;12(1). DOI: 10.1038/s41598-022-18051-9.

Hellsten U, Harland RM, Gilchrist MJ, Hendrix D, Jurka J, Kapitonov V, et al. The Genome of the Western Clawed Frog Xenopus tropicalis. Science (80- ). 2010 Apr;328(5978):633–6.

Hime PM, Lemmon AR, Lemmon ECM, Prendini E, Brown JM, Thomson RC, et al. Phylogenomics reveals ancient gene tree discordance in the Amphibian Tree of Life. Syst Biol. 2021; 70, 49–66. DOI:10.1093/sysbio/syaa034.

James-Zorn C, Ponferrada V, Fisher ME, Burns K, Fortriede J, Segerdell E, et al. Navigating Xenbase: An Integrated Xenopus Genomics and Gene Expression Database. In: Kollmar M, editor. Eukaryotic Genomic Databases. Methods in Molecular Biology. New York: Humana Press; 2018; pp 251–305.

Knytl M, Smolík O, Kubíčková S, Tlapáková T, Evans BJ, Krylov V. Chromosome divergence during evolution of the tetraploid clawed frogs, Xenopus mellotropicalis and Xenopus epitropicalis as revealed by Zoo-FISH. PLoS One. 2017 May;12(5):e0177087.

Knytl M, Kalous L, Rylková K, Choleva L, Merilä J, Ráb P. Morphologically indistinguishable hybrid Carassius female with 156 chromosomes: A threat for the threatened crucian carp, C. carassius, L. PLoS One. 2018 Jan;13(1):e0190924.

Knytl M, Tlapakova T, Vankova T, Krylov V. Silurana Chromosomal Evolution: A New Piece to the Puzzle. Cytogenet Genome Res. 2018 Dec;156(4):223–8.

Knytl M, Fornaini NR. Measurement of Chromosomal Arms and FISH Reveal Complex Genome Architecture and Standardized Karyotype of Model Fish, Genus Carassius. Cells. 2021 Sep;10(9):2343.

Knytl M, Forsythe A, Kalous L. A Fish of Multiple Faces, Which Show Us Enigmatic and Incredible Phenomena in Nature: Biology and Cytogenetics of the Genus Carassius. Int J Mol Sci. 2022 Jul;23(15):8095.

Knytl M, Fornaini NR, Bergelová B, Gvoždík V, Černohorská H, Kubíčková S, et al. Divergent subgenome evolution in the allotetraploid frog Xenopus calcaratus. Gene. 2023 Jan;851:146974.

Kretschmer R, Rodrigues BS, Barcellos SA, Costa AL, Cioffi M de B, Garnero ADV, et al. Karyotype evolution and genomic organization of repetitive dnas in the saffron finch, sicalis flaveola (Passeriformes, aves). Animals. 2021;11(5). DOI: 10.3390/ani11051456

Krylov V, Kubickova S, Rubes J, Macha J, Tlapakova T, Seifertova E, et al. Preparation of Xenopus tropicalis whole chromosome painting probes using laser microdissection and reconstruction of X. laevis tetraploid karyotype by Zoo-FISH. Chromosom Res. 2010 Jun;18(4):431–9.

Liao D. Concerted evolution: Molecular mechanism and biological implications. Am J Hum Genet. 1999;64(1):24–30.

Liu Y, Song M, Luo W, Xia Y, Zeng X. Chromosomal Evolution in the Amolops mantzorum Species Group (Ranidae; Anura) Narrated by Repetitive DNAs. Cytogenet Genome Res. 2019;157(3):172–8.

Milioto V, Vlah S, Mazzoleni S, Rovatsos M, Dumas F. Chromosomal Localization of 18S-28S rDNA and (TTAGGG)n Sequences in Two South African Dormice of the Genus Graphiurus (Rodentia: Gliridae). Cytogenet Genome Res. 2019;158(3):145–51.

Myers PZ. Tandem Repeats and Morphological Variation. Nat Educ. 2007;1(1):1.

Nascimento J, Baldo D, Lourenço LB. First insights on the retroelement Rex1 in the cytogenetics of frogs. Mol Cytogenet. 2015;8(1):1–9.

Oliveira VCS, Altmanová M, Viana PF, Ezaz T, Bertollo LAC, Ráb P, et al. Revisiting the Karyotypes of Alligators and Caimans (Crocodylia, Alligatoridae) after a Half-Century Delay: Bridging the Gap in the Chromosomal Evolution of Reptiles. Cells. 2021 Jun;10(6):1397.

Phimphan S, Aiumsumang S, Tanomtong A. Characterization of Chromosomal and Repetitive Elements in the Genome of “Rana nigrovittata” (Anura, Ranidae): Revealed by Classical and Molecular Techniques. Cytol Genet. 2021;55(6):583–9.

Pucci MB, Nogaroto V, Moreira-Filho O, Vicari MR. Dispersion of transposable elements and multigene families: Microstructural variation in Characidium (Characiformes: Crenuchidae) genomes. Genet Mol Biol. 2018;41(3):585–92.

Roco ÁS, Liehr T, Ruiz-García A, Guzmán K, Bullejos M. Comparative Distribution of Repetitive Sequences in the Karyotypes of Xenopus tropicalis and Xenopus laevis (Anura, Pipidae). Genes (Basel). 2021 Apr;12(5):617.

Schmid M, Steinlein C. Chromosome banding in Amphibia. XXXII. The genus Xenopus (Anura, Pipidae). Cytogenet Genome Res. 2015;145:201–17.

Schott SCQ, Glugoski L, Azambuja M, Moreira-Filho O, Vicari MR, Nogaroto V. Comparative Cytogenetic and Sequence Analysis of U Small Nuclear RNA Genes in Three Ancistrus Species (Siluriformes: Loricariidae). Zebrafish. 2022 Oct;19(5):200–9.

Sember A, Pelikánová Š, de Bello Cioffi M, Šlechtová V, Hatanaka T, Do Doan H, et al. Taxonomic Diversity Not Associated with Gross Karyotype Differentiation: The Case of Bighead Carps, Genus Hypophthalmichthys (Teleostei, Cypriniformes, Xenocyprididae). Genes (Basel). 2020 Apr;11(5):479.

Seroussi E, Knytl M, Pitel F, Elleder D, Krylov V, Leroux S, et al. Avian Expression Patterns and Genomic Mapping Implicate Leptin in Digestion and TNF Signaling, Suggesting that Their Interacting Adipokine Role is Unique to Mammals. Int J Mol Sci. 2019 Sep;20(18):4489.

Session AM, Uno Y, Kwon T, Chapman JA, Toyoda A, Takahashi S, et al. Genome evolution in the allotetraploid frog Xenopus laevis. Nature. 2016 Oct;538(7625):336–43.

Silva DMZA, Utsunomia R, Pansonato-Alves JC, Oliveira C, Foresti F. Chromosomal mapping of repetitive DNA sequences in five species of astyanax (Characiformes, Characidae) reveals independent location of U1 and U2 snRNA sites and association of U1 snRNA and 5S rDNA. Cytogenet Genome Res. 2015;146(2):144–52.

Sinzelle L, Thuret R, Hwang H-Y, Herszberg B, Paillard E, Bronchain OJ, et al. Characterization of a novel Xenopus tropicalis cell line as a model for in vitro studies. Genesis. 2012 Mar;50(3):316–24.

Tymowska J. Polyploidy and cytogenetic variation in frogs of the genus Xenopus. In: Green DM-, Sessions SK-, editors. Amphibian cytogenetics and evolution. San Diego: Academic Press; 1991; pp 259–97.

Valadkhan S. snRNAs as the catalysts of pre-mRNA splicing. Curr Opin Chem Biol. 2005;9(6):603–8.

Valente GT, Mazzuchelli J, Ferreira IA, Poletto AB, Fantinatti BEA, Martins C. Cytogenetic mapping of the retroelements Rex1, Rex3 and Rex6 among cichlid Fish: New insights on the chromosomal distribution of transposable elements. Cytogenet Genome Res. 2011;133(1):34–42.

Vicari MR, Artoni RF, Moreira-Filho O, Bertollo LAC. Colocalization of repetitive DNAs and silencing of major rRNA genes. A case report of the fish Astyanax janeiroensis. Cytogenet Genome Res. 2008;122(1):67–72.

Volff JN, Körting C, Sweeney K, Schartl M. The non-LTR retrotransposon Rex3 from the fish Xiphophorus is widespread among teleosts. Mol Biol Evol. 1999;16(11):1427–38.

Yampolsky LY. Mutation and Genome Evolution. Encyclopedia of Evolutionary Biology. Elsevier; 2016; pp 77–83.

Zattera ML, Gazolla CB, Soares A de A, Gazoni T, Pollet N, Recco-Pimentel SM, et al. Evolutionary Dynamics of the Repetitive DNA in the Karyotypes of Pipa carvalhoi and Xenopus tropicalis (Anura, Pipidae). Front Genet. 2020;11(July):1–10.

Zhang G, Li B, Li C, Gilbert MTP, Jarvis ED, Wang J. Comparative genomic data of the Avian Phylogenomics Project. Gigascience. 2014;3(1):1–8.

Download PDF

Version 1

posted

You are reading this latest preprint version

Consequences of polyploidy and divergence as revealed by cytogenetic mapping of repetitive elements in African clawed frogs (Xenopus, Pipidae)

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Primary cell cultures and metaphase spread preparations

FISH with repetitive DNA probes

Painting FISH

Results

Sequences and genomic locations of probes in available genome assemblies

FISH with U1 and U2 snDNA probes

Painting FISH

FISH with H3 and Rex3 probes

Discussion

U1 and U2 snRNA repeat loci

Histone H3 and Retroelement Rex3 loci

Co-localization of repetitive elements

Conclusion

Declarations

Statement of Ethics

Conflict of Interest Statement

Funding Sources

Author Contributions

The Sanger sequences that support the findings of this study are openly available in the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/genbank/).

References

Status:

Version 1