The family Cervidae is the second most diverse family in the infraorder Pecora and is characterized by a striking variability in the diploid chromosome numbers among species, ranging from 6 to 70. Chromosomal rearrangements in Cervidae have been studied in detail by chromosome painting. There are many comparative cytogenetic data for both subfamilies (Cervinae and Capreolinae) based on homologies with chromosomes of cattle and Chinese muntjac. Previously it was found that interchromosomal rearrangements are the major type of rearrangements occurring in the Cervidae family. Here, we build a detailed chromosome map of a female reindeer (Rangifer tarandus, 2n = 70, Capreolinae) and a female black muntjac (Muntiacus crinifrons, 2n = 8, Cervinae) with dromedary homologies to find out what other types of rearrangements may have underlined the variability of Cervidae karyotypes. To track chromosomal rearrangements and the distribution of nucleolus organizer regions not only during Cervidae but also Pecora evolution, we summarized new data and compared them with chromosomal maps of other already studied species. We discuss changes in the pecoran ancestral karyotype in the light of new painting data. We show that intrachromosomal rearrangements in autosomes of Cervidae are more frequent than previously thought: at least 13 inversions in evolutionary breakpoint regions were detected.

Artiodactyla is a large mammalian order that includes camels, pigs, whales, hippos, and ruminants (the suborder of animals with divided stomachs). Previously, the name Cetartiodactyla [Montgelard et al., 1997] was used for this order, which was recently suggested to be abandoned [Prothero et al., 2022]. The family Cervidae is the second most diverse family in the suborder Ruminantia [Webb, 2000]. Cervidae includes 2 subfamilies: Cervinae and Capreolinae [Heckeberg, 2020]. Representatives of the family are widespread in America and Eurasia and have high economic (farm animals, hunting) and ecosystem values (food source for carnivores, impact on vegetation).

For decades the systematic relationship in Cervidae has been a hotly debated topic. The original phylogeny based on morphology and comparative anatomy [Gentry et al., 1999] was supported by molecular studies [Heckeberg, 2020]. In addition, recent studies, based on sequences of the complete mitochondrial genome [Hassanin et al., 2012] and on all available data on 318 existing and extinct species [Zurano et al., 2019], significantly clarified not only Cervidae but also Artiodactyla phylogeny. There are also studies integrating molecular, morphological, and bioinformatics approaches [Heckeberg, 2020]. Previously the family was divided into 3 subfamilies Cervinae, Capreolinae, and Hydropotinae, but recent phylogenetic data place Hydropotes inermis, a monotypic Hydropotinae species, in the subfamily Capriolinae [Heckeberg, 2020].

The accumulated cytogenetic data for the Cervidae family allow the evolution of karyotypes to be traced. Cervidae karyotypes are characterized by diversity in the diploid chromosome number (2n = 6–70) [Fontana and Rubini, 1990; Duarte and Jorge, 2003] and have evolved by tandem and robertsonian translocations of acrocentric chromosomes [Rubes et al., 2012]. Karyotypes of Capreolinae are conserved and predominantly represented by an ancestral karyotype (2n = 70) [Fontana and Rubini, 1990; Huang et al., 2006a] in all tribes. Whereas Cervinae karyotypes were formed through multiple changes: robertsonian translocations are the most common type of rearrangements in tribe Cervini, and tandem fusions underlie the karyotypic diversity of Muntiacini [Yang et al., 1997a, b, c; Huang et al., 2006a]. Cervidae karyotypes were studied by comparative chromosome painting using mostly 2 sets of painting probes, cattle [Bonnet-Garnier et al., 2003; Frohlich et al., 2017] and Chinese muntjac probes [Yang et al., 1995, 1997a, b, c; Huang et al., 2006a], which allowed tracing the main trends in karyotype evolution of this family. Muntjacs have been extensively studied in terms of comparative cytogenetics using the localization of the Chinese muntjac painting probes and BAC clones to illustrate fusions and syntenic block orientation on chromosomes [Huang et al., 2006b]. Evolutionary rearrangements between ruminant autosomes are rare, except for centric and tandem fusions [Cernohorska et al., 2011, 2013; Rubes et al., 2012; Frohlich et al., 2017]. Still, comparative maps with Chinese muntjac and cattle probes illustrate only interchromosomal rearrangements. On the contrary, dromedary probes (2n = 74) represent more syntenic segments and can uncover smaller rearrangements, including intrachromosomal ones. Only Siberian roe deer chromosomes were studied by dromedary paints [Dementyeva et al., 2010]. In all painting studies, Cervidae karyotypes were compared to the putative pecoran ancestral karyotype (PAK) [Slate et al., 2002; Kulemzina et al., 2014]. Not only intrachromosomal rearrangements were identified with dromedary paints but also evolutionary breakpoint regions in artiodactyl genomes [Kulemzina et al., 2009; Farré et al., 2019; Proskuryakova et al., 2019]. Information about evolutionary breakpoint regions in ruminant genomes was obtained by bioinformatic genome analysis and BAC clone localization [Farré et al., 2019]. In silico bioinformatic approach provides another source of information about genome evolution – the analysis and detection of cryptic chromosomal divergences. However, so far this strategy is limited to the species with sequenced and well-assembled chromosome-level genomes [Bana et al., 2018; Chen et al., 2019; Mudd et al., 2020].

Still, there are several problems with cervid phylogeny, and some cytogenetic markers can help to resolve them. For example, interspecific variation of the X chromosome provides a rich source of phylogenetic information. There is yet another type of phylogenetic marker on chromosomes: the incorporation of repetitive elements. Just recently research proved the presence of satellite DNA blocks in pericentromeric regions of all analyzed deer species, which resulted in the reconstruction of phylogenetic trees based on the satellite DNA sequences [Vozdova et al., 2020]. Further, the phylogenetic topology was compared with phylogeny based on the mitochondrial cytochrome b gene sequences to infer the evolutionary relationships among Cervidae [Vozdova et al., 2020] and Neotropical deer species [Vozdova et al., 2021] and their position within Capreolinae. The phylogenetic trees constructed based on the satellite I–IV DNA relationships generally support the present cervid taxonomy.

In this article, we summarize the available data on chromosomal painting in Cervidae and provide new comparative chromosome painting data for 2 cervid species with dromedary homologies: reindeer (Rangifer tarandus) and black muntjac (Muntiacus crinifrons). We trace the main trends in chromosomal evolution not only in Cervidae but also in Pecora. We reveal intrachromosomal rearrangements and nucleolus organizer region (NOR) mobility that occurred during chromosomal evolution in the Cervidae family. Our data made it possible not only to trace the evolutionary chromosomal rearrangements that occurred in Cervidae but also to amend the ancestral karyotype of Pecora.

Species

The list of studied species, diploid chromosome numbers, and source of cell lines are given in Table 1. All cell lines are cryopreserved in the cell culture collection of general biological purpose (No. 0310-2016-0002) of the Institute of Molecular and Cellular Biology (IMCB) of Siberian Branch, Russian Academy of Sciences (SB RAS).

Table 1.

List of studied Cervidae species and their characteristics

 List of studied Cervidae species and their characteristics
 List of studied Cervidae species and their characteristics

Chromosome Preparation

Metaphase chromosomes were obtained from fibroblast cell lines. Briefly, cells were incubated at 37°C, 5% CO2 in αMEM medium (Gibco), supplemented with 15% fetal bovine serum (Gibco), 5% AmnioMAX-II complete (Gibco), and antibiotics (ampicillin 100 μg/mL, penicillin 100 μg/mL, amphotericin B 2.5 μg/mL). Metaphases were obtained by adding colcemid (0.02 mg/L) and ethidium bromide (1.5 mg/mL) to an actively dividing culture for 3–4 h. Hypotonic treatment was performed with 0.25% KCl, 0.2% sodium citrate for 20 min at 37°C followed by fixation with 3:1 methanol:glacial acetic acid. Metaphase chromosome preparations were made from a suspension of fixed fibroblasts, as described previously [Yang et al., 1999; Yang and Graphodatsky, 2017]. G-banding on metaphase chromosomes prior to fluorescence in situ hybridization (FISH) was performed using a standard procedure [Seabright, 1971]. Chromomycin A3-DAPI-after G-banding (CDAG) staining procedure was performed as described earlier [Lemskaya et al., 2018].

FISH Probes and Procedure

The characteristics of the set of dromedary chromosome-specific probes were also reported previously [Balmus et al., 2007]. Probes containing ribosomal DNA and telomere repeated sequences were described earlier [Kulemzina et al., 2016; Proskuryakova et al., 2018]. The protocol of selection of BAC clones was reported in previous research [Proskuryakova et al., 2017]. In this study, for validation of the syntenic group of NOR localization, 4 cattle BAC-clones from the CHORI-240 library (218H17, 28J13, 297A4, 437K9) were used.

Dual-color FISH experiments on G-banded metaphase chromosomes were conducted as described by Yang and Graphodatsky [2017]. Digoxigenin-labeled probes were detected using anti-digoxigenin-Cy3 (Jackson Immunoresearch), whereas biotin-labeled probes were detected with avidin-FITC (Vector Laboratories) and anti-avidin FITC (Vector Laboratories). Images were captured and processed using VideoTesT 2.0 Image Analysis System and a Baumer Optronics CCD Camera mounted on an Olympus BX53 microscope (Olympus).

Comparative Chromosome Maps with Dromedary Homologies

To trace chromosome evolution in Cervidae, comparative chromosome maps with dromedary probes were prepared for 2 species from different subfamilies: the black muntjac (Cervinae) and reindeer (Capreolinae). To establish the genome-wide comparative chromosome map of cervids, dromedary painting probes were localized by FISH. Chromosome identification was performed using GTG-banding or inverted DAPI staining.

The karyotype of the female black muntjac (M. crinifrons, MCR), 2n = 8, consists of 6 autosomes and a pair of acrocentric X chromosomes translocated onto pair 4 of autosomes. The Y chromosome is present only in the male karyotype [Yang et al., 1997a]. The low diploid number in MCR reflects the evolutionary tendency of Muntiacini chromosomes to form tandem or centric fusions [Yang et al., 1997a; Frohlich et al., 2017]. The painting with 35 dromedary (CDR) autosomal paints revealed 62 conserved segments in the black muntjac karyotype (Fig. 1a).

Fig. 1.

Comparative chromosome maps with dromedary (Camelus dromedarius, CDR) homologies. a Black muntjac (M. crinifrons). b Reindeer (R. tarandus). c, d Localization of nucleolus organizer regions (18S, 5.8S, and 28S rDNA) and telomeric repeats (TTAGGG) on reindeer (RTA) (с) and on black muntjac (MCR) chromosomes (d). e Chromomycin A3 (CMA3)-DAPI after GTG-banding (CDAG) staining of black muntjac metaphase chromosomes (upper panel) and GTG-banding or inverted DAPI (lower panel). NOR, nucleolus organizer region; H, heterochromatin; tel, telomeric repeats.

Fig. 1.

Comparative chromosome maps with dromedary (Camelus dromedarius, CDR) homologies. a Black muntjac (M. crinifrons). b Reindeer (R. tarandus). c, d Localization of nucleolus organizer regions (18S, 5.8S, and 28S rDNA) and telomeric repeats (TTAGGG) on reindeer (RTA) (с) and on black muntjac (MCR) chromosomes (d). e Chromomycin A3 (CMA3)-DAPI after GTG-banding (CDAG) staining of black muntjac metaphase chromosomes (upper panel) and GTG-banding or inverted DAPI (lower panel). NOR, nucleolus organizer region; H, heterochromatin; tel, telomeric repeats.

Close modal

The reindeer karyotype (R. tarandus, RTA) comprises 70 chromosomes: 34 pairs of acrocentric autosomes, a metacentric Х, and an acrocentric Y chromosome [Graphodatsky et al., 2020]. This diploid number of chromosomes is common for the Capreolinae subfamily [Fontana and Rubini, 1990; Graphodatsky et al., 2020]. Previously, cattle painting probes detected 1 tandem fusion and 6 fissions that formed the reindeer karyotype corresponding to bovine chromosome equivalents [Frohlich et al., 2017]. To check for hidden intrachromosomal rearrangements, dromedary paints were hybridized to RTA chromosomes and this revealed 66 conserved segments (Fig. 1b). Chromosomal maps of black muntjac and reindeer with dromedary homologies are summarized in Figure 1a, b.

NOR and Telomere Repeat Localization in Cervids

The FISH analysis revealed the localization of telomere repeats and ribosomal DNA sequences on black muntjac and reindeer chromosomes (Fig. 1c, d). To localize ribosomal genes and telomeric repeats on the chromosomes of both cervids, hybridization of a probe containing 18S, 5.8S, and 28S rDNA [Maden et al., 1987] and a telomeric probe obtained by template-less synthesis was carried out [Ijdo et al., 1991].

Two NOR signals on MCR1, and 4 signals on RTA1 and RTA2 chromosome pairs were identified. On RTA chromosomes, NORs were identified in subtelomeric regions, whereas in MCR1 they were found on the distal regions of the q arm. Telomeric repeats were situated in the terminal regions of chromosomes in both species.

The distribution of different repeated elements in reindeer chromosomes was investigated previously [Lee et al., 1998; Frohlich et al., 2017]. To reveal the distribution of AT- and GC-enriched heterochromatin, CDAG staining [Lemskaya et al., 2018] on black muntjac chromosomes was performed. Pericentromeric heterochromatic blocks on MCR are enriched by GC-rich sequences (Fig. 1e, GC-enriched heterochromatic blocks are highlighted in green). AT-rich heterochromatin, stained in bright blue, was not detected on the chromosomes of black muntjac. Previously according to the C-banding data, the accumulation of heterochromatin blocks (chromosomes 4, 15, 17, 20, 23, Y) in the tufted deer has been shown [Huang et al., 2006b]. In the Indian muntjac (Muntiacus muntjak), accumulation of heterochromatin is observed only on the X chromosome [Hsu and Benirschke, 1977; Biltueva et al., 2020]. The data obtained in this study and published data indicate a tendency for the accumulation of heterochromatin on the chromosomes of the tribe Muntiacini.

Evolution of Cervidae Karyotypes

Drastic differences in diploid chromosome numbers in muntjacs were noticed in the 1970s, and the cause of such extensive chromosomal exchanges in a short evolutionary time has puzzled cytogeneticists ever since. Black muntjac has the second lowest chromosome number in eutherian mammals after Indian muntjac. The giant chromosomes of black muntjac were formed by centric and tandem fusions as revealed by comparison of banding patterns, Chinese muntjac and Indian muntjac paints [Yang et al., 1995, 1997a], and subsequent mapping of BAC clones [Huang et al., 2006a, b]. The reindeer (RTA) G-banded karyotype was described previously [O’Brien et al., 2006]. Six fissions and 1 fusion were revealed in RTA karyotype using cattle microdissected chromosomes [Frohlich et al., 2017]. Here, by including black muntjac (subfamily Cervinae) and reindeer (subfamily Capreolinae) in a comparative cytogenetics framework based on dromedary/cattle/human paints, we made a complete high-resolution comparative map for both cervid karyotypes using dromedary chromosome-specific probes.

Capreolinae karyotypes are characterized by a high degree of conservation. Previously, studies using dromedary chromosome-specific probes were performed for over a dozen ruminant species but only for 1 Capreolinae species – the Siberian roe deer (Capreolus pygargus, CPY) [Dementyeva et al., 2010]. Thus, the comparative chromosome map of the reindeer obtained by us matches the one obtained earlier for the Siberian roe deer [Dementyeva et al., 2010]. The order of the conserved segments is the same as for the PAK, with the exception of some fissions and fusions [Kulemzina et al., 2014].

The retention of the order of conserved segments on chromosomes corresponding to PAK E (CDR22/3/22/3/22/3), PAK S (CDR30/24/30), PAK L (CDR10/33/10) was shown for reindeer and previously for Siberian roe deer [Dementyeva et al., 2010]. But in black muntjac, we can see another order of conserved segments: MCR4 (CDR24/30), MCR1 (CDR10/33), and MCR3 (CDR22/3/22/3) corresponding to the elements of the Ruminantia ancestral karyotype [Kulemzina et al., 2011]. A similar phenomenon has been described previously for bovids – the ancestral condition of chromosome homologies to PAK E (CDR22/3/22/3/22/3) [Proskuryakova et al., 2019]. To track the evolutionary history of these particular homologous regions we localized painting probes containing dromedary chromosomes 10, 33, 22, 3, 30, and 24 in a range of cervid species (gray mazama, white-tailed deer, sika deer, red deer, tufted deer) and analyzed the results with previous data [Balmus et al., 2007; Murmann et al., 2008; Kulemzina et al., 2009, 2011, 2014; Dementyeva et al., 2010; Cernohorska et al., 2013; Proskuryakova et al., 2019]. The summarized data for different Ruminantia karyotypes are presented in Figure 2.

Fig. 2.

Schematic representation of intrachromosomal rearrangements on the Ruminantia phylogenetic tree [Zurano et al., 2019] (left) based on dromedary chromosome painting data [Balmus et al., 2007; Kulemzina et al., 2009, 2011, 2014; Proskuryakova et al., 2019], ENSEMBL genome browser data, and new data obtained in this study. For other types of rearrangements see Frohlich et al. [2017]. Newly found ancestral associations are highlighted in purple, and inverted regions are highlighted in orange. In the frames, a putative ruminant ancestral karyotype (RAK) [Kulemzina et al., 2011], pecoran ancestral karyotype (PAK) [Kulemzina et al., 2014], and edited pecoran ancestral chromosome associations with dromedary homologies are presented based on our new data and data from Proskuryakova et al. [2019]. In the edited pecoran ancestral karyotype (right), human (HSA) and dromedary (CDR) homologies are shown on the left and on the right of the ancestral blocks, respectively.

Fig. 2.

Schematic representation of intrachromosomal rearrangements on the Ruminantia phylogenetic tree [Zurano et al., 2019] (left) based on dromedary chromosome painting data [Balmus et al., 2007; Kulemzina et al., 2009, 2011, 2014; Proskuryakova et al., 2019], ENSEMBL genome browser data, and new data obtained in this study. For other types of rearrangements see Frohlich et al. [2017]. Newly found ancestral associations are highlighted in purple, and inverted regions are highlighted in orange. In the frames, a putative ruminant ancestral karyotype (RAK) [Kulemzina et al., 2011], pecoran ancestral karyotype (PAK) [Kulemzina et al., 2014], and edited pecoran ancestral chromosome associations with dromedary homologies are presented based on our new data and data from Proskuryakova et al. [2019]. In the edited pecoran ancestral karyotype (right), human (HSA) and dromedary (CDR) homologies are shown on the left and on the right of the ancestral blocks, respectively.

Close modal

Comprehensive analyses of published data [Balmus et al., 2007; Kulemzina et al., 2009, 2011, 2014; Dementyeva et al., 2010; Proskuryakova et al., 2019], ENSEMBL genome browser data, and our new comparative maps show that in pecoran karyotypes more ancient conservative block associations are observed. Most likely, the karyotype of the black muntjac preserved the ancestral condition of chromosomes: PAK L – MCR1 (CDR10/33), as in okapi, giraffe, Siberian musk deer, and cattle; PAK E – MCR3 (CDR22/3/22/3), as in cattle, musk ox, and sheep; and PAK S – MCR4 (CDR24/30), as in musk deer, sika deer, fallow deer, white-tailed deer (Fig. 2). Our new data allow us to amend the previously published PAK [Kulemzina et al., 2014; Farré et al., 2019]. The new putative ancestral conditions of pecoran ancestral elements PAK E, PAK S, and PAK L are shown in the frame in Figure 2.

Previously it was hypothesized that intrachromosomal rearrangements were not characteristic for the evolution of cervid karyotypes [Frohlich et al., 2017] driven by tandem fusions, robertsonian fusions, and fissions [Yang et al., 1995]. Here, we find that inversions in cervids, as well as in other pecoran karyotypes, repeatedly occur in hotspots that likely correspond to evolutionary breakpoint regions [Larkin et al., 2009] situated on chromosome homologs of PAK E (BTA7), PAK S (BTA24), and PAK L (BTA15) [Farré et al., 2019]. Combined data for PAK E, PAK S, and PAK L homologs in both bovids and Muntiacini indicate the convergent nature of these repeated rearrangements with the reuse of the same evolutionary breakpoints.

NOR Distribution in Pecora

Hybridization of probes containing ribosomal genes showed their localization in chromosome 1 in black muntjac and in chromosomes 1 and 2 in reindeer. These genome regions are mobile, and by mapping them, one may reveal how evolutionarily close the studied species are [Gerbault-Seureau et al., 2017]. It was suggested that the distribution of NOR sites could be phylogenetically informative not only for Bovidae but also for Pecora [Gallagher et al., 1999]. Based on an available dromedary and human (Homo sapiens, HSA) comparative chromosome map [Balmus et al., 2007], we established that the NORs in the black muntjac are localized interstitially between HSA1 (CDR23/21/13/21/23) and HSA7 (CDR7); also, in reindeer on HSA1 (CDR21/9/13) and HSA7 (CDR7). There are a lot of comparative and NOR location data of different Artiodactyla species [Di et al., 1993; Gallagher et al., 1998, 1999; Iannuzzi et al., 2001; Balmus et al., 2007; Nguyen et al., 2008; Biltueva et al., 2020]. New and previous data on NOR location are presented in Table 2. We validated homologies with Bos taurus by colocalization experiments with cattle BACs and NOR-containing probes.

Table 2.

Summary of NOR distribution in different pecoran species

 Summary of NOR distribution in different pecoran species
 Summary of NOR distribution in different pecoran species

In the majority of Artiodactyla, NORs are located at the ends of chromosomes. The interstitial position of the NOR in muntjacs could be a result of tandem fusions. Two interstitial NORs were described in the Indian muntjac (M. muntjak, MMU) on MMU1 (between HSA1 and HSA19/5), MMUХ, and Y1 (between HSA1 and HSA7) [Biltueva et al., 2020]. Our summary in Table 2 indicates that some NOR localization sites occur on the same syntenic groups in Pecora homologous to the following human chromosomes: HSA7, HSA1, HSA2/1, HSA9/5, and HSA10.

One common place of NOR location in Pecora is in the HSA7 homeolog. It is described for cattle [Gallagher et al., 1999], sheep [Di et al., 1993; Iannuzzi et al., 2001], and 3 cervid species: Indian muntjac [Biltueva et al., 2020], black muntjac, and reindeer. NORs are located on another synteny – HSA1 (BTA3, CDR21/9/13) in cattle (B. taurus, Bovidae) [Gallagher et al., 1999], sheep (Ovis aries, Caprinae) [Iannuzzi et al., 2001], reindeer, and Indian muntjac [Biltueva et al., 2020]. Localization near another region of HSA1 (BTA16, CDR23/21/13/21/23) as in MCR is observed in sika deer (Cervus nippon, Cervinae) [Gallagher et al., 1999].

It is possible that the NOR was located on the putative artiodactyl (сetartiodactyl) ancestral chromosome 1 (HSA1) [Kulemzina et al., 2009, 2011]. During karyotype evolution, this ancestral element has undergone multiple inversions, a fusion, and a fixation of NOR in different parts of the HSA1 syntenic block. A similar location of NOR on homologs of HSA1 in distantly related artiodactyl species (boar and dromedary) [Balmus et al., 2007] indicates the conserved localization of NORs inside the order, as well as the possible location of NOR in the common ancestor of Artiodactyla. In Pecora, NOR shows on HSA1 homologs (CDR23/21/13/21/23, CDR21/9/13, CDR13) at least in 8 species from different families. The NOR in Pecora also repeatedly occurs on several other syntenic groups: HSA7, HSA19/5, and HSA10 (Table 2). The NOR occurrence in multiple places in Pecora may be related to the retrotransposon insertion in an intergenic spacer of the ribosomal gene clusters as suggested by Raskina et al. [2008].

High-resolution chromosome maps of 2 cervid species, i.e., reindeer and black muntjac, provide unique information about the evolution of intrachromosomal rearrangements. In this research, we traced intrachromosomal rearrangements and NOR distribution not only in Cervidae but also in pecoran karyotype evolution. By summarizing the newly and previously published data, we discuss changes in the PAK in the light of new data. We also show that intrachromosomal rearrangements on autosomes in Cervidae are more frequent than previously thought: at least 13 inversions repeated in several hotspots during cervid karyotype evolution were detected.

We kindly acknowledge Mary Thompson for establishing the cell lines at the Laboratory of Genomic Diversity, NCI-Frederick, MD, USA. We would like to acknowledge the Director of Catoctin Wildlife Preserve and Zoo, Richard Hahn. We kindly acknowledge Olga Uphyrkina Federal Research Center for Biodiversity of the Terrestrial Biota of East Asia, Vladivostok, Russia for sika deer biopsy and Lutz Froenicke from the Davis Genome Center, CA, USA and Roscoe Stanyon from Institute of Florence, Florence, Italy for the white-tailed deer cell line.

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All protocols (all studies/manipulations etc.) were approved by the Ethics Committee on Animal and Human Research of the Institute of Molecular and Cellular Biology (Novosibirsk, Russia), protocol No. 01/21 from January 26, 2021. This article does not contain any studies with human participants performed by any of the authors.

The authors have no conflicts of interest to declare.

The work was supported by a research grant of the Russian Science Foundation (RSF, 19-14-00034-П).

Conceptualization: Alexander S. Graphodatsky, Anastasia A. Proskuryakova. Investigation: Anastasia A. Proskuryakova, Ekaterina S. Ivanova. Methodology: Anastasia A. Proskuryakova, Ekaterina S. Ivanova, Polina L. Perelman. Formal analysis: Anastasia A. Proskuryakova, Ekaterina S. Ivanova. Funding acquisition: Alexander S. Graphodatsky. Resources: Malcolm A. Ferguson-Smith, Fentang Yang, Polina L. Perelman, Innokentiy M. Okhlopkov. Supervision: Alexander S. Graphodatsky, Anastasia A. Proskuryakova. Visualization: Anastasia A. Proskuryakova, Ekaterina S. Ivanova. Writing – original draft: Anastasia A. Proskuryakova. Writing review and editing: Anastasia A. Proskuryakova, Ekaterina S. Ivanova, Polina L. Perelman, Malcolm A. Ferguson-Smith, Fentang Yang. All authors have read and agreed to the published version of the manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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