Comparative Chromosome Mapping of Musk Ox and the X Chromosome among Some Bovidae Species

Bovidae, the largest family in Pecora infraorder, are characterized by a striking variability in diploid number of chromosomes between species and among individuals within a species. The bovid X chromosome is also remarkably variable, with several morphological types in the family. Here we built a detailed chromosome map of musk ox (Ovibos moschatus), a relic species originating from Pleistocene megafauna, with dromedary and human probes using chromosome painting. We trace chromosomal rearrangements during Bovidae evolution by comparing species already studied by chromosome painting. The musk ox karyotype differs from the ancestral pecoran karyotype by six fusions, one fission, and three inversions. We discuss changes in pecoran ancestral karyotype in the light of new painting data. Variations in the X chromosome structure of four bovid species nilgai bull (Boselaphus tragocamelus), saola (Pseudoryx nghetinhensis), gaur (Bos gaurus), and Kirk’s Dikdik (Madoqua kirkii) were further analyzed using 26 cattle BAC-clones. We found the duplication on the X in saola. We show main rearrangements leading to the formation of four types of bovid X: Bovinae type with derived cattle subtype formed by centromere reposition and Antilopinae type with Caprini subtype formed by inversion in XSB1.

chromosome fusions [14] in musk ox karyotype. Moreover, there is a huge pool of data describing cetariodactyl karyotypes using chromosome painting [14,28], especially using human and dromedary probes [5,29,30]. Here, we used the combination of these human and dromedary painting probes to establish a detailed comparative chromosome map for musk ox to interpret the descent of this species' genome organization in an evolutionary context.

Species
The list of studied species, diploid chromosome number, and the source of cell lines are presented in Table 1

FISH Probes
The protocol for the selection of BAC-clones was reported previously [15]. Briefly, we selected 26 BAC clones highly conserved among Cetartiodactyla from bovine CHORI-240 library using bioinformatic tools. BAC DNA was isolated using the Plasmid DNA Isolation Kit (BioSilica, Novosibirsk, Russia) and amplified with GenomePlex Whole Genome Amplification kit (Sigma-Aldrich Co., St. Louis, MO, USA). Labeling of BAC DNA was performed using GenomePlex WGA Reamplification Kit (Sigma-Aldrich Co., St. Louis, MO, USA) by incorporating biotin-16-dUTP or digoxigenin-dUTP (Roche, Basel, Switzerland). The list of BAC-clones is shown in Table 2. Plasmid containing ribosomal DNA [35] was amplified and labeled as described above. Telomere repeats were synthesized and labeled in non-template PCR using primers (TTAGGG)5 and (CCCTAA)5 [36]. Human and dromedary chromosome-specific probes were described previously [6,32] and were labeled by DOP-PCR [37] with biotin-16-dUTP or digoxigenin-dUTP (Roche, Basel, Switzerland).

FISH Procedure
Dual-color FISH experiments on G-banded metaphase chromosomes were conducted as described by Yang and Graphodatsky [32]. Tripsin-treated chromosomes were immobilized in 0.5% formaldehyde in PBS followed by formamid denaturing and overnight probe hybridization at 40 • C. Digoxigenin-labeled probes were detected using anti-digoxigenin-CyTM3 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, whereas biotin-labeled probes were identified with avidin-FITC (Vector Laboratories) and anti-avidin FITC (Vector Laboratories, Inc., Burlingame, CA, USA). Images were captured and processed using VideoTesT 2.0 Image Analysis System (Zenit, St. Petersburg, Russia) and a Baumer Optronics CCD camera mounted on a BX53 microscope (Olympus, Shinjuku, Japan).

Comparative Chromosome Map of Musk Ox, Dromedary, and Human
The musk ox karyotype includes six submetacentric and 17 acrocentric autosomes and one sex chromosomal pair (2N = 48) ( Figure 1). The fundamental number of autosomal arms in musk ox is 58, which in general is characteristic for karyotypes of the Bovidae family [18]. To establish the genome-wide chromosome comparative map of the musk ox, human and dromedary painting probes were used. The chromosome map ( Figure 1) and additional comparison with cattle chromosomes (the reference karyotype for ruminants) and with pecoran ancestral karyotype (PAK) are summarized in Table 2. The painting probes from 22 human (HSA) and 35 dromedary (CDR) autosomal paints revealed 51 and 61 conserved segments on the musk ox karyotype, respectively.   We studied the distribution of repeated sequences in the musk ox karyotype using several methods. FISH analysis revealed the localization of telomere repeat and ribosomal DNA sequences. Six nucleolar organizing regions (NORs) with telomeric localization on OMO 1, OMO 2 and OMO 8 chromosome pairs were identified ( Figure 1). Telomeric repeats are situated on terminal regions of chromosomes. The CDAG differential staining revealed centromeric and telomeric GC-enriched heterochromatin with prominent AT-and GC-enriched pericentromeric blocks of heterochromatin on acrocentric chromosomes ( Figure 2). Smaller blocks of heterochromatin were observed on metaand submetacentric chromosomes, except chromosome 6, where a large block of heterochromatin was identified. Enlarged telomeric blocks were observed on at least 3 pairs of autosomes, while only two pairs of small acrocentrics appeared to have repeated sequences distributed over the whole chromosome.

Mapping of the X Chromosome in Bovidae
To investigate the order of conserved syntenic segments on X chromosomes in the Bovidae family, 26 BAC-clones were localized using FISH on X chromosomes of four species (nilgai bull, saola, gaur, and Kirk's Dikdik) in a series of pairwise FISH experiments (Table 3). In all Bovinae species, similar order of BAC-clones was observed. The same order was observed earlier in Antilopinae subfamily, except for the marker inversion in Caprini [15]. In total, comparative analysis of BAC order revealed identical syntenic blocks: X Syntenic Block 1 (13 BACs, XSB1), X Syntenic Block 2 (7 BACs, XSB2), and X Syntenic Block 3 (6 BACs, XSB3) [15]. Two types of chromosome X changes were identified in a course of Bovidae evolution: a centromere reposition, and inversions of an entire syntenic block. Interestingly, a segmental duplication in XSB3 containing CH-108D16 was detected on saola X ( Figure 3).  We studied the distribution of repeated sequences in the musk ox karyotype using several methods. FISH analysis revealed the localization of telomere repeat and ribosomal DNA sequences. Six nucleolar organizing regions (NORs) with telomeric localization on OMO 1, OMO 2 and OMO 8 chromosome pairs were identified ( Figure 1). Telomeric repeats are situated on terminal regions of chromosomes. The CDAG differential staining revealed centromeric and telomeric GC-enriched heterochromatin with prominent AT-and GC-enriched pericentromeric blocks of heterochromatin on acrocentric chromosomes ( Figure 2). Smaller blocks of heterochromatin were observed on metaand submetacentric chromosomes, except chromosome 6, where a large block of heterochromatin was identified. Enlarged telomeric blocks were observed on at least 3 pairs of autosomes, while only  Table 3. The order of 26 CHORI-240 BACs on Bovidae X chromosomes. The color of the cells corresponds to a given conserved syntenic segment. To display the complete scheme of evolution in Bovidae, the X chromosome maps published previously are also presented [15]. The region duplicated in saola and inverted in Caprini is labeled with a lighter colour.  Table 3. The order of 26 CHORI-240 BACs on Bovidae X chromosomes. The color of the cells corresponds to a given conserved syntenic segment. To display the complete scheme of evolution in Bovidae, the X chromosome maps published previously are also presented [15]. The region duplicated in saola and inverted in Caprini is labeled with a lighter colour.

Syntenic Block X BAC's Order in Bovinae Subfamily X BAC's Order in Antilopinae Subfamily In most Bovinae
In saola Caprini tribe Hippotragini and Antilopini tribe
Here, a complete high-resolution comparative map for musk ox karyotype was obtained using human and camel chromosome specific probes and compared to cattle karyotype ( Table 2). Our results are in agreement with previous publications showing the origin of musk ox submetacentric chromosomes [14].
We show the presence of prominent heterochromatin blocks at centromeric positions in the musk ox karyotype. Many bovid species are characterized by prominent pericentromeric blocks of heterochromatin and their karyotype evolution is marked by frequent occurrence of Robertsonian fusions [2,18]. Several hypotheses point to the role that repetitive sequences may have in driving chromosome evolution in bovids by increasing the occurrence of Robertsonian translocations due to the physical proximity of centromeres of acrocentric chromosomes during meiosis [38]. As shown previously, repetitive sequences were involved in formation of Robertsonian translocations in mice [38]. Therefore, the presence of heterochromatic blocks on acrocentric chromosomes in bovid species may contribute to the high variability of bovid karyotypes, including the occurrence of cytotypes in many species, high frequency of Robertsonian fusions, and autosome to the X chromosome translocations.
The musk ox is a representative of subfamily Antilopinae, Caprini tribe. Both comparative linkage and FISH maps showed one major distinction between ovine (Antilopinae, Caprini) and bovine (Bovinae) karyotypes. This difference resulted from a translocation involving segments homologous to BTA 9 and BTA 14 [4,7,39]. However, this association is not observed in musk ox. Presumably, this is determined by the basal position of Ovibovina [40], suggesting that the BTA 9/14 translocation occurred only in Caprina subtribe. confirmed by ENSEMBLE genome browser data, also blast data of alpaca RH markers from the chromosome homologous to CDR22 show homology with BTA20 (unpublished data). The order of conservative segments on BTA7 is HSA19p/5 and CDR22/3/22/3. These data differ from research published previously reporting HSA5/19p/5 and CDR3/22/3/22 [6].
The ancestral form of PAC A2 is similar to pronghorn (AAM) 1q [5]. However, other bovid species and Moschidae (Siberian musk deer) [30] showed an inversion changing the order of homologous segments into HSA 21/3/21 ( Figure 4). Therefore, this inversion likely represents a cytogenetic marker for at least Bovidae+Moschidae. The verification of this human syntenic association is needed in Cervidae where the fission of the synteny was shown for Muntiacus muntjac [41]. Although Cervidae have been well studied with bovid [42] and muntjac [41] probes, comparison to human probes is still unknown, hindering the deduction of ancestral rearrangements.
During FISH experiments on the localization of CDR 22 on OMO chromosomes, an additional small region of homology on OMO 22 was detected. This region was also detected in other Bovidae species: cow, sheep ( Figure 5), and saola [5]. The sequence homologies of HSA5 = BTA20 are confirmed by ENSEMBLE genome browser data, also blast data of alpaca RH markers from the chromosome homologous to CDR22 show homology with BTA20 (unpublished data). The order of conservative segments on BTA7 is HSA19p/5 and CDR22/3/22/3. These data differ from research published previously reporting HSA5/19p/5 and CDR3/22/3/22 [6].

Bovine X Chromosome Evolution
The family Bovidae includes two major branches: Bovinae and Antilopinae [40]. Earlier cytogenetic studies identified three types of morphological diversity of the X chromosome in Bovidae: an antilopinae type (acrocentric), a tragelaphines type (acrocentric), and a cattle type (submetacentric) [20]. Tragelaphines chromosome X was likely formed from the ancestral pecoran X by two inversions. This type is ancestral in Bovinae and presented in nilgai, saola (Figure 6), and domestic river buffalo [44]. The X chromosome in nilgai and saola are marked by several morphological features. The first one is an autosomal translocation onto chromosome X in nilgai karyotype. In Tragelaphini and Boselaphini tribes, independent autosomal translocations were observed [9,23,45,46]. Such rearrangements have an impact on the behavior of chromosomes in meiosis, manifested as a lowering of synapsis in the pseudoautosomal region [45]. The second one is a segmental duplication of sequences homologous to CH-108D16 in saola (PNG) ( Table 3). Unfortunately, we cannot determine whether this segmental duplication is characteristic for the entire species due to the lack of information of other individuals. However, the duplicated region contains genes responsible for intrauterine development and may have an adaptive value.
The cattle subtype of the Х chromosome is formed by centromere reposition of the ancestral X chromosome in Bovinae. This type of the X chromosome is presented in cattle, American bison [15], and gaur ( Figure 6). Thus, it appears to be characteristic not only of cattle, but of the whole subtribe Bovina.
The centromere reposition and one inversion resulted in the formation of an acrocentric caprine type of the X chromosome [15]. This type of the X chromosome is retained in Kirk's Dikdik and sable antelope [15]. We suggest calling this type of bovid the X -Antilopine type. Another inversion, which occurred within the XSB3 in the Caprini lineage, is an apomorphic phylogenetic marker for this tribe [15] and marks the formation of the Caprini subtype of X. The mapping of Panthalops hodgsonii X chromosome would assert this type for the whole Caprini tribe.
In general, the X chromosome is highly conserved in eutherians [19], but several different types The split of the ancestral element PAK U is shown in musk ox and also in PNG, BTA, OAR, and DHU karyotypes, thus suggesting that this fission is a marker for the Bovidae lineage [5].

Bovine X Chromosome Evolution
The family Bovidae includes two major branches: Bovinae and Antilopinae [40]. Earlier cytogenetic studies identified three types of morphological diversity of the X chromosome in Bovidae: an antilopinae type (acrocentric), a tragelaphines type (acrocentric), and a cattle type (submetacentric) [20]. Tragelaphines chromosome X was likely formed from the ancestral pecoran X by two inversions. This type is ancestral in Bovinae and presented in nilgai, saola (Figure 6), and domestic river buffalo [44]. The X chromosome in nilgai and saola are marked by several morphological features. The first one is an autosomal translocation onto chromosome X in nilgai karyotype. In Tragelaphini and Boselaphini tribes, independent autosomal translocations were observed [9,23,45,46]. Such rearrangements have an impact on the behavior of chromosomes in meiosis, manifested as a lowering of synapsis in the pseudoautosomal region [45]. The second one is a segmental duplication of sequences homologous to CH-108D16 in saola (PNG) ( Table 3). Unfortunately, we cannot determine whether this segmental duplication is characteristic for the entire species due to the lack of information of other individuals. However, the duplicated region contains genes responsible for intrauterine development and may have an adaptive value.
Genes 2019, 10 FOR PEER REVIEW 11 Overall, we can distinguish four types of the X chromosome in Bovidae: Bovinae type with derived cattle subtype; and Antilopinae type with Caprini subtype. The Bovinae type was formed from the ancestral pecoran X by two inversions, whereas the Antilopinae type was formed by inversion and centromere reposition. The Cattle and Caprini subtypes were created by centromere repositions and inversion in XSB3, respectively. Figure 6. Changes in the structure of the Bovidae X chromosome are depicted on the phylogenetic tree of the family (the tree topology is from [40]). PAX is Pecoran ancestral X chromosome [15]. Major conservative segments are shown in yellow, blue, and pink. Centromere positions are designated by a black circle. White arrowheads show the orientation of the conservative segments. Chromosome changes are shown on phylogenetic tree near respective branches: CR-centromere reposition; and Inv-inversion. Frames show types and subtypes of the bovid X chromosome. The timescale is in million years (MY) of evolution. Nilgai bull X chromosome is shown without autosomal translocation.

Conclusions
Detailed comparative maps were obtained for musk ox karyotype and X chromosomes of four bovids: Kirk's Dikdik, gaur, saola, and nilgai bull. Large structural rearrangements leading to the formation of the karyotype of the musk ox were shown. In general, its karyotype is close to the putative ancestral karyotype of Pecora infraorder. The detailed analysis of the BAC-clones order across four species and published data allowed illustrating chromosomal rearrangements during the Figure 6. Changes in the structure of the Bovidae X chromosome are depicted on the phylogenetic tree of the family (the tree topology is from [40]). PAX is Pecoran ancestral X chromosome [15]. Major conservative segments are shown in yellow, blue, and pink. Centromere positions are designated by a black circle. White arrowheads show the orientation of the conservative segments. Chromosome changes are shown on phylogenetic tree near respective branches: CR-centromere reposition; and Inv-inversion. Frames show types and subtypes of the bovid X chromosome. The timescale is in million years (MY) of evolution. Nilgai bull X chromosome is shown without autosomal translocation.
The cattle subtype of the Хchromosome is formed by centromere reposition of the ancestral X chromosome in Bovinae. This type of the X chromosome is presented in cattle, American bison [15], and gaur ( Figure 6). Thus, it appears to be characteristic not only of cattle, but of the whole subtribe Bovina.
The centromere reposition and one inversion resulted in the formation of an acrocentric caprine type of the X chromosome [15]. This type of the X chromosome is retained in Kirk's Dikdik and sable antelope [15]. We suggest calling this type of bovid the X -Antilopine type. Another inversion, which occurred within the XSB1 in the Caprini lineage, is an apomorphic phylogenetic marker for this tribe [15] and marks the formation of the Caprini subtype of X. The mapping of Panthalops hodgsonii X chromosome would assert this type for the whole Caprini tribe.
In general, the X chromosome is highly conserved in eutherians [19], but several different types of chromosome rearrangements on the cetartiodactyl X have been shown [15]. It was suggested that the evolutionary chromosome rearrangements may reduce gene flow by suppressing recombination and contributing to species isolation [47]. However, in ruminant species, several evolutionary breakpoint regions (EBR) on the X chromosome associated with enhancers were described that may change gene expression [16]. Therefore, these rearrangements may have an adaptive value and an evolutionary meaning.
Overall, we can distinguish four types of the X chromosome in Bovidae: Bovinae type with derived cattle subtype; and Antilopinae type with Caprini subtype. The Bovinae type was formed from the ancestral pecoran X by two inversions, whereas the Antilopinae type was formed by inversion and centromere reposition. The Cattle and Caprini subtypes were created by centromere repositions and inversion in XSB1, respectively.

Conclusions
Detailed comparative maps were obtained for musk ox karyotype and X chromosomes of four bovids: Kirk's Dikdik, gaur, saola, and nilgai bull. Large structural rearrangements leading to the formation of the karyotype of the musk ox were shown. In general, its karyotype is close to the putative ancestral karyotype of Pecora infraorder. The detailed analysis of the BAC-clones order across four species and published data allowed illustrating chromosomal rearrangements during the formation of four main types of X chromosomes in the Bovidae family. In summary, conservation in BACs order was shown in the Bovinae and Antilopinae subfamilies.