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

Abstract

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 XSB3.

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G C A T
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Article
Comparative Chromosome Mapping of Musk Ox and
the X Chromosome among Some Bovidae Species
Anastasia A. Proskuryakova 1, * , Anastasia I. Kulemzina 1, Polina L. Perelman 1,2 ,
Dmitry V. Yudkin 3, Natalya A. Lemskaya 1,3, Innokentii M. Okhlopkov 4, Egor V. Kirillin 4,
Marta Farré5,6, Denis M. Larkin 5, Melody E. Roelke-Parker 7, Stephen J. O’Brien 8,9,
Mitchell Bush 10 and Alexander S. Graphodatsky 1,2
1
Institute of Molecular and Cellular Biology, SB RAS, 630090 Novosibirsk, Russia; zakal@mcb.nsc.ru (A.I.K.);
polina.perelman@gmail.com (P.L.P.); lemnat@mcb.nsc.ru (N.A.L.); graf@mcb.nsc.ru (A.S.G.)
2Novosibirsk State University, 630090 Novosibirsk, Russia
3
State Research Center of Virology and Biotechnology “Vector”, Federal Service for Surveillance on Consumer
Rights Protection and Human Well-being (FBRI SRC VB “Vector”, Rospotrebnadzor),
630559 Koltsovo, Novosibirsk Region, Russia; yudkin_dv@vector.nsc.ru
4Institute for Biological Problems of Cryolithozone Siberian Branch of RAS, 677980 Yakutsk, Russia;
imo-ibpc@yandex.ru (I.M.O.); e.kir@mail.ru (E.V.K.)
5The Royal Veterinary College, Royal College Street, University of London, London NW1 0TU, UK;
mfarrebelmonte@gmail.com (M.F.); dlarkin@rvc.ac.uk (D.M.L.)
6School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK
7Frederick National Laboratory of Cancer Research, Leidos Biomedical Research, Inc.,
Frederick, MD 21701, USA; melody.roelke-parker@nih.gov
8Theodosius Dobzhansky Center for Genome Bioinformatics, Saint-Petersburg State University,
Sredniy Av. 41A, 199034 Saint-Petersburg, Russia; lgdchief@gmail.com
9Oceanographic Center, Nova Southeastern University, Fort Lauderdale 3301 College Ave,
Fort Lauderdale, FL 33314, USA
10 The Center for Species Survival, Department of Reproductive Sciences, Smithsonian Conservation Biology
Institute, Smithsonian’s National Zoological Park, 1500 Remount Road, Front Royal, VA 22630, USA;
giraffedoc@hotmail.com
*Correspondence: andrena@mcb.nsc.ru; Tel.: +79-60-7995-653
Received: 17 September 2019; Accepted: 24 October 2019; Published: 29 October 2019


Abstract:
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.
Keywords: BAC-clones; chromosome painting; Kirk’s Dikdik; musk ox; saola; nilgai bull; gaur
Genes 2019,10, 857; doi:10.3390/genes10110857 www.mdpi.com/journal/genes

Genes 2019,10, 857 2 of 14
1. Introduction
Cetartiodactyla is a large mammalian order, including camels, whales, pigs, hippos,
and ruminants—the suborder of animals with divided stomach. Bovidae is the most specious
family in Ruminantia comprising 143 species with 50 genera [
1
]. Bovidae is generally subdivided into
2 subfamilies: Bovinae (the bovids: tribes Bovini, Tragelaphini and Boselaphini), represented by nilgai,
four-horned antelope, wild cattle, bison, Asian buffalo, African buffalo, and kudu; and Antilopinae
(antelopes and caprini: Neotragini, Aepycerotini, Cephalophini, Oreotragini, Hippotragini, Alcelaphini,
Caprini, Antilopini, and Reduncini tribes), represented by antelopes, gazelles, goats and their
relatives [
2
]. Bovids include several domesticated species (cattle, goat, and sheep) with high
economic significance. Although recent advances have been made in the genomic inference of these
species, phylogenetic relationships of species within the family are complex and remain somewhat
unresolved [3].
Accumulated cytogenetic data for the Bovidae family allow tracing the trends in evolution of
karyotypes of the Bovinae [
4
–
7
] and Antilopinae [
7
–
11
] subfamilies. Bovid karyotypes are characterized
by tandem and Robertsonian translocations of acrocentric chromosomes, producing wide variation
in (2n) chromosome numbers [
12
,
13
]. Previously, some 43 bovid species have been studied by
comparative chromosome painting, mostly with cattle painting probes [
14
]. Due to its economic
importance, the cattle genome has been widely studied, identifying interchromosome rearrangements
between species but resolving few intrachromosomal rearrangements. The use of high-resolution
dromedary probes [
5
] and BAC mapping [
9
,
15
] has provided increased precision with respect to
syntenic segments orientation relative to centromeres and inversions. Proposed ancestral Bovidae and
pecoran karyotypes were imputed based on genomic [
16
] and cytogenetic data [
5
,
16
,
17
]. Although the
diploid chromosome number of Bovidae species ranges from 30 to 60, the number of autosomal arms
in karyotypes is stable at 58 for most karyotyped species [
18
]. Comparative chromosome painting
data have been applied to phylogenetic analyses of relationships between tribes. For example, a clear
marker association was detected for Tragelaphini: the translocation of cattle chromosomes 1;29 [
8
],
which is shared by all members of this group. Because large chromosomal rearrangements by and
large correspond in a parsimony sense to morphology-based phylogenies for the group, it seems that
chromosomal rearrangements played an important role in the speciation of the Bovidae family [12].
The X chromosome in the Bovidae family presents a special case in Bovidae evolution. displaying
marked karyotypic variability between species, in contrast to highly conserved X chromosome
morphology seen for the majority of eutherian mammals [
19
]. Surprisingly, unlike conserved X
chromosome of the majority of eutherian mammals noted by Ohno [
19
], bovid chromosome X
displayed variability. Several types of the X chromosome were identified: the cattle type appeared to
be submetacentric, while the tragelaphines and antilopinae was acrocentric [20].
Cytogenetic maps of Bos taurus,Bubalus bubalis, and Ovis aries X chromosomes, when compared with
that of Homo sapiens, revealed the conservation of pseudoautosomal region position in the Antilopinae
subfamily, which was detected using a molecular cytogenetics approach [
21
,
22
]. In Ruminants and,
especially, in the Bovidae family, a substantial number of intrachromosomal rearrangements, including
inversions and centromere repositions, have been identified previously by using comparative bacterial
artificial chromosome (BAC) mapping [
15
]. The cited research, however, described species from only
three tribes: Bovini, Hippotragini, and Caprini. Moreover, several independent bovid lineages show the
presence of compound sex chromosomes resulting from gonosome and autosome fusions [
8
,
14
,
20
,
21
,
23
].
Here, we extend the list of 18 species studied by detailed X chromosome BAC mapping to include
species from three tribes: nilgai bull (Boselaphini), saola, gaur (Bovini), and Kirk’s Dikdik (Antilopini)
to reveal intrachromosomal rearrangements that occurred on the X chromosome in Bovidae family.
Among species of Bovidae family, musk ox (Ovibos moschatus), deserves special attention.
This species belongs to a basal branch of the Caprini tribe [
3
], and along with reindeer, they are
the only ungulates of the Arctic to survive the late Pleistocene extinction associated the most recent
retreat from glaciation [
24
]. There are classic cytogenetic [
25
–
27
] and molecular cytogenetic data on

Genes 2019,10, 857 3 of 14
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.
2. Material and Methods
2.1. Species
The list of studied species, diploid chromosome number, and the source of cell lines are
presented in Table 1. All cell lines belong to the cell culture collection of general biological purpose
(No. 0310-2016-0002) of the Institute of Molecular and Cellular Biology (IMCB) of the Siberian Branch,
Russian Academy of Sciences (SB RAS).
Table 1. The list of Bovidae cell lines used in this study.
Scientific Name,
Abbreviation
Common
Name Subfamily Diploid
Number Sample/Cell Line Source/Acknowledgment
Ovibos moschatus, OMO Musk ox Antilopinae
(Caprini) 48, XX Allaikhovsky District, Sakha Republic, Yakutia, Russia. IMCB SB
RAS, Novosibirsk
Ovis aries musimon, OAR Sheep Antilopinae
(Caprini) 54, XX
Melody Roelke and June Bellizzi, Catoctin Wildlife Preserve and
Zoo, Maryland, USA; Laboratory of Genomic Diversity,
NCI-Frederick, MD, USA
Madoqua kirkii, MKI Kirk’s Dikdik Antilopinae
(Antilopini) 48, XY
Mitchell Bush, Conservation and Research Center, National
Zoological Park, Virginia, USA; Laboratory of Genomic Diversity,
NCI-Frederick, MD, USA
Bos taurus, BTA Cattle Bovinae (Bovini) 60, XX IMCB SB RAS, Novosibirsk.
Bos gaurus, BGA Gaur Bovinae
(Bovini) 58, XX
Doug Armstrong, Henry Doorly Zoo, Omaha, NE, USA; Laboratory
of Genomic Diversity, NCI-Frederick, MD, USA
Pseudoryx nghetinhensis, PNG
Saola Bovinae
(Bovini) 50, XX [5]
Boselaphus tragocamelus, BTR Nilgai bull Bovinae
(Boselaphini) 44, X+14, X+14
Melody Roelke and June Bellizzi, Catoctin Wildlife Preserve and
Zoo, Maryland, USA; Laboratory of Genomic Diversity,
NCI-Frederick, MD, USA
2.2. Chromosome Preparation
Metaphase chromosomes were obtained from fibroblast cell lines. Briefly, cells were incubated
at 37
◦
C in 5% CO2 in medium
α
MEM (Gibco), supplemented with 15% fetal bovine serum (Gibco),
5% AmnioMAX-II complete (Gibco) and antibiotics (ampicillin 100
µ
g/mL, penicillin 100
µ
g/mL,
amphotericin B 2.5
µ
g/mL). Metaphases were obtained by adding colcemid (0.02 mg/L) and ethidium
bromide (1.5 mg/mL) to actively dividing culture for 3–4 hours. Hypotonic treatment was performed
with 3 mM KCl, 0.7 mM sodium citrate for 20 min at 37
◦
C and followed by fixation with
3:1 methanol:glacial acetic acid (Carnoy’s) fixative. Metaphase chromosome preparations were
made from a suspension of fixed fibroblasts, as described previously [
31
,
32
]. G-banding on metaphase
chromosomes prior to fluorescence in situ hybridization (FISH) was performed using the standard
procedure [
33
]. Heterochromatin analysis was performed by the Combined Method of Heterogeneous
Heterochromatin Detection (CDAG) [
34
]. AT- and GC-enriched repetitive sequences were detected
by DAPI (40-6-diamidino-2-phenylindol) and CMA3 (chromomycin A3) fluorescent dyes following
formamide denaturation and renaturation in hot salt solution.
2.3. 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

Genes 2019,10, 857 4 of 14
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).
2.4. 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).
3. Results
3.1. 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.
Genes 2019, 10 FOR PEER REVIEW 5
Figure 1. Comparative chromosome painting map of musk ox with homologies to human (HSA) and
dromedary (CDR). Nucleolar organizing regions (NOR) show the localization of the nucleolar
organizing region.
Figure 2. Chromomycin A3-DAPI after G-banding (CDAG) staining performed on metaphase
chromosomes of musk ox: GTG-banding (left) and CMA3/DAPI-staining after denaturation and
renaturation procedure (right).
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 meta-
and 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
Figure 1.
Comparative chromosome painting map of musk ox with homologies to human (HSA)
and dromedary (CDR). Nucleolar organizing regions (NOR) show the localization of the nucleolar
organizing region.

Genes 2019,10, 857 5 of 14
Table 2.
Correspondence between conserved chromosomal segments in musk ox (OMO), human (HSA),
dromedary (CDR), cattle (BTA) and Pecoran ancestral karyotype (PAK) [
5
] revealed by chromosome
painting. The order of conservative segments is started from centromere.
OMO HSA CDR BTA PAK
1p 22q’/12q”/4pq 32/2 17 N1
1q 21/3/21 1 1 A2
2p 6p 20 23 R
2q 2q”/1 5/13 2 B2
3p 16p/7 18 25 T
3q
12pq’/22q”/12pq’/22q”
12/34/12 5 C2
4p 4/8p” 26 27 V
4q 2pq/9 28/15/28/15/4 11 C1
5p 11 10/33/10 29 W
5q 18 30/24/30 24 S
6p 10q 11 28 U
6q 3 17 22 Q
7 4pq 2 6 F
8 19p/5 22/3/22/3 7 E
9 1 21/9/13 3 A1
10 8p’/9/8p’/9 31/4/31/4 8 B1
11 1 23/21/13/21/23 16 K
12 7 7 4 D
13 15/14/15/14 6 10 G
14 6q 8 9 H1
15 16q/19p 9 18 M
16 15/14 27/6 21 P
17 13 14 12 I
18 20/10p/20 19/35/19 13 J
19 11 10/33/10 15 L
20 17 16 19 N2
21 8q 25/29/25 14 H2
22 5 22/3 20 O
23 10q 11 26 U
X X X X X
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 meta-
and 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.
3.2. 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

Genes 2019,10, 857 6 of 14
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).
Genes 2019, 10 FOR PEER REVIEW 5
Figure 1. Comparative chromosome painting map of musk ox with homologies to human (HSA) and
dromedary (CDR). Nucleolar organizing regions (NOR) show the localization of the nucleolar
organizing region.
Figure 2. Chromomycin A3-DAPI after G-banding (CDAG) staining performed on metaphase
chromosomes of musk ox: GTG-banding (left) and CMA3/DAPI-staining after denaturation and
renaturation procedure (right).
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 meta-
and 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
Figure 2.
Chromomycin A
3
-DAPI after G-banding (CDAG) staining performed on metaphase
chromosomes of musk ox: GTG-banding (
left
) and CMA3/DAPI-staining after denaturation and
renaturation procedure (right).
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.
Genes 2019, 10 FOR PEER REVIEW 7
Figure 3. The duplication of the X chromosome segment in Pseudoryx nghetinhensis (PNG) shown by
dual color FISH of cattle BAC-clones (pink and green) from CHORI-240 library. Three FISH
experiments illustrate the revealed order of BACs on the X chromosome: 386M8, 108D16, 54D24,
93K24, 108D16. White arrows indicate the duplicated region corresponding to 108D16: 1, 2 –54D24
and 93K24 are between duplicated regions, 3 –386M8 is outside of duplicated region.
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
X syntenic block
1 (XSB1)
CH240-514O22 CH240-514O22 CH240-66H2 CH240-66H2
CH240-287O21 CH240-287O21 CH240-155A13 CH240-155A13
CH240-128C9 CH240-128C9
CH240-106A3
CH240-90L14 CH240-90L14
CH240-106A3 CH240-373L23 CH240-373L23
CH240-229I15 CH240-229I15 CH240-62M10 CH240-62M10
CH240-103E10 CH240-103E10 CH240-122P17 CH240-122P17
CH240-386M8 CH240-386M8 CH240-252G15 CH240-252G15
X syntenic block
2 (XSB2)
CH240-108D16 CH240-108D16 CH240-375C5 CH240-375C5
CH240-54D24 CH240-54D24 CH240-130I15 CH240-130I15
CH240-93K24 CH240-93K24 CH240-118P13 CH240-118P13
CH240-108D16
CH240-122N13 CH240-122N13 CH240-25P8 CH240-25P8
CH240-195J23 CH240-195J23 CH240-14O10 CH240-14O10
CH240-316D2 CH240-316D2 CH240-214A3 CH240-214A3
X syntenic block
3 (XSB3)
CH240-214A3 CH240-214A3 CH240-386M8 CH240-386M8
CH240-14O10 CH240-14O10 CH240-103E10 CH240-103E10
CH240-25P8 CH240-25P8 CH240-128C9 CH240-229I15
CH240-118P13 CH240-118P13 CH240-106A3 CH240-106A3
CH240-130I15 CH240-130I15 CH240-229I15 CH240-128C9
CH240-375C5 CH240-375C5 CH240-287O21 CH240-287O21
CH240-252G15 CH240-252G15
CH240-122P17
CH240-514O22 CH240-514O22
CH240-122P17 CH240-316D2 CH240-316D2
CH240-62M10 CH240-62M10 CH240-195J23 CH240-195J23
CH240-373L23 CH240-373L23 CH240-122N13 CH240-122N13
CH240-90L14 CH240-90L14 CH240-93K2 CH240-93K2
CH240-155A13 CH240-155A13 CH240-54D24 CH240-54D24
CH240-66H2 CH240-66H2 CH240-108D16 CH240-108D16
4. Discussion

Genes 2019,10, 857 7 of 14
Genes 2019, 10 FOR PEER REVIEW 7
Figure 3. The duplication of the X chromosome segment in Pseudoryx nghetinhensis (PNG) shown by
dual color FISH of cattle BAC-clones (pink and green) from CHORI-240 library. Three FISH
experiments illustrate the revealed order of BACs on the X chromosome: 386M8, 108D16, 54D24,
93K24, 108D16. White arrows indicate the duplicated region corresponding to 108D16: 1, 2 –54D24
and 93K24 are between duplicated regions, 3 –386M8 is outside of duplicated region.
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
X syntenic
block 1
(XSB1)
CH240-514O22
CH240-514O22
CH240-66H2
CH240-66H2
CH240-287O21
CH240-287O21
CH240-155A13
CH240-155A13
CH240-128C9
CH240-128C9
CH240-106A3
CH240-90L14
CH240-90L14
CH240-106A3
CH240-373L23
CH240-373L23
CH240-229I15
CH240-229I15
CH240-62M10
CH240-62M10
CH240-103E10
CH240-103E10
CH240-122P17
CH240-122P17
CH240-386M8
CH240-386M8
CH240-252G15
CH240-252G15
X syntenic
block 2
(XSB2)
CH240-108D16
CH240-108D16
CH240-375C5
CH240-375C5
CH240-54D24
CH240-54D24
CH240-130I15
CH240-130I15
CH240-93K24
CH240-93K24
CH240-118P13
CH240-118P13
CH240-108D16
CH240-122N13
CH240-122N13
CH240-25P8
CH240-25P8
CH240-195J23
CH240-195J23
CH240-14O10
CH240-14O10
CH240-316D2
CH240-316D2
CH240-214A3
CH240-214A3
X syntenic
block 3
(XSB3)
CH240-214A3
CH240-214A3
CH240-386M8
CH240-386M8
CH240-14O10
CH240-14O10
CH240-103E10
CH240-103E10
CH240-25P8
CH240-25P8
CH240-128C9
CH240-229I15
CH240-118P13
CH240-118P13
CH240-106A3
CH240-106A3
CH240-130I15
CH240-130I15
CH240-229I15
CH240-128C9
CH240-375C5
CH240-375C5
CH240-287O21
CH240-287O21
CH240-252G15
CH240-252G15
CH240-122P17
CH240-514O22
CH240-514O22
CH240-122P17
CH240-316D2
CH240-316D2
CH240-62M10
CH240-62M10
CH240-195J23
CH240-195J23
CH240-373L23
CH240-373L23
CH240-122N13
CH240-122N13
CH240-90L14
CH240-90L14
CH240-93K2
CH240-93K2
CH240-155A13
CH240-155A13
CH240-54D24
CH240-54D24
CH240-66H2
CH240-66H2
CH240-108D16
CH240-108D16
Figure 3.
The duplication of the X chromosome segment in Pseudoryx nghetinhensis (PNG) shown by
dual color FISH of cattle BAC-clones (pink and green) from CHORI-240 library. Three FISH experiments
illustrate the revealed order of BACs on the X chromosome: 386M8, 108D16, 54D24, 93K24, 108D16.
White arrows indicate the duplicated region corresponding to 108D16: 1, 2 –54D24 and 93K24 are
between duplicated regions, 3 –386M8 is outside of duplicated region.
4. Discussion
4.1. Evolution of Musk Ox and Bovid Karyotypes
Prior G-banding karyotypes of musk ox [
25
,
26
] revealed five fusions that formed submetacentric
chromosomes described using cattle microdissected chromosomes [
14
]. 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.
Overall, the obtained comparative map indicates that musk ox karyotype is nearly homologous to
the pecoran ancestral karyotype [
5
] (Table 2). The comparison of the ancestral elements of the musk
ox with other pecoran species demonstrates the rearrangements that formed its karyotype, but also
more events occurring in different lineages (Figure 5). The musk ox karyotype has evolved from
PAK through six fusions (CDR 1 +2/32, CDR 20 +5/13, CDR 18 +12/34/12, CDR 26 +28/15/28/15/4,
CDR 33/10 +24/30, and CDR 4+17), one fission (CDR 11), and three inversions (on HSA 21/3/21, CDR
22/3/22/3, HSA 12pq’/22q”12pq’/22q”) (Figure 4). It is characterized by inversions on ancestral elements
A2, C2, E, and a split of U.
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.

Genes 2019,10, 857 8 of 14
Genes 2019, 10 FOR PEER REVIEW 9
(Giraffidae) [30], while ancestral conditions were retained in Java mouse deer, pronghorn, Siberian
musk deer and saola, mark another hot spot of chromosome evolution that requires further study.
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].
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].
Figure 4. A scheme depicting chromosome homologies of pecoran species to the ancestral karyotype
chromosomes (PAK) [5] with human homologies on the left and dromedary on the right. Presented
species include AAM (Antilocapra americana) [5], MMO (Moschus moschiferus) [30], and
representatives of different Bovidae tribes: PNG (Pseudoryx nghetinhensis) [5], BTA (Bos taurus) [6]
(Bovini), OAR (Ovis aries) [10], OMO (Ovibos moschatus) (Caprini), DHU (Damaliscus hunteri) [11]
(Alcelaphini). Centromere positions are shown by an asterisk. New data obtained in this study are
marked by a black circle in cell corners.
On another ancestral chromosome, PAK N1 centromere reposition events occurred
independently on homologous chromosomes in several bovid lineages (Pseudoryina, Ovibovina,
Alcelaphini) (Figure 5). Further refinement of the ancestral chromosome N1 was achieved (Table 4),
Figure 4.
A scheme depicting chromosome homologies of pecoran species to the ancestral karyotype
chromosomes (PAK) [
5
] with human homologies on the left and dromedary on the right. Presented
species include AAM (Antilocapra americana) [
5
], MMO (Moschus moschiferus) [
30
], and representatives
of different Bovidae tribes: PNG (Pseudoryx nghetinhensis) [
5
], BTA (Bos taurus) [
6
] (Bovini),
OAR (Ovis aries) [
10
], OMO (Ovibos moschatus) (Caprini), DHU (Damaliscus hunteri) [
11
] (Alcelaphini).
Centromere positions are shown by an asterisk. New data obtained in this study are marked by a black
circle in cell corners.
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.
The ancestral pecoran synteny PAK C2 represents an interesting case. An inversion in HSA
12pq’/22q” occurred independently in different phylogenetic lineages in Pecora (Antilocapridae,
Moschidae, Bovina, Caprina) (Figure 4) [
16
]. Therefore, there is a hot spot of chromosome evolution in
the region homologous to HSA12pq’/22q” in Pecora. Additional investigation is required to verify if
this inversion occurred in the same region, with an in-depth analysis of the DNA sequence surrounding
this region needed to elucidate the genomic elements causing repeated rearrangements. Contrary to

Genes 2019,10, 857 9 of 14
what has been previously suggested [
5
], this ancestral PAK chromosome C2 was composed of HSA
12/22, and not of HSA 12/22/12/22, because the outgroup and many species from basal lineages have
the HSA 12/22 association (whales, Java mouse deer, giraffe, okapi, saola, hirola) [
5
,
11
,
30
,
43
]. Similarly,
the independent inversions in PAK chromosome E (CDR 22/3/22/3/22/3) in BTA 7 (Bovina), OAR 5,
OMO 8 (Caprini) (CDR 22/3/22/3), and giraffe (Giraffidae) [
30
], while ancestral conditions were retained
in Java mouse deer, pronghorn, Siberian musk deer and saola, mark another hot spot of chromosome
evolution that requires further study.
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].
Genes 2019, 10 FOR PEER REVIEW 10
showing that the order of human syntenic regions on the PAK ancestral chromosome N1 is HSA
22’/12’’/4pq and not 12’/22’’/4pq, as was reported earlier [5]. OMO 1p (HSA 22q/12pq’’/4pq) retained
the ancestral order of conserved segments N1 but was then tandemly fused with PAK A2 based on
the position of the ancestral centromere.
Figure 5. Localization of CDR 22 (green) and CDR 3 (pink) on BTA (Bos taurus), OAR (Ovis aries), and
OMO (Ovibos moschatus) metaphase chromosomes by FISH showing additional previously
unreported by painting fragment homologous to CDR22.
4.2. 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
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.
Figure 5.
Localization of CDR 22 (green) and CDR 3 (pink) on BTA (Bos taurus), OAR (Ovis aries),
and OMO (Ovibos moschatus) metaphase chromosomes by FISH showing additional previously
unreported by painting fragment homologous to CDR22. White arrows indicate chromosomes with
specific signal.
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].
On another ancestral chromosome, PAK N1 centromere reposition events occurred independently
on homologous chromosomes in several bovid lineages (Pseudoryina, Ovibovina, Alcelaphini)
(Figure 5). Further refinement of the ancestral chromosome N1 was achieved (Figure 4), showing that
the order of human syntenic regions on the PAK ancestral chromosome N1 is HSA 22’/12”/4pq and not
12’/22”/4pq, as was reported earlier [
5
]. OMO 1p (HSA 22q/12pq”/4pq) retained the ancestral order
of conserved segments N1 but was then tandemly fused with PAK A2 based on the position of the
ancestral centromere.
4.2. 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

Genes 2019,10, 857 10 of 14
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.
5. 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
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,

Genes 2019,10, 857 11 of 14
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.
5. 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.
Author Contributions:
Conceptualization, A.I.K. and A.S.G.; Data curation, A.A.P. and P.L.P.; Formal analysis,
A.A.P.; Funding acquisition, D.M.L. and A.S.G.; Investigation, A.A.P., A.I.K. and N.A.L.; Methodology, A.A.P. and
A.I.K.; Project administration, A.S.G.; Resources, P.L.P., D.V.Y., N.A.L., I.M.O., E.V.K., M.F., D.M.L., M.B., M.E.R.-P.
and S.J.O.; Software, M.F. and D.M.L.; Supervision, A.I.K.; Visualization, A.A.P.; Writing—original draft, A.A.P.;
Writing—review & editing, P.L.P., M.F., D.M.L. and S.J.O.
Funding:
The work was supported by a research grant of the Russian Science Foundation (RSF, 19-14-00034),
the Biotechnology and Biological Sciences Research Council (Grant BB/P020062/1 to D.M.L.), and Russian
Foundation for Basic Research (RFBR) grant 17-00-00145 (D.M.L.).
Acknowledgments:
We would like to sincerely thank Malcolm A. Ferguson-Smith, Fengtang Yang and Patricia
CM O’Brien for kindly providing the sets of human and dromedary camel chromosome-specific painting probes.
We would like to sincerely thank Nicolai Mamaev (Institute for Biological Problems of Cryolithozone Siberian
Branch of RAS, Russia) to helping in providing musk ox samples. We would like to sincerely thank Mitchell
Bush (Conservation and Research Center, National Zoological Park, Virginia, USA), Doug Armstrong (Henry
Doorly Zoo, OMAHA, NE, USA), Melody Roelke (NIH, Bethesda, USA), June Bellizzi and director Richard Hahn
(Catoctin wildlife Zoo and Preserve, MD, USA) for kindly providing samples. We would like to thank Mary
Thompson (NCI-Frederick, USA) for establishing cell lines.
Conflicts of Interest: The authors declare no conflict of interest.
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