diversity
ArticleComparative Analysis of the Polymorphism of the Casein
Genes in Camels Bred in Kazakhstan
Makpal Amandykova 1,2, * , Kairat Dossybayev 1,2 , Aizhan Mussayeva 2 , Bakytzhan Bekmanov 1,2
and Naruya Saitou 3
1

2

3

*



Citation: Amandykova, M.;
Dossybayev, K.; Mussayeva, A.;
Bekmanov, B.; Saitou, N.
Comparative Analysis of theFaculty of Biology and Biotechnology, Al-Farabi Kazakh National University, Al-Farabi Ave., 71,
Almaty 050040, Kazakhstan; kairat1987_11@mail.ru (K.D.); bobekman@rambler.ru (B.B.)
Laboratory of Genetics and Cytogenetics, Institute of Genetics and Physiology SC MES RK, Al-Farabi Ave., 93,
Almaty 050060, Kazakhstan; aimus_@mail.ru
Division of Population Genetics, National Institute of Genetics, 1111 Yata, Mishima 411-8540, Shizuoka, Japan;
saitounr@nig.ac.jp
Correspondence: makpal_30.01@mail.ru; Tel.: +7-7473609475

Abstract: Caseins play an important role in determining the technological properties and quantitative
characteristics of camel milk. To date, only a few studies on the genetic polymorphism of casein genes
have been reported in the camel populations of Kazakhstan. Therefore, this work aimed to identify
the genetic polymorphism level of casein genes among camel populations of the Almaty region of
Kazakhstan. The PCR-RFLP method was used for this purpose and the following genotypes wererevealed as a result: CSN3 gene—CC, CT, TT, where the T allele predominated in all populations,with a frequency of 0.60; CSN2 gene—AA, AG, GG, with the predomination of A allele (0.64); andCSN1S1 gene—GG and GT, with the predomination of G allele (0.94). Statistical analysis was carriedout using the POPGENE and GenAlEx software. The χ2 values were equal to 12.1 (CSN3), 8.6(CSN2), and 14.5 (CSN1S1). As a result, three out of 53 animals were designated as the “core” of the
population—animals with the desired genotypes: CC genotype for the CSN3 gene and AA genotype
for the CSN2 gene. Such animals can be selected for further use with an increase in the number oflivestock with high productivity rates.

Polymorphism of the Casein Genes inCamels Bred in Kazakhstan. Diversity

Keywords: camel; milk productivity; casein genetic variability; CSN3; CSN2; CSN1S1; PCR-RFLP

2022, 14, 285. https://doi.org/
10.3390/d14040285
Academic Editors: Luc Legal andTatiana Deniskova
Received: 24 February 2022
Accepted: 7 April 2022
Published: 11 April 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affiliations.

Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/

1. Introduction
One-humped (Camelus dromedarius) and two-humped (Camelus bactrianus) camels arewidely spread in Africa and Asia. Namely, the dromedary inhabits in the northern part ofAfrica, the Middle East part of Asia, and the Indian subcontinent, while the Bactrian camel
occupies inner, central, and East Asia: China, Mongolia, Kazakhstan, Kyrgyzstan, andAfghanistan, northern areas of Iran, India, Pakistan and up to eastern Turkey [1]. Recently,
it was revealed that the Bactrians have Central Asian origin from where they immigrated
eastward to Mongolia, where the wild Bactrian camels inhabit today [2]. On the other hand,the dromedaries were domesticated in the southeast of the Arabian Peninsula [3].
Previously, the use of camels in the production of dairy products was a non-specialized
and secondary activity. It is worth noting that camel milk appears to contain more common
proteins such as immunoglobulins, lactoferrins, and the useful enzymes lysozyme andlactoperoxidase, which aid the body in fighting infection [4,5]. There are a large number ofstudies currently underway looking into whether camel milk can be an effective preventive
measure against diabetes and heart diseases [6,7]. According to research in India, there
are no cases of type 1 diabetes among populations that consume camel milk regularly,
compared to 5.5% in other populations that do not consume this milk [8]. Camel milk
does not clot in an acidic environment and has a high buffering capacity, controlling4.0/).

Diversity 2022, 14, 285. https://doi.org/10.3390/d14040285

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Diversity 2022, 14, 285

2 of 12hyperglycemia in type 1 diabetes [9,10]. Camel milk fat contains very little or no βlactoglobulin and is rich in immunoglobulins compatible with breast milk [11]. Therefore,
its products are positioned as medicinal, hypoallergenic, and dietary.
Increased interest in camel milk in a more urbanized world has prompted research
into the breeding of dairy camels [12]. Along with the investigation of non-genetic factors influencing milk composition, research was conducted on the evaluation of genetic
factors [13]. Considering the significance of camels for dairy production in many desert
regions, genomic tools are being developed to assess milk yield, milk composition, andudder morphology as key targets for future genomic breeding [14].
Advanced proteomic methods were recently used to analyze the whey protein complex
of one-humped and two-humped camels. Caseins (CN) account for roughly 80% of cow
milk proteins [15], while the casein content of camel milk is 52–87% [16], consisting of
αs1-, αs2-, β-, γ- and κ-CN [17–19]. While β- and γ-CN are monomorphic, four distinct
genetic variants (A, B, C, and D) have been identified in αs1-CN [20]. Some studies arelooking into the genetic factors that influence milk yield increase. For example, oxytocin,a neurohypophyseal peptide involved in a variety of biological functions, affects milksecretion, temperament, and reproduction [21].
Kappa-caseins are important in the stabilization of casein micelles, which determine
the specific properties of milk [22]; its breakdown by chymosin is responsible for milk
coagulation. They prevent casein micelle aggregation and, thus, help to keep calcium phosphate in solution, as well as provide milk calcium and phosphorus bioavailability, whichis especially important for human nutrition [23]. The study of genetic polymorphisms incasein proteins can be used to increase population diversity, preserve the genetic structure,and understand its relationships with the characteristics of dairy production.
As a result of the study on the putative regulatory regions of the camel κ-casein gene,two polymorphic sites were detected in camel promoters. The first is a transition g.975A> G in position −112 which does not affect any putative regulatory regions. The latter,at nucleotide −17, is a transition g.1029T > C. The presence of thymine is responsible forthe creation of an extra putative consensus sequence for the hepatocyte nuclear factor-1(HNF-1). Another member of hepatocyte nuclear factors (HNF-3) is well known to be apotential regulator of casein genes expression [24]; thus, it is suggested that there is theinfluence of HNF-1 allelic variant in camel κ-casein gene regulation. Though the influenceof HNF-1 allelic variants on CSN3 needs to be further substantiated, the presence of C
allele at g.1029 locus in the 50 UTR of CSN3 gene provides the opportunity for the rapid
directional selection in favour of such alleles [25].
β-casein is the most abundant protein in camel milk, and its coding gene (CSN2) is
considered the “main” gene in other species, the alleles of which are associated with various
levels of protein gene expression [26]. It has been reported that the presence of various
levels of β-casein phosphorylation affects the availability and distribution of calcium, as
well as the stability of micelles [27], implying that β-CN plays an important role in nutrition
and the development of the technological properties of milk and dairy products. Several
studies in ruminants have found an association between β-casein gene polymorphism andeconomically important characteristics of milk [28].
Previously, Pauciullo et al. identified a transition g.2126A > G in CSN2 promoter regionof four Sudanese Camelus dromedarius populations [26]. This 2126A/G SNP is located three
nucleotides downstream of the TATA box and might modify the binding affinity of RNApolymerase and change the gene expression. Association among these three genotypes withmilk composition traits suggests a positive effect of A/A genotype on acidity and protein
percentage. Higher protein and acidity values were observed in the milk of individuals
carrying the A/A genotype [29].
In ruminants, the αS1-CN fraction has been extensively studied and characterized atboth the protein and DNA levels. Many alleles associated with different rates of protein
synthesis have been identified in the corresponding coding gene (CSN1S1) [30,31]. Two
genetic variants of αS1-CN have been described in one-humped camels [32]. αs1-casein

Diversity 2022, 14, 285

3 of 12

polymorphism affects the milk lipids and protein compositions, so it has a strong impact
on the nutritional quality and technological properties of milk. Currently, at least four
protein variants (A, B, C, and D) have been detected in the dromedary αs1-CN, and themolecular event responsible for these phenotype variations was clarified in three out of fourcases. The allele C is due to a single nucleotide polymorphism (c.150G > T) occurring at theexon 5 and resulting in the amino acid substitution p.30Glu > Asp in mature protein [33].
Recently, Erhardt et al. reported a new variant, named D. Even though the CSN1S1 gene
polymorphism is actively studied in camels, its effect on the technological properties of milk
has not yet been identified, and the role of these SNPs remains to be studied in comparison
with the phenotypic data of animals [17].
αs2-casein is the most hydrophilic of all milk casein proteins. The proportion ofs2-CN in milk varies greatly between species, and this protein is completely absent in
the composition of human milk [34]. The CSN1S2 gene, which encodes this protein, hasrarely been studied in most mammals, including camels. To date, no information has beenfound about any single-nucleotide polymorphisms (SNP) found in this gene affecting the
productive qualities of camel milk.
SNP genotyping technologies are valuable resources for animal breeding programs.
Genomic selection by PCR-RFLP analysis is a simple and accessible method for selecting
the best breeding animals, as well as a useful tool in small-scale basic research studies ofthe economic properties associated with single nucleotide polymorphisms [35].
This work aimed to identify the genetic polymorphism of casein genes and to determine the allelic variants frequencies in Camelus dromedarius, Camelus Bactrianus, and hybridsof the Kazakh camel populations, as well as identifying animals with desirable genotypes
according to the studied genes. For the first time, we conducted genotyping of Kazakhstan
camels for three SNPs occurring in CSN3, CSN2, and CSN1S1 casein genes, the resultsof which can be used for breeding management and the selection of highly productive
animals. This paper aims to present and summarize the results of genotyping of camels
and identify the “core” of populations: animals that have the «desired» genotypes for thestudied SNPs.
2. Materials and Methods
2.1. Collection of Materials
All procedures performed in studies involving animals followed the ethical standardsof the Institute of Genetics and Physiology (Almaty, Kazakhstan), at which the studies wereconducted. Blood samples were collected using Venosafe tubes with K2EDTA (Leuven,
Belgium) in the Ile district of the Almaty region of Kazakhstan (Figure 1). Camel breeding
has been a traditional branch of productive livestock breeding in this region for centuries,
as a result, there are a large number of camel breeding farms (Otemis Makan Farm, DauletBeket LLP, etc.). Thus, 53 camels of the age of 2–3 years from four distinct herds wereselected for our study. Moreover, family ties of camels bred in the same herd are possible.
There are eight one-humped camels (n = 6 ♀; n = 2 ♂)—dromedaries (Camelus dromedarius)
in the first population; eight hybrids (n = 5 ♀; n = 3 ♂) and one dromedary (♀) in thesecond population; fourteen hybrids (n = 10 ♀; n = 4 ♂) and six two-humped camels(n = 4 ♀; n = 2 ♂)—Bactrians (Camelus bactrianus) in the third population; and sixteen
Bactrians (n = 8 ♀; n = 8 ♂) in the fourth population. The samples were refrigerated in
special containers and transported to the laboratory of Animal Genetics and Cytology,
where they were stored in a freezer until further use for DNA extraction.

Diversity
Diversity2022,
2022,14,
14,285x FOR PEER REVIEW

44 of 1212FigureFigure1.
1. Location
Location ofof sample
sample collection
collection from
fromKazakhstan.
Kazakhstan.

2.2. Genomic DNA Extraction and PCR-RFLP Analysis
2.2. Genomic
and PCR-RFLP
GenomicDNA
DNAExtraction
was extracted
according Analysis.
to the protocol supplied by the manufacturer

DNAkitwasaccordingthe protocol
suppliedby the manufacin theGenomic
DNA-sorb-B
forextracted
DNA isolation
fromtobloodand tissues(AmpliSens,
Moscow,
◦
turer
in
the
DNA-sorb-B
kit
for
DNA
isolation
from
blood
and
tissues(AmpliSens,
MosRussia). Prepared DNA samples were stored in a freezer at −20 C. The DNA quality
cow,determined
Russia). Prepared
DNA samples
were stored inanda freezer
at −20 °C. TheofDNA
wasusing agarose
gel electrophoresis,
the concentration
DNAqualiwas
ty was determinedusing agarose
electrophoresis,Waltham,and the concentrationof DNAwasdeterminedusing Nanodrop
One gel(ThermoScientific,
MA, USA). The
genomic
determinedusing Nanodrop
Onepolymerase(ThermoScientific,
Waltham,
MA,USA).inTheDNA
were amplified
through thechain reaction(PCR)
methodthe genomic
thermalDNA Mastercycler
were amplified
throughthe(Eppendorf,
polymeraseHamburg,
chain reaction(PCR) PCR
methodthe thercycler
nexus
gradient
Germany).
was inperformedin
10cyclerµL final
volume solution
the (Eppendorf,
Master Mix (PCR
MasterMix, ThermoScientific,mal
Mastercycler
nexus using
gradient
Hamburg,
Germany).
PCR was perWaltham,USA).
1 shows
forusing
genestheCSN3,
CSN2
CSN1S1:
formed inMA,
10 µL
final Table
volumesolution
MasterMixand(PCR
MasterPCR
Mix,fragment
Thermosizes,
oligonucleotide
primers,
annealing
temperature,
andCSN3,
restriction
Scientific,
Waltham, MA,
USA).
Table 1 shows
for genes
CSN2enzymes.
and CSN1S1: PCR
fragment sizes, oligonucleotide primers, annealing temperature, and restriction enTable
1. PCR-RFLP analysis conditions, restriction enzymes and oligonucleotide primers for casein
zymes.

№

№1
2

1
3

2
3

SNP

genes genotyping.
Table 1. PCR-RFLP analysis conditions, restriction enzymes and oligonucleotide primers for caFragment
Annealing
Restriction
sein genes genotyping.Primers
References
LengthFragment
CSN3
488 bpSNP
g.1029T > C Length
CSN2

CSN3
g.2126A > G
g.1029T>C
CSN1S1c.150G > T

CSN2
g.2126A > G
CSN1S1c.150G > T

Temperature

50 -CACAAAGATGACTCTGCTATCG-30
Primers
50 -GCCCTCCACATATGTCTG-30 .

50 -GTTTCTCCATTACAGCATC-30
659 bp 5′-CACAAAGATGACTCTGCTATCG50 -TCAAATCTATACAGGCACTT-30 .

488 bpEnzymes

AnnealingTem- Restriction En60 ◦ C
AluI[6]References
peraturezymes
53 ◦ C

HphI

[26]

3′
60 °C
AluI
[6]
50 -TGAACCAGACAGCATAGAG-30◦C930 bp 5′-GCCCTCCACATATGTCTG-3′.
55
SmoI
[17]
50 -CTAAACTGAATGGGTGAAAC-30
5′-GTTTCTCCATTACAGCATC-3′
659 bp
53 °C
HphI
[26]
5′-TCAAATCTATACAGGCACTT-3′.
The PCR products were visualized using 2% agarose gel electrophoresis, which was
5′-TGAACCAGACAGCATAGAG-3′
930 bp stained with ethidium bromide. Restriction products
55 °C were analyzed
SmoI
[17]
using 5% polyacry5′-CTAAACTGAATGGGTGAAAC-3′
lamide gel electrophoresis stained with SYBR Gold Nucleic Acid Gel Stain (ThermoScientific, Waltham, MA, USA) in 1 × TBE buffer. The results were visualized by using a
The PCR products were visualized using 2% agarose gel electrophoresis, which was
gel-documenting system (Quantum-ST5-1100 Vilber Lourmat, France).
stained with ethidium bromide. Restriction products were analyzed using 5% polyacrylamide
electrophoresis
2.3.
Statisticalgel
Data
Processing stained with SYBR Gold Nucleic Acid Gel Stain (ThermoScientific,
Waltham,
MA, USA)in 1 × TBE buffer.equilibrium
The results (χ2)
werewerevisualized
by using
The allele frequency
and Hardy–Weinberg
calculated
in allagel-documenting
system(Quantum-ST5-1100
Vilber Lourmat,
France). of occurrence ofstudiedpopulations.
Statisticalanalysis of such indicators
as the frequency
genotypes,
theDataobserved
and effective number of alleles, Nei’s gene diversity, and Shannon’s
2.3. Statistical
Processing
Information
index
was
carriedusing the GeneAlex[36] and(χPOPGENE
Softwarein[37].
2) were calculated
all
The allele frequency and out
Hardy–Weinberg
equilibrium
These programs calculate the above statistics based on Nei’s standard genetic distance,
studied populations. Statistical analysis of such indicators as the frequency of occurrence
which has the property that if the rate of genetic change is constant per year or generationof genotypes, the observed and effective number of alleles, Nei’s gene diversity, andDiversity 2022, 14, 285

5 of 12then Nei’s standard genetic distance (D) increases in proportion to the divergence time.
This measure assumes that genetic differences are caused by mutation and genetic drift [38].
A phylogenetic tree based on the standard Nei’s genetic distance was constructed by
MEGA-X, which is based on the principle of the Neighbor-joining Method, to find pairs ofneighbors that minimize the total branch length at each stage of clustering of neighbors,
starting with a star-like tree [39,40].
3. Results
Four populations of camels bred in the Almaty region were genotyped on polymorphic SNPs in three genes (g.942G > T, CSN1S1 gene; g.2126A > G, CSN2 promoter; andg.1029T > C, CSN3 promoter). As a result, the following genotypes were identified among
the studied populations: CC, CT, and TT for CSN3; AA, AG, and GG for CSN2; GG and GT
for CSN1S1 gene (Table 2).
Table 2. CSN3, CSN2, and CSN1S1 genes polymorphism study.
CSN3

CSN2
AlleleFrequency

Identified
Genotypes

CSN1S1AlleleFrequency

Identified
Genotypes

AlleleFrequency

Population

Overall

CC

CT

TT

C

T

AA

AG

GG

A

G

GG

GT

TT

G

T

1
2
3
4

8
9
20
16

1
1
2
8

3
2
10
3

4
6
8
5

0.30
0.18
0.37
0.44

0.70
0.82
0.63
0.56

5
4
18
1

2
2
2
6

1
3
0
9

0.75
0.60
0.95
0.25

0.25
0.40
0.05
0.75

6
8
20
14

2
1
0
2

0
0
0
0

0.88
0.94
1.00
0.94

0.12
0.06
0
0.06

53

12

18

23
χ2 = 12.1

0.40

0.60

28

12

13
0.64
χ2 = 8.6

0.36

48

5

0
0.94
χ2 = 14.5

0.06

Overall

Identified Genotypes

As a result of camel genotyping for the CSN3 gene, the T allele predominated in all
four populations, with a frequency of 0.61 ranging from 0.56 to 0.82 (Table 1). Regarding
CSN2 gene polymorphism, the frequency of A allele was higher in the 1st, 2nd, and 3rd
populations, with values of 0.75, 0.6, and 0.95, respectively. In the fourth population,in contrast, allele G accounted for the majority of genotypes (0.75). For CSN1S1 gene,it can be seen that G allele predominated in all four populations, while T allele had aminor frequency.
These research results were used to calculate the Hardy–Weinberg equilibrium (χ2).
The χ2 values in the studied camel groups were equal to 12.1 (g.1029T > C, CSN3 promoter,p = 0.146798; non-significant at 5% probability level of significance), 8.6 (g.2126A > G,CSN2 promoter, p = 0.377154; non-significant at 5% probability level of significance), and14.5 (g.942G > T, allele C of CSN1S1, p = 0.069629; non-significant at 5% probability levelof significance).
Next, an identical analysis of the frequency of occurrence of various genotypes andalleles for the studied genes was performed, dividing the studied individuals by species:
one-humped camels—dromedary (Camelus dromedarius), two-humped camels—Bactrian(Camelus bactrianus), and their hybrids.
As shown in Table 3, the «desired allele» C for the kappa-casein gene was mostfrequently found in Bactrians (0.52), while in dromedaries and hybrids, this indicator had
nearly the same value (0.3 and 0.33). In the case of the β-casein gene, the «desired allele» A
was found most frequently in hybrids (0.8), then in dromedaries (0.66), and least frequently
in Bactrians (0.48).

Diversity 2022, 14, 285

6 of 12

Table 3. CSN3, CSN2, and CSN1S1 genotype distribution in one-humped, two-humped camels andtheir hybrids.
CSN3
Populations

One-humped
camels(Camelus
dromedarius)
Twohumped
camels(Camelus
bactrianus)
Hybrids
Overall

Identified
Genotypes

Overall

CSN2
AlleleFrequency

Identified
Genotypes

CSN1S1AlleleFrequency

Identified
Genotypes

AlleleFrequency

CC

CT

TT

C

T

AA

AG

GG

A

G

GG

GT

TT

G

T

9

1

4

4

0.33

0.67

5

2

2

0.66

0.33

7

2

0

0.89

0.11

22

8

7

7

0.52

0.48

7

7

8

0.48

0.52

20

2

0

0.95

0.05

22

3

7

12

0.30

0.70

16

3

3

0.80

0.20

21

1

0

0.98

0.02

53

12

18

23

0.40

0.60

28

12

13

0.64

0.36

48

5

0

0.94

0.06

χ2 = 0.40

χ2 = 0.98

χ2 = 36.32

The obtained data of all studied animals were analyzed to determine several statisticalindicators: the identified and effective number of alleles, Nei’s gene diversity, Shannon’s information index, and original measures of Nei’s genetic identity and genetic distance (Table 4).
Table 4. Summary of the genetic variation statistics for all loci.
Locus

Na

Ne

h

I

CSN3

CC
CT
TT

2.00002.00002.00001.5392
1.8134
1.96570.35030.4486
0.49130.53490.64080.6844

CSN2

AA
AG
GG

2.00002.00002.00001.9824
1.58791.49020.49560.37020.32890.68870.55710.5107CSN1S1GG
GT
TT

2.00002.00001.00001.20611.20611.00000.17090.17090.00000.31250.31250.0000Mean

1.88891.53230.31410.4713

Statistical error

0.33330.34830.16840.2255Na = Observed number of alleles [41]. Ne = Effective number of alleles [42]. h = Nei’s gene diversity [43].
I = Shannon’s Information index [44].

The average value of the observed number of alleles (Na), which represents the actual
number of alleles found in the studied populations, was the same (2.0000) for all genotypes
identified in this study, except the TT genotype for the CSN1S1 gene (1.0000 ± 0.3333),
which is attributable to the complete absence of this genotype in the studied populations.
For the abovementioned reasons, the average effective number of alleles (Ne) was
highest for the AA genotype of the CSN2 gene (1.9824 ± 0.3483) and lowest for the TT
genotype of the CSN1S1 gene (1.0000 ± 0.3483). This indicator describes the number ofalleles with the same frequency required to achieve the same expected heterozygosity as in
the population under study. In terms of gene diversity, this indicator had the highest value
for the AA genotype of the CSN2 gene (0.4956 ± 0.1684) and the lowest value for the TT
genotype of the CSN1S1 gene (0.4956 ± 0.1684), indicating the complete absence of genetic
diversity for this gene. The Shannon’s index, which measures gene diversity, was also highfor the CSN2 AA genotype and equal to zero for the CSN1S1 TT genotype.
Next, we constructed a phylogenetic tree to assess the related proximity of populationsin terms of the diversity of CSN3, CSN2, and CSN1S1 genes (Figure 2). It can be seen that,

Diversity 2022, 14, 285

of genetic diversity for this gene. The Shannon’s index, which measures gene diversity,
was also high for the CSN2 AA genotype and equal to zero for the CSN1S1 TT genotype.
7 of 12
Next, we constructed a phylogenetic tree to assess the related proximity of populations in terms of the diversity of CSN3, CSN2, and CSN1S1 genes (Figure 2). It can beseen that, according to the studied SNPs, the 1st and 3rd populations are the most closely related toother andare thesimilarto thepopulation,
the 4thpopulationisaccordingto eachthe studied
SNPs,
1st and
3rd2ndpopulations
arewhile
the most
closely
related tolocatedon andthe branchof thephylogenetic
tree, whichthemostdistant fromall populaeachotherare similar
to the
2nd population,
whileisthe
4th
population
is locatedon thebranchtions. of the phylogenetic tree, which is the most distant from all populations.

Figure 2. Phylogenetic tree based on the standard Nei’s genetic distance between four camel populations.
Figure 2. Phylogenetic tree based on the standard Nei’s genetic distance between four camel populations.
To check the reliability of the constructed phylogenetic tree, we used data from thecalculation of genetic identity and genetic distance between populations, presented in the
To check the reliability of the constructed phylogenetic tree, we used data from thetable below (Table 5).
calculation of genetic identity and genetic distance between populations, presented in
the table
below(Table5).
original
measuresof genetic identity and genetic distance [37]. Nei’s genetic identity
Table
5. Nei’s(above diagonal) and genetic distance (below diagonal).
Table 5. Nei’s original measures of genetic identity and genetic distance [37]. Nei’s genetic identity(abovediagonal) and genetic1distance (below diagonal).
Population
2
3
4
1
Population
21
32
43
4

–1

0.956720.9603
30.8684
40.0443–0.04430.04050.04050.14110.1411–0.9567–
0.05690.05690.10880.10880.94470.96030.9447––0.21740.21740.89690.86840.89690.80460.8046––

The genetic identity of the 1st and 3rd populations is 0.9603, and the genetic distanceThe genetic identity of the 1st and 3rd populations is 0.9603, and the genetic disis 0.0405, demonstrating their close relationship, as shown in the phylogenetic tree. Furtance is 0.0405, demonstrating their close relationship, as shown in the phylogenetic tree.
thermore, the most related populations were the 1st and 2nd, as well as the 2nd and 3rd,
Furthermore, the most related populations were the 1st and 2nd, as well as the 2nd andwith genetic identities of 0.9567 and 0.9447, respectively. The genetic identity of the 1st,
3rd, with genetic identities of 0.9567 and 0.9447, respectively. The genetic identity of the
2nd, and 3rd populations is approximately the same as that of the 4th population (0.8684;
1st, 2nd,and3rd populations
is andapproximately
the same
thatdistantof the from4th population
0.8969,and0.8046).
Thus, the 3rd
4th populations
are theasmost
each other,(0.8684;0.8969,and0.8046).
Thus,
the
3rd
and
4th
populations
are
the
most
distant fromas their genetic distance has the highest rate of 0.2174.
each other, as their genetic distance has the highest rate of 0.2174.
4. Discussion
4. Discussion
Heterozygosity is considered the most widely used criterion for assessing genetic

Heterozygosity
is considered
the most
widelyfor assessing
genetic
variation
in a population.
The effective
numberof usedallelescriterion
is assumed
to be a measure
variationinapopulation.
The
effective
numberofallelesisassumedtobeameasureofof genetic diversity in both individual species and populations, and it is determined bygenetic
diversity
both individual
and populations,
it isanddeterminedby thetheproportionofin
polymorphic
loci, species
the numberof alleles perandlocus,
the evennessofproportionof polymorphic
loci,number
the numberof alleles
perandthetheinverse
evennessof alallele
frequencies.
The effectiveof alleles
is usedtolocus,calculatemeasurelelehomozygosity.
frequencies. The
effective
number of ofallelesused toincalculate
the inverse
measureofWhen
the frequency
theseisalleles
the populationis thesame,
heterozygosity will be equal to the actual one [45].
The presence of thymine in the studied CSN3 gene SNP is responsible for creating an
additional putative consensus sequence for hepatocyte nuclear factor-1 (HNF-1). According
to reports, this transcription factor regulates several genes involved in innate immunity,
lipid and glucose transport, metabolism, along with others [46]. However, as the g.1029T> C transition occurs in the C. dromedarius promoter just in front of the exon 1, allele C is

Diversity 2022, 14, 285

8 of 12also responsible for the creation of an additional putative site for the transcription factor
HNF-1, which is why this allele is considered desirable in selection of the CSN3 gene
polymorphism. However, the frequency of the desired C allele was less significant, ranging
from 0.18 to 0.44 among studied populations, which is lower than that of in Sudanese camel
populations, where this indicator ranged from 0.30 to 0.46 [6]. Thus, based on the dataobtained, it is clear that there is a need for targeted selection in favor of the C allele in all of
the studied populations. On average, the frequency of allele T was observed to be 0.60 andthat of C was observed to be 0.40. The present results are in agreement with the results ofthe genotyping of Indian camel populations, where the T allele was equal to 0.763 and the
C allele was observed to be 0.237 [25]. This trend is also observed in the Egyptian camel
population, where the C allele showed the lowest frequency of occurrence (0.32) comparedto the T allele (0.68) [47].
SNP (g.2126A > G) occurs three nucleotides downstream of the TATA box and modifies
the putative consensus site for the non-canonical TATA box (TFmatrix: M00252), lowering
the binding affinity from 87.7 to 86.2%. The lower frequency of the G allele indicates that
this genetic variant, which is responsible for the low affinity of the TATA box-binding site,
may be less facilitated during gene transcription [26]. As a result, we can suggest that allele
A is “desirable” in this SNP. The frequency of the desired A allele was 0.64 on average in all
studied populations, while the frequency of the G allele was 0.36. These indicators coincidewith the results of genotyping of 180 Sudanese camels, where the A allele frequency wasestimated to be 0.65 and the G allele frequency was 0.35 [26], as well as with the genotyping
data of Egyptian camels, where the frequency of the A allele indicated 0.63, and the G allele
was 0.37, respectively [29].
According to the results of several studies, the PCR-RFLP method for typing SNP
g.942G > T can be used to determine the variability in the camel CSN1S1 gene, regardlessof age, sex, or lactation stage [33]. This can be useful for a broader analysis of the variabilityof camel milk protein and enables the analysis of the relationship between milk protein
variability and milk production characteristics in camels, as has already been carried out
for several farm animals [48,49]. According to the findings of our study, the TT genotype
did not occur in all four populations; thus, the frequency of the T allele is much lower than
the frequency of the G allele (0.06 and 0.94, respectively). The T allele did not exist in thethird population, even as a heterozygous genotype. It should be noted that the “desired”
genotype for the studied SNP was not established yet, and future research should focus on
determining the relationship of the identified genotypes with the technical properties orother characteristics of milk.
The use of χ2 allows us to determine the extent to which the actual genotype distribution corresponds to its theoretical value. According to the Hardy–Weinberg equilibrium
law, any equilibrium ratio of alleles can occur in infinite genetic populations in the absenceof mutation, migration, and selection, and the relative frequencies of each allele remain
constant from generation to generation at the same time. It is well known that shifts indynamic equilibrium in favor of a specific allele or genotype are caused by the combined
action of four factors: mutations, migrations, selection, and scholastic fluctuations in allele
concentration due to small population size (genetically automatic processes or genetic
drift) [26]. The χ2 values in the studied camel groups indicate a deviation in the actual
allele frequency of the studied genes from the theoretical one. It demonstrates the lack ofgene balance for these loci. The latter can be explained by the action of artificial selection in
the population.
Summarizing the data on the frequency of occurrence of alleles and genotypes for
the CSN2 and CSN3 genes, the so-called «core» of populations: animals with the desired
genotypes for both SNPs under consideration, can be identified. Thus, three out of fiftythree animals can be assigned to the «core» of populations, as they have the corresponding«desirable» CC genotype for the CSN3 gene and AA genotype for the CSN2 gene. All three
animals are hybrids; two of them are from the third population and one is from the second.

Diversity 2022, 14, 285

9 of 12

Such animals can be selected for further use with an increase in the number of livestockwith high productivity rates.
Furthermore, it was interesting for us to compare the results of our study with camels
belonging to certain species: one-humped, two-humped camels, and hybrids. According
to the literature, dromedary milk production is quite high—over 2000 kg per lactation
period (up to 16–18 months). Bactrians are distinguished by a lower milk yield but a higher
fat content [50]. Nar-Maya and Kospak hybrids of the first generation are intermediate
between Bactrians and dromedaries [51]. As a result, we can assume a relationship between
the frequency of occurrence of the «desirable» genotypes of certain species based on our
research findings, and their milk yield indicators based on literature data. As our study
results show, the data on genotyping does not fully correspond to the data on quantitative
indicators of camel productivity. However, even though milk from two-humped camels
has a higher fat content than milk from one-humped camels and hybrids, it can be assumed
that the high content of the C allele of kappa-casein in two-humped camels affects this milk
quality trait. This assumption can be confirmed by performing a combined genotyping andbiochemical analysis of milk from the same camel population.
Concerning the phylogenetic tree, the observed discrepancy can be explained by thesimilarity of allele frequencies for the studied genes. For example, the first and third
populations closest to each other in the phylogenetic tree have the closest allele frequencies
for all studied SNPs (Table 2), which shows their similarity at genetic polymorphism levelof these genes. This may also indicate the presence of closely related relationships between
these populations. A similar explanation can be given regarding the divergence of theremaining populations on the phylogenetic tree. The measurement of genetic changes
is determined by two parameters: genetic identity (I), which estimates the proportion ofgenes that are identical in two populations, and genetic distance (D), which estimates the
proportion of gene changes that have occurred during the evolution of two populations.
The I value can range from 0 to 1, which corresponds to extreme situations in which noneor all genes are identical, respectively; the D value can range from zero to infinity. D
can exceed 1, as each gene can change more than once in one or both populations, sinceevolution continues for many generations.
5. Conclusions
Camel genotyping by milk productivity genes is assumed to be a more effective
method of targeted breeding for improving milk quantitative and qualitative parameters.
The results of this work show that the genotyping of camels for casein protein genes using
the PCR-RFLP method can be successfully used to determine the potential of the studied
animals for use in breeding work with an increase in the milk productivity of populations.
The results of our research can be used to describe the genetic potential of the studied
populations for the CSN3, CSN2, and CSN1S1 genes of milk productivity, allowing us
to make recommendations to camel farms for future breeding management within the
populations. Furthermore, the obtained data can be used as a basis for comparative analysis
with the data from subsequent studies conducted in this area. Further research should
be carried out by way of a comparative analysis of the obtained genetic data with thephenotypic parameters of animals (milk yield, biochemical properties of milk, etc.), as
well as a description of the studied genes using high-throughput sequencing methods, toobtain more extensive data on the relationship of the studied SNPs with the technologicalproperties of milk.
Author Contributions: Conceptualization, M.A. and K.D.; methodology, M.A.; software, M.A.;
validation, A.M., B.B., and N.S.; formal analysis, M.A. and K.D.; investigation, M.A.; resources, A.M.
and B.B.; data curation, M.A.; writing—original draft preparation, M.A.; writing—review and editing,
K.D.; visualization, A.M. and B.B.; supervision, A.M., B.B., and N.S.; project administration, M.A.;
funding acquisition, B.B. All authors have read and agreed to the published version of the manuscript.

Diversity 2022, 14, 285

10 of 12Funding: This research was funded by the Ministry of Education and Science of the Republic of
Kazakhstan, grant number OR11465435 «Development and application of new genomic technologies
for protecting organisms from mutagenic effects, increasing the productivity of natural resources andimproving the quality of life of the population».
Institutional Review Board Statement: The animal study protocol was approved by the Ethics
Committee of the Non-profit joint-stock company «Kazakh National Agrarian University» (14September 2020).
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We express our sincere thanks to the heads of the camel farms of the Ili districtof the Almaty region for their assistance in collecting research materials and providing data on thespecies of camels. We are most grateful to Vsevolodov E.B. for the consultation on preparing themanuscript of the paper.
Conflicts of Interest: The authors declare no conflict of interest.

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