A total of 75 individuals from five sheep populations in Kazakhstan
were investigated based on 12 STR (short tandem repeat, also known as microsatellite) markers in order to study their genetic
structure and phylogenetic relationship based on genetic distances. These
sheep had a high level of genetic diversity. In total, 163 alleles were
found in all the populations using 12 microsatellite loci. The mean
number of alleles, effective number of alleles, and polymorphism information content (PIC) values per loci were
13.4, 5.9, and 0.78, respectively. Comparing the allelic diversity between
the populations, the highest genetic diversity was observed in the Edilbay-1 sheep breed (
Sheep breeding is the most ancient branch of animal husbandry in Kazakhstan. The country has more than 20 distinct sheep breeds to date. Among them, the sheep breeds which first appeared in Kazakhstan by origin and history are Edilbay, Kazakh Arkhar-Merino, Kazakh Finewool, and Kazakh fat-tailed coarse wool. These breeds are well adapted to the various climatic conditions in Kazakhstan. Various breeds of sheep in one way or another are different in terms of biological efficiency. This study aims at investigating the genetic diversity of local sheep breeds. The animals studied belong to purebred and farms' own selection and tribal cards. The Kazakh Arkhar-Merino sheep breed was investigated on Kumtekey breeding farm, where the best sheep of this breed are kept. The Kazakh Arkhar-Merino is based on the interspecific hybridization of wild Arkhar rams with fine wool ewes of Novo-Caucasian Merino, Précoce, and Rambouillet breeds that occurred during 1934–1950. R-Kurty farm is also a breeding farm where the highly productive animals of the Kazakh Finewool breed are concentrated. Edilbay sheep breed is bred in Birlik breeding centre, where the purebred and highly productive sheep of this breed are kept. The Kazakh fat-tailed coarse wool sheep breed is the product of popular selection which has lasted many years. They are widely bred in many sheep farms all over Kazakhstan. However, highly productive animals typical of this breed are concentrated in Kabyl-Nur farm. Thus, four sheep breeds selected by us for molecular genetic studies are, firstly, the most common sheep breeds in Kazakhstan, which are well adapted to different climatic conditions of breeding and housing. Secondly, these breeds differ from each other in their origin and method of breeding, and thus they are excellent subjects for comparative molecular genetics research. Thirdly, the specification of breeding farms from which sheep were studied is extremely important, since we use the results of the molecular genetics study of these breeds in these farms to improve breeding work and speed up the selection process in order to create a highly productive breeding core in a short period of time. Since research started in 2010, the molecular database has grown, and the characterization of genetic diversity in farm animals has become particularly pertinent (Groeneveld et al., 2010).
Today, the FAO and the ISAG–FAO Advisory Group on Animal Genetic Diversity recommend specific sets of STR (short tandem repeat, also known as microsatellite) loci for genetic analyses, such as for horse, cattle, and pig breeds. Of the different types of molecular markers, STRs are suitable for studying genetic diversity because of their abundance – the large amount of allelic variation at each locus is highly polymorphic, their distribution throughout the genome is random, and inheritance is codominant (Rekha et al., 2016; Barcaccia et al., 2013; Putman et al., 2014). In addition, STRs are able to generate information for the planning of crossings and further selection of genotypes in genetic breeding programs (Faleiro et al., 2007; Crispim et al., 2014). The objective of the current research was to investigate 12 STR loci based on genetic diversity in sheep herds as well as the differentiation and relationship among the number of alleles and genetic links between Kazakh sheep breeds.
Blood samples were taken from five sheep populations: two of them were
Edilbay-1, though of two various Edilbay-1 sheep herds, and three
others were different sheep breeds in the categories Kazakh Finewool, Kazakh
Arkhar-Merino, and Kazakh fat-tailed coarse wool. The 15 animals were chosen
randomly from each population. Genomic DNA was extracted using a commercial
kit (GeneJET Genomic DNA Purification Kit, ThermoFisher Scientific, USA).
Both the quality and concentration of DNA were verified by spectrophotometric
and agarose gel electrophoresis. In this study, 12 STR primers were used, and
all of them are recommended by the International Society of Animal Genetics
(ISAG, 2017). Amplification was carried out using a Tetrad 2
thermal cycler (Bio-Rad). Polymerase chain reaction (PCR) products were attached in the ABI310 Genetic Analyser, and
GeneMapper software was used to determine fragment size. The number of
alleles, effective number of alleles, polymorphism information content (PIC)
values, observed and expected heterozygosities, Wright's
All examined markers were polymorphic for all the populations examined. In
total, 161 alleles were found in five native Kazakh sheep breeds based on
12 STR loci (Table 1). The highest number of alleles was 16 for markers
Genetic diversity analysis of sheep populations based on the 12
microsatellite markers. Number of alleles (
The effective number of alleles for each marker varied between 3.2 and 9.1, with a mean value of 5.9. The PIC values ranged from 0.65 to 0.88, with a mean of 0.78. Observed heterozygosity values varied from 0.52 to 0.89, with an average of 0.73, while the expected heterozygosity values ranged from 0.69 to 0.89 with a mean value of 0.81 (Table 1). Our study demonstrated a high genetic polymorphism in the investigated sheep breeds. These estimates are higher than those reported for other sheep breeds (Ferrando et al., 2014, in France and Spain; Salamon et al., 2014, in Croatia and Bosnia and Herzegovina; Al-Atiyat et al., 2015, in Australia; Gaouar et al., 2016, in Morocco).
Genetic diversity within the five sheep populations. Mean number of
alleles (MNA), effective number of alleles (
The average number of alleles, the effective number of alleles, and the expected and observed heterozygosity for each breed are shown in Table 2. The average
number of alleles for the Edilbay-1 breed is 8.33. This analysis showed that
there was no significant differentiation among the groups from the following
populations: Kazakh Arkhar-Merino (7.08), Kazakh Finewool (7.91), Edilbay-2
(7.58), and Kazakh fat-tailed coarse wool (7.41). With the exception of
The phylogenetic tree constructed from Nei's standard genetic distances among five sheep populations.
The highest
The neighbour joining tree for all samples was constructed using pairwise population matrix of Nei's genetic distances in order to represent the relationships between the sheep breeds (Fig. 1). Edilbay-1 and Edilbay-2 were initially classified as sub-clusters, which were further clustered into Kazakh fat-tailed coarse wool, whereas Kazakh Arkhar-Merino and Kazakh Finewool sheep breeds were grouped around the same node. Moreover, in the factorial correspondence analysis, the distinction of three clusters is illustrated by three axes showing variances of 37.93 %, 24.35 %, and 22.49 %, respectively (Fig. 2).
The factorial correspondence analysis of five sheep populations studied on the bases of 12 STR loci.
In this study, 12 STR (microsatellite) markers were used to evaluate genetic
diversity in five populations. The analysis revealed no significant
differences in the main genetic characteristics of the interbred population:
number of alleles, effective number of alleles, and expected and observed
heterozygosities. A high level of genetic diversity was observed in loci
Pairwise population matrix of Nei's genetic distances (above the
diagonal) and pairwise population
The overall average of
The fixation index was estimated on a per locus basis, and due to negative
assortative mating, an excess of heterozygosity was found at markers
In addition, to estimate the genetic variability of the studied sheep
breeds, we calculated the expected and observed heterozygosity values.
Except for
Previously, a study was conducted by Ozerov et al. (2008) on four sheep
breeds of Kazakhstan (Degeres Mutton-wool, Kazakh Arkhar-Merino, Kazakh Finewool, and Edilbaev) using 20 microsatellite loci. As a result, it was
determined that all studied sheep breeds showed a high level of polymorphism
in all 20 microsatellite loci and were in a state of genetic equilibrium according to the Hardy–Weinberg ratio. However, in our studies of the Kazakh Arkhar-Merino
sheep breed, there were significant differences between the expected and
observed heterozygosity (
Furthermore, in order to analyse population differentiation and structure, Wright's
In the present study, we used pairwise population
Nei's genetic distances (Nei, 1972) between the five populations of sheep were calculated using 12 STR loci (Table 3), which varied from 0.469 to 0.217. The results of genetic distance of the present study were lower than the findings of Bai et al. (2015), who reported that the genetic distance ranged from 0.21 to 0.62 for Chinese indigenous sheep breeds, which was higher than that of Egyptian sheep breeds (Rushdi et al., 2015). The values of genetic distances showed that the investigated sheep populations are characterized as having a high range of variability of the allele pool, presence or absence of certain alleles, and differences in frequency of occurrence alleles. The largest genetic distance was observed between Kazakh Arkhar-Merino and Edilbay-1 (0.469). These two breeds were absolutely different from each other originally: by phenotype, according to the different branches of stock breeding and geographical places. Kazakh Arkhar-Merino is a meat-woolly breed with fine wool. Kazakh Arkhar-Merino is well adapted to breeding at high altitudes, and these sheep differ favourably from other breeds in conditions of mountain pasture. In contrast, the Edilbay-1 sheep breed is classified as the coarse wool sheep from the meat-fatty category. They are well adapted to the severe desert and semi-desert conditions of Kazakhstan. Also, Kazakh Arkhar-Merino is geographically most distant from Edilbay-1. In comparison with other populations, a closer relationship was found between Kazakh Arkhar-Merino and Kazakh Finewool (0.25), which could be attributed to the low geographical distance between these two populations. The same result has been found between Kermani and Lori-Bakhtiari Pakistani sheep (0.25), which both breed in Iran (Vajed Ebrahimi et al., 2017). Both of them (Kazakh Arkhar-Merino and Kazakh Finewool) refer to the meat and wool types of sheep breeds. Kazakh fat-tailed coarse wool and Edilbay-2 had less similarity to the Kazakh Finewool than Edilbay-1.
Further, to assess the genetic relationships among the population, a phylogenetic tree was constructed using the neighbour joining method (Saitou and Nei, 1987) based on Nei's genetic distance. The results of the phylogenetic tree indicated that the Edilbay-1 and Edilbay-2 sheep breeds were clustered into Kazakh fat-tailed coarse wool, which are coarse wool breeds and have a common origin. However, Kazakh Finewool and Kazakh Arkhar-Merino were grouped in the same node, both being fine wool sheep, as these breeds have the same ancestral background. Likewise, these two breeds were categorized together on the same branch of the phylogenetic tree in the previous study (Ozerov et al., 2008).
In addition, factorial correspondence analysis demonstrated that Kazakh Arkhar-Merino and Kazakh Finewool were isolated from the other studied populations due to the high level of differentiation and no sharing of alleles. Edilbay-2 is an admixture of both Edilbay-1 and Kazakh fat-tailed coarse wool. According to the FCA findings, this is connected with historical origin of these Kazakh sheep breeds.
In this study, within and among herds, the genetic diversity of five Kazakh sheep populations was assessed using 12 microsatellite markers. Based on our results, all five populations examined show high genetic diversity through a high effective number of alleles, a large mean number of alleles, high PIC values, and 12 completely polymorphic tested microsatellites. Moderate differentiation was found between Kazakh Arkhar-Merino and Edilbay-1, whereas differentiation between Edilbay-1 and Edilbay-2 was lower. Therefore, the evaluation of the results of Nei's genetic distance, neighbour joining, and FCA agreed with the historical origin of animals. As our study showed, although 10 years have passed since the research by Ozerov et al. (2008), it has been discovered that genetic diversity remains in sheep breeds other than Kazakh Arkhar-Merino. The main reasons for this fact are as follows. Scientists and livestock breeders have always been working on the improvement of these breeds. The advantages of this research also include the ability of the breeds to transmit all useful economic traits to their offspring. Breeding work is carried out mainly along the lines using several herds. Of course, not only the best males, but also elite queens, are selected to replenish livestock. Lines of sheep are created according to some outstanding qualities – precocity, weight, size of fat tail, and quality of wool. The work consists mainly of mating animals with distant degrees of kinship in the line. In addition, based on the data obtained, it is possible to recommend the Kazakh Arkhar-Merino breed for the selection against homozygous individuals. Further, the results achieved on STR loci are proposed to be used to control and conserve the genetic diversity of native sheep breeds.
The materials used the current study are available from the corresponding author on reasonable request.
KD and ZO participated in the collection of all materials and conducted all the experiments. KD and AM wrote the paper. RZh, BB, NS, and BM participated in the editing of the paper. All authors read and approved the final paper.
The authors declare that they have no conflict of interest.
The protocol for the use of materials was agreed with the Commission on Bioethics of the Kazakh National Agrarian University, Almaty, Kazakhstan.
The authors thank Gulnur Zhunussova for helping in all experiments and for technical support.
This research has been supported by the Ministry of Education and Science of the Republic of Kazakhstan (grant no. 541).
This paper was edited by Steffen Maak and reviewed by Semir Bechir Suheil Gaouar and one anonymous referee.