Domestic sheep (Ovis aries) have played important roles in diverse human societies as a source of food, hide, and wool, and are one of the major components of agro-pastoral societies since the Neolithic (Chen et al., 2006). In the 21st century, where various industries emerged and technology developed, Mongolia continues to play a socio-economic role in animal husbandry (Yu, 2012). In Mongolia, compared to other types of livestock, such as goats, horse, cattle, and camels, Mongolian sheep have been a dominant domestic animal since ancient times. The sheep population comprised > 60% of total domestic animals in Mongolia (Onolragchaa et al., 2019). Mongolia supported 32.2 million heads of sheep in the 2019 census, sheep account for 45.5% of all livestock, the thirteenth largest population in the world (NSO, 2019). In addition, Mongolian indigenous sheep (MIS) are well adapted to the harsh environment in diverse natural zones (i.e., mountain, steppe, and forest). Ten breeds or strains are currently recognized in Mongolia as morphologically and genetically distinct, although it remains unclear whether these represent breeds or strains (Onolragchaa et al., 2019).

The Mongolian native (MN) sheep is widely distributed throughout the country. The Orkhon sheep is the first Merino breed derived in Mongolia. This semi-fine wool sheep of the central steppe zone has been developed from the F1 progeny of Mongolian fat-tailed ewes and precocious Soviet Merino or Tsigai and Altay rams, which were used on successive generations of cross-breeds (Batsukh et al., 1991). The Altanbulag sheep was produced through improving the Mongolian native sheep by Hisar and Edelbay rams. Hybridization was conducted up to the F2 and F3 hybrid generations in local forest-steppe region. These hybrid generations were hybridized with each other to establish meat-fat tailed type breed. The Suffolk breed was imported from France to Mongolia. These high-grade sheeps will be used in Mongolian agriculture in accordance with the breeding strategy and policy.

As the importance of genetic resources of living organisms increases worldwide, countries around the world have recognized the economic value of genetic resources and signed the Convention on Biological Diversity (CBD) for biodiversity conservation and sustainable use of resources (Park, 2007; Choi, 2012). As such, the importance of animal genetic resources is emerging worldwide, and genetic studies related to it have been conducted. Therefore, efforts are needed to protect and secure genetic resources for sheep breeds that currently exist in Mongolia. No studies on the genetic relationships between these breeds/strains and native sheep. We found only two study that considered some common breeds of Mongolia (Luo et al., 2005; Onolragchaa et al., 2019). Mitochondrial DNA (mtDNA) polymorphisms have played a significant role in tracing the origin of specific breeds and the genetic diversity of domestic sheep and other livestock species due to their maternal mode of inheritance, have a high copy number and have a greater rate of substitution on average than nuclear genes, making them particularly useful for resolving intra-species branching (Moore, 1995). Studies performed on the control region (CR; also called the Displacement loop or D-loop) and Cytochrome B (CytB) are more variable than are other mtDNA regions. For this reason, the majority of phylogenetic studies have used these markers to investigate the genetic relationships among related breeds within species (Meadows et al., 2011). D-loop and CytB gene of mtDNA of modern sheep from a wide geographical range describe five different haplogroups (A, B, C, D, and E) (Wood et al.,1996; Hiendleder et al., 1998b; 2002; Pedrosa et al., 2005; Guo et al., 2005; Meadows et al., 2005; 2007). According to those initial studies, haplogroup A predominates in Asian sheep, while haplogroup B predominates in European sheep (Guo et al., 2005; Meadows et al., 2007). In addition haplogroups D and E, the most recent lineages, were only found in the Caucasus and Turkey (Tapio et al., 2005; Meadows et al., 2007), whereas relatively rare haplotypes from Haplogroup C have a wide geographical distribution (Chen et al., 2006). A recent study on the complete mitochondrial genome of eighty eight Mongolian native sheep examined the relationship between two sheep breeds. The phylogenetic analysis confirms the division of domestic sheep in to the three (A, B, and C) haplogroups, but T-insertion, deletion and heteroplasmy were not found (Onolragchaa et al., 2019). In this context, the purpose of our study was to evaluate the genetic diversity and to clarify the origin of the MIS using the analysis of the mitochondrial markers D-loop and CytB. By comparative analysis of specific mitochondrial markers and complex molecular phylogeny, this study aims to identify the clear descendants and interrelation of the MIS with Europe sheep.

Sample collection and DNA extraction

In the present study, whole blood samples of Mongolian native (n = 24), Orkhon (n = 23), Altanbulag (n = 25) and Europe sheep (Suffolk) (n = 22) were collected from Darkhan province, Mongolia and stored at -40°C until further using. DNA samples were extracted from the blood a using the QuickGene DNA Whole blood Kit (KURABO, Japan) according the manufacturer’s instruction methods. The extracted genomic DNA was used for experiments after measuring the concentration and purity using the ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). For genetic diversity and phylogenetic analysis, we amplified from the gene that or mtDNA (mitochrondrial DNA) CytB and D-loop (control region) area. CytB were amplified 743bp using primers F: 5’-GATCTCCCAGCTCCATCAAA -3’ and R: 5’-TGAGGGGGAGTGTTAAGTGG -3’(Dudu et al., 2016), D-loop area were by amplified 1,055bp using primers: F: 5’-AACTCCCAAACATACAACACGG 3’and R: 5’-ATTTGAGTATTGAGGGCGGGAT-3’(Li et al., 2006). PCR was performed in a total reaction volume of 15 μL containing 50-100 ng/uL of genomic DNA, 0.5 μL of each primer, 1.5 μL of 10x PCR buffer, 1.2 μL of dNTPs, 0.2 μL of Hot Start Taq DNA Polymerase and DW (distilled water) was used to perform PCR using the GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). The PCR amplification was conducted using following conditions: pre-denaturation at 95°C for 10 minutes followed by 35 cycles of 95°C for 45 seconds, 55°C-59°C for 45 seconds, and 72°C for 60 seconds. After 15 minutes of extension at 72°C, the reaction was terminated at 4°C. Before sequencing, the PCR products were purified using AccuPrep PCR/Gel DNA Purification Kit (BIONEER, Korea), amplified PCR product fragments were directly sequenced. The heteroplasmy was confirmed by 2% Agarose gel, and the nucleotide sequence was also confirmed.

Data analysis

The sequence was processed using DNA sequencing Analysis 5.1 Software (AppliedBiosystems), and sorted and edited using BioEdit software v.7.2.5 (Hall, 1999). DNA Sequence Polymorphism (DnaSP) v5.1 (Librado et al., 2009) was used to calculate the haplotype diversity and nucleotide diversity, number of haplotypes and mtDNA (CytB and D-loop). Ten sheep mtDNA control reference sequences belonging to the five known haplogroups (Meadows et al., 2011) were also includes in the analysis, to facilitate the recognition of haplogroup statutus of each individual Haplogroup A (HM236174.1, HM236175.1), Haplogroup B (HM236176.1, HM236177.1), Haplogroup C (HM236178.1, HM236179.1), Haplogroup D (HM236180.1, HM236181.1) and Haplogroup E (HM236182.1, HM236183.1). We also used HaploView software (https://www.broadinstitute.org/haploview/downloads) to check the sequence and frequency of haplotypes. A phylogenetic tree was constructed with FigTree v1.4.3 Software (http://tree.bio.ed.ac.uk/software/figtree/). The Median joining (MJ) networks (Bandelt et al., 1999) was drawn using the program Network V.5.0.0.3 software to investigate the possible relationships among haplotypes and to determine the number of mutations between haplogroups.

Mitochondrial CytB analysis

To estimate the origin of the MIS the determined CytB sequences were edited and linked using Bioedit software and compared to already identified (Meadows et al., 2011) sheep mtDNA haplogroup reference sequences. Ovine 743bp of CytB sequences were examined in 94 sheep samples (72 MIS, 22 Europe sheeps), which are representing four population of sheeps raised in Mongolia (Fig. 1). In detection of total 17 haplotypes in CytB region of four sheep populations, 7 belong to Altanbulag sheep, 10 to MN sheep. In each population there were identified 5-10 haplotypes of CytB region. In the case of CytB, there were found 28 SNPs, haplotype diversity was 0.7610, which was relatively lower than D-loop. For accurate results, we used MJ network figuration to identify the phylogeny of haplotypes. The phylogenetic tree results were consistent. There were identified 3 haplogroups (A, B and C) in 17 types of haplotypes as in reference haplotypes. As a result, haplogroup A was dominanted (n = 46 of 94 sheeps), followed by haplogroup B (n = 36) and haplogroup C (n = 12) (Table 1). In addition, the haplogroup results of CytB were few haplotypes and reference haplogroups were not found (Fig. 2).

Table 1. Distribution of mtDNA CytB haplogroups of Mongolian sheep.

Breed/population	No. of sample	No. of haplotype (CytB)	Haplogroups
			A	B	C
Altanbulag	25	7	15 (60.0%)	6 (24.0%)	4 (16.0%)
Mongolian native	24	10	10 (41.7%)	8 (33.3%)	6 (25.0%)
Orkhon	23	5	10 (43.5%)	11 (47.8%)	2 (8.7%)
Suffolk	22	7	11 (50.0%)	11 (50.0%)	0 (0.0%)
Total	94	29	46	36	12

The cases with same haplotypes were from different breeds. When the haplotypes were classified by each breed, the number of haplotypes was higher..

Figure 1.Sheep examined in this study. Mongolian sheep Altanbulag sheep (A), MN sheep (B), Orkhon sheep (C), Suffolk sheep (D).

Figure 2.Median-joining network of Mongolian sheep based on mtDNA CytB region.

Mitochondrial D-loop analysis

In detection of total 76 haplotypes in D-loop region of four sheep populations, 24 haplotypes belong to Altanbulag sheep, 24 to MN sheep. In each population there were identified 21-24 haplotypes, 220 SNPs as known as hypervariable regions. We have identified 3 haplogroups (A, B and C) in 76 haplotypes as in reference haplotypes using MJ network figuration (Fig. 3). As a result, haplogroups of each sheep population in D-loop region were consistent with the result of CytB (Table 2). The haplotype diversity and nucleotide diversity in the mtDNA D-loop region are shown on Table 3. The haplotype diversity of Orkhon sheep (1) was relatively higher than MN sheep and Suffolk sheep (0.996). The highest nucleotide diversity was found in MN sheep (0.02105), the lowest in Suffolk sheep (0.01678). In D-loop region, SNPs were 7.8 times, haplotype was 4.5 times, and diversity was 0.2338 higher than CytB’s. Furthermore, in the process of creating haplogroups using the MJ network, there were presented Heteroplasmy, T insertion and deletion in haplogroup A (Table 3). Sequence analysis showed that T deletion and T insertion in D-loop occurred at a high rate in MIS population (T insertion = 47, T deletion = 83, Table 3). T deletion in Suffolk sheep was 100% (the highest), in Orkhon sheep 91.3%, in Altanbulag sheep 88.0%, in MN sheep 75.0% (the lowest). Among the reference haplogroups, T deletion was occurred only in A, B and D haplogroups. T deletions did not exist in haplogroups C and E, but T deletions was appeared in all Suffolk sheep. It seems to be likely that it was derived from haplogroup C and E.

Table 2. Distribution of mtDNA CytB haplogroups of Mongolian sheep.

Breed/population	No. of sample	No. of haplotype (CytB)	Haplogroups
			A	B	C
Altanbulag	25	7	15 (60.0%)	6 (24.0%)	4 (16.0%)
Mongolian native	24	10	10 (41.7%)	8 (33.3%)	6 (25.0%)
Orkhon	23	5	10 (43.5%)	11 (47.8%)	2 (8.7%)
Suffolk	22	7	11 (50.0%)	11 (50.0%)	0 (0.0%)
Total	94	29	46	36	12

The cases with same haplotypes were from different breeds. When the haplotypes were classified by each breed, the number of haplotypes was higher..

Table 3. Distribution of mtDNA D-loop nucleotide, Haplotyde diversity, T insertion, deletion and heteroplasmy in Mongolian sheep.

Breed/population	No. of sample	Indel		Nucleotide diversity	Haplotype diversity	Heteroplasmy (ratio)
		Insertion	Deletion
Altanbulag	25	15 (60.0%)	22 (88.0%)	0.01806	0.997	3 (12.0%)
Mongolian native	24	10 (41.7%)	18 (75.0%)	0.02105	0.996	2 (8.3%)
Orkhon	23	11 (47.8%)	21 (91.3%)	0.01771	1	1 (4.2%)
Suffolk	22	11 (50.0%)	22 (100.0%)	0.01678	0.996	2 (9.1%)
Total	94	47 (50.0%)	83 (88.3%)			8 (8.5%)

Figure 3.Median-joining network of Mongolian sheep based on mtDNA D-loop region.

Heteroplasmy was found in Mongolian sheep population, but has not been reported in previous studies. In the result of sequencing of each type, heteroplasmy was generated by deletion of 1 repeat in 75nt tandem repeat of diversity in the D-loop, and appeared in both of the short mutant genome and the normal genome. Heteroplasmy was discovered in 8 of 94 Mongolian sheeps (3 Altanbulag sheeps, 2 MN sheeps, 1 Orkhon sheep, 2 Suffolk sheeps; Table 3). From this result, mutation has occurred in Mongolian sheep from ancient times. Therefore, in the future, more studies on Mongolian sheep breeds are needed to utilize mtDNA heteroplasmy as a breed specificity. For accurate results, to improve our understanding of the phylogenetic relationships among haplotypes with in lineages, within the sheep populations, as well as between MIS and Europe sheep, we constructed MJ network for each lineage in population with 10 references sequences retrieved from the NCBI (Genbank). When we used MJ network to combine the reference sequence’s data with Mongolian sheep’s data, only A, B and C haplogroups were matching. Haplogroup A was found in mainly breeds from Asia, haplogroup B observed at the highest frequency in breeds sourced from Europe (Guo et al., 2006). Haplogroup C which was first identified in Chinese sheep (Luo et al., 2005; Guo et al., 2006), occurred more often in Near East (West Asia), but were found in three population distributed in the Mongolia. D and E haplogroups did not appear in our study, which are very rare, and have been reported to occur only in Turkish sheep and Caucasus regions. Therefore, Mongolian sheep, which the rest of breeds except Suffolk sheep can be derived from Asia, Europe and Near East (West Asia). D and E haplogroups showed in Fig. 2, 3 and 4 are taken from reference data. In comparison to previously well-defined sheep mtDNA lineages A, B (Hiendleder et al., 1998b), and C (Guo et al., 2005; Pedrosa et al., 2005), all these 93 haplotypes of MIS and Europe sheep can be clearly grouped into these three lineages A, B, and C with 46, 36 and 12 haplotypes, respectively. Finally, the result of Haplogroup showed that the number of haplotypes of D-loop was greater than CytB, but the result of haplogroup was consistent in D-loop and CytB (Table 2). Altanbulag sheep’s haplogroupA was predominant at 60.0%, haplogroupB was found at 24.0%, and haplogroupC at 16.0%. MN sheep’s haplogroupA was also predominant at 41.7%, haplogroupB at 33.3% and haplogroupC at 25.0%. Unlike the two breeds, Orkhon sheep’s haplogroupB was predominant at 47.8%, haplogroupA was found at 43%, and haplogroupC at 8.7%. Suffolk sheep’s haplogroupA and haplogroupB were equal, and haplogroupC was not appeared.

As the importance of genetic resources increase around the world, it is recognized that animals’ genetic resources have industrial economic value. For this reason, efforts are being made to improve genetically modified breeds with good genetic resources around the world and establish them as unique breed. In modern Mongolia, sheep are very important animal, as they still lead the industry around animal husbandry. A sheep breeds in Mongolia withstand cold and dry climate conditions and have good genetic resources. They are very important through genetic characteristics and establishment of breeds through maintaining genealogy, and efforts to secure the gene source of breeds. For this, it is essential to estimate and confirm the origin through the genetic diversity and structural studies of Mongolian sheep. This study analyzed mtDNA two regions of CytB and D-loop, which have many mtDNA mutations to estimate the origin of Mongolian sheep and to investigate the genetic diversity and structure. The data indicated an abundant genetic diversity in Mongolian sheep population. This finding is consistent with other genetic diversity studies of MIS (Luo et al., 2005; Onolragchaa et al., 2019). In this study, the haplotype diversity was higher than the results of previous studies (Chen et al., 2006; Pardeshi et al., 2007; Onolragchaa et al., 2019), whereas the nucleotide diversity was lower than the results of previous study (Chen et al., 2006). As a result of confirming MJ network and Phylogenetic tree of the Mongolian and Europe sheep, compared with ten sheep mtDNA control reference sequences belonging to the five haplogroups (Meadows et al., 2011) (Fig. 5 -7). Mongolian sheep are thought to be derived from haplogroupA (Asia), B (Europe) and C (West Asia). In general, the haplogroup prevalence in present study is consistent with the previous studies on domestic sheep breeds in Asia (Luo et al., 2005; Chen et al., 2006; Pardeshi et al., 2007; Wang et al., 2007; Zhao et al., 2011; Arora et al., 2013; Singh et al., 2013).

Heteroplasmy, which caused by the diversity of 75 tandem repeat, has not been reported in previous studies of Mongolian sheep, but has been found in other animals. Studies of other mammals, such as monkey 160bp (Hayasaka et al., 1991), bat 81bp (Wikinson et al., 1991), rat 79bp (Stewart et al., 1994) and sturgeon 74-82bp (Ludwig et al., 2000) have confirmed that heteroplasmy occurs due to a deletion mutation of a repeating sequence in the D-Loop region of mtDNA. Heteroplasmy is stably inherited from mother to offspring. In the case of monkeys, heteroplasmy was found in the majority of monkeys in a specific region. Based on this, heteroplasmy was maintained for several generations of the monkeys (Hayasaka et al., 1991; Wilkinson et al., 1991). Therefore, heteroplasmy is inherited, so it can be used as an indicator to identify the lineage. In the case of Mongolian sheep, there is a difference in the occurrence of heteroplasmy in each breed, but it is considered to be insignificant and difficult to be judged due to the specificity of the breed. This is not related to haplogroupC as shown in the result of T deletion. Different distribution patterns exist for each haplogroup (A, B, and C) of three population of Mongolian sheep breeds. Since, Mongolia is adjacent to Asian China and European Russia, MIS are crossing over two neighboring countries and considered to belong to various haplogroups. Our findings were consistent with the similar results of previous studies on domestic sheep breeds, in which haplogroup A and B were predominant in most of breeds, whereas haplogroup C was at low frequency in Mongolian sheep (Luo et al., 2005; Onolragchaa et al., 2019) and in other countries (Meadows et al., 2007; Singh et al., 2013; Gorkhali et al., 2015). Only in one Mongolian sheep (Altanbulag No. 44) was found haplogroup A as in reference haplogroups, while the rest were belonged to the haplogroup, but the same haplotype was not found.

In this study, we investigated the genetic diversity and origin of three MIS population and one Europe sheep population raised in Mongolia. It is vital to report that indigenous sheep populations of Mongolia retain high levels of genetic diversity based on the results from analysis of two mtDNA markers (D-loop and CytB). Phylogenetic analysis indicated that three maternal lineages (haplogroups A, B and C) were found in these breeds except Europe breed, which only had A and B haplogroups. From this result, Mongolian sheep populations were in hybridization with European sheep varieties. The heteroplasmy, which has never been found in Mongolian sheep, has been newly discovered in this study. Heritability was high, as the mutation has occurred in MIS from the ancient times. Further research will incorporate ancient sheep samples from archeological sites in Mongolia with a much large number of samples from the border area in Mongolia to contribute to a better understanding of the origin of MIS. Also additional molecular studies are required using domestic sheep from neighbor countries such as Russia and China.

The authors are grateful to Prof. Baatartsogt Oyungerel, Department of Biotechnology, Mongolian University of Life Sciences, Ulaanbaatar, Mongolia, for his helpful provide samples, comments and discussions.

No potential conflict of interest relevant to this article was reported.

The study was approved by the Hankyong National University Animal Ethics Committee (No.2018-6).

Conceptualization: Hong Sik Kong

Data curation: Yi Seul Kim

Formal analysis: Yi Seul Kim, Badamsuren Batsukh

Funding acquisition: Hong Sik Kong

Investigation: Yi Seul Kim, Jiyeon Seong

Methodology: Yi Seul Kim, Jiyeon Seong

Project administration: Hong Sik Kong, Jiyeon Seong

Resources: Khaliunaa Tseveen, Badamsuren Batsukh

Software: Yi Seul Kim

Supervision: Hong Sik Kong

Validation: Yi Seul Kim, Jiyeon Seong

Visualization: Yi Seul Kin, Khaliunaa Tseveen

Writing - original draft: Yi Seul Kin, Khaliunaa Tseveen

Writing - review & editing: Khaliunaa Tseveen, Jiyeon Seong