在来家畜研究会報告 30：155–167（2021） Rep. Soc. Res. Native Livestock 30：155–167（2021）

Report on Genetic Characteristics of Kazakhstan Original Kushum

1 2 3 4,10 Trung Ba Nguyen , Ripon C. Paul , Yu Okuda , Thu Nu Anh Le , Phuong Thi Kim Pham 1, Kushaliye J Kaissar 5, Akhmedenov Kazhmurat 6, Sarsenova Bibigul 6, Meirat Bakhtin 7, Polat Kazymbet 7, Suleimenov Zh Maratbek 8, Alikhan Meldebekov 8, Masahide Nishibori 9, Takayuki Ibi 10, Takehito Tsuji 10 10,11 and Tetsuo Kunieda

1 An Giang University, Vietnam National University Ho Chi Minh city, An Giang, Vietnam

2 Patuakhali Science and Technology University, Barishal, Bangladesh

3 Okayama University of Science, Okayama, 700-0005, Japan

4 University of Agriculture & Forestry, Hue University, Hue, Vietnam

5 Zhangir Khan West Kazakhstan Agrarian-Technical University, Uralsk, Kazakhstan

6 M. Utemisov West Kazakhstan State University, Uralsk, Kazakhstan

7 Radiobiological Research Institute, JSC Astana Medical University, Astana, Kazakhstan

8 Institute of Zoology, Almaty, Kazakhstan

9 Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8511, Japan

10 Graduate School of Environmental and Life Science Okayama University, Okayama 700-8530, Japan

11 Faculty of Veterinary Medicine, Okayama University of Science, Imabari 794-8555, Japan

Abstract A total of 22 animals of Kazakhstan original Kushum horse were sampled and investigated the genetic characteristics by using 247 bp of D-loop region of mitochondrial DNA and four SNPs (rAX, rW, rA and rD) in male-specific euchromatic region of the Y chromosome and functional genes associated with coat color, body composition and locomotion traits. We detected 10 mitochondrial DNA haplotypes that fell into 8 of the 17 major haplogroups of the horse mitochondrial DNA indicating a unique haplotype composition with high genetic diversity. We also found two Y-chromosomal haplotypes in Kushum which likely originated from Trotter and/or Don breeds. The findings regarding the mitochondrial DNA and Y-chromosomal haplotypes are concordant with the documented maternal and paternal origins of the Kushum horses. The allele frequencies of ASIP, MC1R and MATP associated with coat color were consistent with the coat color variations of the Kushum horses. The allele frequencies of MSTN associated with endurance performance and DMRT3 associated with gait suggested that the

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observed allele frequencies of these genes were results of selective breeding for these traits. These findings will be useful information for a better understanding of the origin and breeding history of the Kushum horse breeds.

Introduction Horse is domesticated animal from the Turpan, and horse is assumed to be domesticated in central Asia 5,500 years ago, then spread to entire Eurasian continent. However, exact process of horse domestication and spread is still unclear. Kazakhstan is assumed to be the center of horse domestication. (Warmuth et al., 2012; Ludwig et al., 2009; Outram et al., 2009). And Kazakhstan is one of the major countries that produce horses and export horse meat and many people in Kazakhstan produce koumiss a popular fermented liquor made from horse milk. Horses are also commonly used for riding herding and racing in this country. Therefore, many original horse breeds have been established and raised in different regions of Kazakhstan. The Kushum is one of the Kazakhstan original horse breeds established in the Ural region of West Kazakhstan (Fig. 1) between 1931 and 1976 through crosses between mares of local horse and stallions of Trotter and Russian Don breeds (Dmitriez et al., 1989b). The original goal of the breeding of Kushum horses was to improve the body size endurance performance and gait of the local horses to match the military demand for war horses before World War II. Then the Kushum horses have mainly been used for producing milk and meat as well as for herding cattle and sheep after World War II (Dmitriez et al., 1989b). The total number of heads of Kushum horse was 4829 as of 1980 (Dmitriez et al., 1989b). The average withers height of Kushum stallions and mares is 159 and 154 cm respectively (Dmitriez et al., 1989b) and their coat colors include mainly and and occasionally black. Kushum horses are well-adapted to herd maintenance in semi-desert pastures of West Kazakhstan. However, the genetic characteristics of this breed such as genetic diversity phylogeny and allele frequencies of gene associated with important traits of horse are not well understood. Y-chromosomal markers and haplotypes of mitochondrial DNA (mtDNA) are commonly used for investigating genetic diversity and phylogenetic relations among breeds or populations of animals in paternal and maternal lineages respectively. Previous studies have indicated remarkable genetic diversity in mtDNA haplotypes among global horse populations suggesting multiple origins of maternal lineages during horse domestication (Achilli et al., 2012; McGahern et al., 2006; Vilà et al., 2001). Conversely during the breeding of domestic animals, a limited number of stallions are used and genetic introgression between breeds or populations is mainly realized through paternal lineages rather than maternal lineages since effective genetic introgression can be achieved by introducing few stallions. Recently Y-chromosomal haplotypes have also been used for phylogenetic analyses (Felkel et al., 2018; Wallner et al., 2017; Wallner et al., 2013). Particularly Wallner et al. (2017) identified various single-nucleotide polymorphisms

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Russia

Kaztal Zhanibek Kazakhstan

China

Uzbekistan Kyrgyz

Türkmenistan Fig. 1. A map of Kazakhstan indicating the sampling places (Zhanibek and Kaztal). (Nguyen et al., 2020)

(SNPs) of the horse Y chromosome and these SNPs have been utilized these for phylogenetic analysis of the paternal lineage (Felkel et al., 2018; Han et al., 2015b; Kakoi et al., 2018). In horses, phenotype of coat color is a crucial allele that plays an important role in the characterization of specific horse breeds and correct animal identification. The basic coat colors namely bay chestnut and black are determined by the melanocortin 1 receptor (MC1R) and agouti-signaling protein (ASIP) genes. Horses homozygous for MC1R and ASIP mutations show chestnut and black coat colors respectively whereas horses harboring the wild type alleles of both genes show bay coat color (Marklund et al., 1996; Rieder et al., 2001). In addition to these basic coat colors cream dilution is caused by a mutation of the solute carrier family 45 member 2 (MATP) gene. A missense mutation of MATP results in back skin or palomino coat color in heterozygous horses and a double-dilute coat color in homozygous horses (Mariat et al., 2003). Since horses are mainly used because of their physical performance breeds have been intensively selected for physical performance-related traits such as locomotion traits. Recently the genes associated with physical performance of horses have been identified. For example, a genetic variant of the myostatin (MSTN) gene is associated with the racing performance of horses (Hill et al., 2010; Tozaki et al., 2010) and a nonsense mutation of the double-sex and mab-3 related transcription factor 3 (DMRT3) gene shows a major effect on locomotion patterns of horses including ambling (Andersson et al., 2012). Therefore, selective breeding for these physical performance related traits might result in the changes of the allele frequency of these genes in the breeds. In this report, we summarized recently published our paper describing the genetic characterization of Kushum horses in Kazakhstan based on haplotypes of mtDNA and Y chromosome, and genes associated with important traits of the horses (Nguyen et al., 2020).

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Materials and Methods Sampling and DNA extraction There was a total of 22 blood samples from 7 males and 15 female Kushum horses in the Zhanibek and Kaztal regions of West Kazakhstan (Fig. 1) during 2017 to 2018. These horses were selected from local herds of Kushum horse to avoid the sampling from the horses sharing kinship. The blood samples were collected from the jugular vein and DNA was extracted from whole blood cells according to the standard phenol–chloroform extraction method.

MtDNA haplotype analysis The DNA fragment containing the D-loop region of mtDNA (15494 to 15740; 247 bp) was amplified by PCR using primers (5’-CTAGCTCCACCATCAACACC-3’ and 5’-ATGGCCCTGAA GAAAGAACC-3’) (Hill et al., 2002) and the amplified fragments were directly sequenced using the dideoxy method to determine mtDNA haplotypes variations as described in Nguyen et al. (2020). The parameters of the mtDNA sequences including haplotype number haplotype diversity and nucleotide diversity were calculated using DnaSP 5.10.1 (Librado et al., 2009). The obtained sequence data were aligned using ClustalW in MEGA 7.1 (Kumar et al., 2016) and a dataset of 97 haplotypes of the D-loop region including 10 Kushum horse haplotypes and 87 haplotypes reported by Cieslak et al. (2010) was generated. A phylogenetic tree was constructed using the neighbor-joining method in MEGA 7.1 with 1000 random bootstrap replicates.

Y-chromosomal haplotype analysis Four SNPs namely rAX, rA, rW and rD on the male-specific region of Y chromosome was determined via direct sequencing of DNA fragments amplified by PCR using primers described in Nguyen et al. (2020) to analyze Y-chromosomal haplotypes (Han et al., 2015b; Wallner et al., 2017). These four SNPs were selected from various Y-chromosomal SNPs reported by Wallner et al. (2017), Wallner et al. (2013) and Filkel et al. (2018) to classify the Y- chromosomal haplotypes into five different groups.

Genotyping of genes associated with coat color and locomotion traits For regarding the genotyping of functional genes associated with body composition, locomotion traits and coat color genotypes of DMRT3 and ASIP were determined via direct sequencing of DNA fragments amplified by PCR as described in Nguyen et al. (2020) using primers described in Kakoi et al. (2009) and Han et al. (2015a) respectively. MC1R MATP and MSTN were genotyped by PCR–RFLP using primers described in Wagner and Reissmann. (2000), Kakoi et al. (2009) and Polasik et al. (2015) respectively. PCR was performed as described in Nguyen et al. (2020). To confirm the accuracy of the genotyping results of PCR–RFLP the amplified fragments from at least two samples for each genotype of these genes were directly

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sequenced using the dideoxy method.

Results and Discussion Variation in mitochondrial DNA haplotypes We investigated mtDNA haplotypes of the Kazakhstan Kushum horses by sequencing and comparing with a 247-bp segment of D-loop region between 15494 and 15740 of reference mtDNA of Equus caballus sequence X97547. As shown in Table 1, 10 haplotypes (KT1−10) with 19 segregated sites were observed in these horses. The obtained genotypic diversity was 0.922 ± 0.034 nucleotide diversity was 0.02365 ± 0.00239 and average nucleotide difference was 5.818.

Table 1. Haplotypes of mtDNA D-loop region observed in Kushum horses (Nguyen et al., 2020).

, .

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 et al 4 4 4 5 5 5 6 6 6 6 6 6 6 6 6 7 7 7 7 (2010) 9 9 9 3 4 9 0 0 1 1 3 4 5 6 6 0 2 2 4 horses haplotype Numbers of of Numbers Kazakhstan Kazakhstan Ceislak Ceislak 4 5 6 4 2 8 2 3 6 7 5 9 9 6 7 3 0 6 0 in Haplotypes T T A C C T C T A T C A T G A T G G A X79547a . C ...... KT-1 5 D2 . C . . . . T ...... A . . KT-2 2 A . C . . . . T . . C . . C . . . A . . KT-3 3 B1 . C . . . . T ...... G C A . . KT-4 2 K3 − . C . . . . T . . C . . C . . C A . . KT-5 1 . C . . . C T . G . . . . C A . . KT-6 1 − . C . . . . T ...... C A A G KT-7 2 K2b C C G T . . T C . . . G . . . . A . . KT-8 3 X2 . C . . . . T . . . . G . . . C A . G KT-9 1 − . C . . T . T . . . T . . A . C A . . KT-10 2 X3c1 a Reference sequence.

According to the previous studied, we also constructed a phylogenetic tree with 97 horse mtDNA haplotypes including 10 Kushum horse haplotypes and 87 haplotypes reported by Cieslak et al. (2010) using the neighbor-joining method (Fig. 2) to estimate the phylogenetic relationships of Kushum horse haplotypes with previously reported haplotypes. Among the 10 haplotypes, 7 haplotypes namely KT1, 2, 3, 4, 7, 8 and 10 were the same as previously reported haplotypes D2, A, B1, K3, K2b, X2 and X3c1 respectively. These haplotypes are known to be ancestral haplotypes survived in modern breeds of horse and widespread among various primitive breeds of the Eurasian continent (Cieslak et al., 2010). The remaining 3 haplotypes namely KT5, 6 and 9 were not included in the data set of the 87 previously reported haplotypes (Cieslak et al., 2010) but were reported to be present in some primitive breeds or native horse populations in the Eurasian continent (Hristov et al., 2018; Lei et al., 2009). Based on the nucleotide sequences of the D-loop regions Cieslak et al. (2010) classified horse mtDNA

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haplotypes into 17 haplogroups. The 10 haplotypes of Kushum horses fell into 8 (A, B, D, K, K3, X2, X3 and X7) of the 17 haplogroups and the distribution of these haplogroups in Kushum horses was essentially similar to that in primitive horse breeds in Central Asia including Akhal-Teke Caspian and Vyatskaya (Cieslak et al., 2010).

Fig. 2. A Neighbor Joining-based phylogeny tree constructed using the 247-bp (15494 to 15740) mtDNA D-loop sequence of 97 haplotypes including 10 Kushum horse haplotypes (indicated by circles) and 87 haplotypes of Cieslak et al. (2010). (Nguyen et al., 2020)

These findings of mtDNA haplotypes indicated high genetic diversity and unique genetic composition of the maternal lineage of Kushum horses. The high genetic diversity of Kushum horses observed in present study suggested that the founder of this breed was not from limited maternal lineages. Since the Kushum breed was established by cross-breeding between mares of Kazakhstan local horse and stallions of exotic breeds the mtDNA haplotypes of Kushum horses must have originated from the population of local horses in the early 20th century. Therefore, the high diversity of mtDNA haplotypes in Kushum horses likely reflects high genetic diversity of the maternal lineage of the local horse population in Central Asia.

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Variation in Y-chromosomal haplotype Regarding the Y-chromosomal haplotype, four SNPs (rAX, rW, rA and rD) in male-specific euchromatic region of the Y chromosome in seven male Kushum horses were investigated. As shown in Table 2 we could classify the Y-chromosomal haplotypes into five groups by the genotypes of these four SNPs (Felkel et al., 2018; Wallner et al., 2017). One group (HG1) was composed of haplotypes which are mainly observed in Asian local breeds as well as in northern European primitive breeds including Icelandic horses. The remaining four groups (HG2, HG3, HG4 and HG5) were composed of haplotypes mainly observed in European and American breeds English Thoroughbred and part of European and American warmblood breeds -influenced breeds and draft breeds and other some local breeds such as Lipizzan respectively.

Table 2. Haplotypes of the Y chromosome observed in Kushum horses (Nguyen et al., 2020).

Group of Y-chromosomal markers Number of haplotypes rAX rA rW rD horses HG1 T T G T 0 HG2 C A G T 5 HG3 C A G − 0 HG4 C T A T 2 HG5 C T G T 0

The genotyping results of the four SNPs in Y-chromosome indicated that five of the seven males possess a haplotype belonged to HG2 and the remaining two possess a haplotype belonged to HG4. Since Kushum horses have been established through three-way crosses of mares of Kazakhstan local horse with stallions of Thoroughbred Trotter and Don breeds the Y-chromosomal haplotypes of Kushum horses must have originated from these exotic breeds. According to Wallner et al. (2017) most Thoroughbred horses possess a haplotype belong to HG3 and Standardbred Trotter horses possess a haplotype belong to HG2. While no information about the Y-chromosomal haplotype is available for Don horses this breed is genetically influenced by Arabian horses (Dmitriez et al., 1989a). Therefore, the HG2 haplotype of Kushum horses has likely originated from Trotter and HG4 from Don whereas no evidence for contribution of Thoroughbred to the Y chromosome of Kushum horses was observed in the horses analyzed in this study.

Variation in genes associated with coat color and locomotion traits In this study, we also investigated the genotypes of genes associated with coat color (ASIP, MATP and MC1R) and locomotion traits (MSTN and DMRT3) of the 22 Kushum horses (Table 3). Regarding coat color-related genes, the genotyping results indicated that all 22 horses carried the wild type C allele of MATP associated with normal coat color and no mutant Ccr allele associated

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with dilute coat color suggesting that MATP is fixed or nearly fixed for C allele in the population of Kushum horses. For the ASIP and MC1R both wild type A and E alleles and mutant a and e alleles respectively were observed in these 22 horses with a relative abundance of wild type alleles. The coat colors estimated from the genotypes of these genes were concordant with the observed coat colors with few exceptions that might be caused by misjudgment of coat color upon observation. These allele frequencies of coat color related genes were essentially consistent with the coat color variation of the Kushum horses namely mainly bay and chestnut and occasionally black (Dmitriez et al., 1989b). Regarding locomotion traits, the frequency of the T allele of MSTN in Kushum horse was high (Table 3). The horses with the TT genotype were reported to show high endurance stamina at long-distance races while those with the CC genotype show high speed at short-distance races (Hill et al., 2010). Since the initial goal of breeding of Kushum horses was mainly for military use endurance for long-distance locomotion might be essential to this breed. While T allele is major allele in many horse breeds the relatively high frequency of the T allele of MSTN is concordant with such demand for the Kushum horses.

Table 3. Genotype distributions and allele frequencies of genes associated with coat color and locomotion traits (Nguyen et al., 2020). c2 values for Genes Genotype distributions Alleles frequencies HWE test AA Aa aa A a ASIP 1.9 12 10 0 0.77 0.23 CC CCCr CCCr C CCr MATP NA 22 0 0 1 0 EE Ee ee E e MC1R 0.88 10 11 1 0.7 0.3 TT TC CC T C MSTN 0.22 18 4 0 0.91 0.09 CC CA AA C A DMRT3 1.14 17 4 1 0.86 0.14 HWE test: Hardy–Weinberg equilibrium test, NA: Not applicable.

Analyzing DMRT3, the frequency of the A allele associated with ambling gait was lower than that of the C allele associated with normal gait (Table 3). Horses homozygous for the nonsense mutation (A allele) of DMRT3 can perform ambling gaits which are characterized by the symmetrical lateral movement of the footfall pattern (Andersson et al., 2012; Promerová et al., 2014). However, the frequencies of A alleles in most horse breeds are generally very low except in those with an ability of ambling gait (Promerová et al., 2014), the observed frequency of A allele (0.14) in Kushum horse is relatively high. While we have no information on the ability of ambling gait of Kushum horses one of the initial purposes of the cross with Trotter during the breeding was to improve the gait of Kushum horses (Dmitriez et al., 1989b). Therefore, the relatively high

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frequency of the A allele might be the result of the introgression of this allele from Trotter stallions which are known to perform ambling gait and show high frequency of the A allele (Jäderkvist et al., 2014; Promerová et al., 2014). Moreover, gaited horses may have been selected at the early stages of breeding to improve their locomotion traits as military horses although such a selection may have no longer been performed in later stages of breeding since these horses were mainly used for milk and meat production subsequently. Thus the observed frequency of the A allele of DMRT3 might be the trace of such breeding history.

Conclusion The breeding of Kushum horses started in 1931 just before World War II in a major horse producing region of former Soviet Union Kazakhstan. At that time producing numerous military horses with high endurance performance good locomotion traits and strong adaptability to cold climates might be required to prepare for a possible war in the European continent. Presumably to match such military demands Kushum horses were bred through systematic breeding programs using mares of local horse and stallions of exotic breeds under strong selection pressure for the traits desired as a military horse. The allele frequencies of genes associated with locomotion traits observed in the present study might reflect these historical situations. Furthermore, the observed composition of mtDNA and Y-chromosomal haplotypes are concordant with the documented maternal and paternal origins of this breed. The results of the present study demonstrate that we can obtain useful information for better understanding of the origin and breeding history of horse breeds using molecular markers.

Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (B) (17H04637, MN) from the Japan Society for the Promotion of Science.

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カザフスタン固有のクシュームウマの遺伝学的特徴に 関する報告

1 2 3 4,10 Trung Ba Nguyen ・Ripon C Paul ・奥 田 ゆ う ・Thu Nu Anh Le ・Phuong Thi Kim Pham 1・Kushaliye J Kaissar 5・Akhmedenov Kazhmurat 6・Sarsenova Bibigul 6・Meirat Bakhtin 7・Polat Kazymbet 7・Suleimenov Zh Maratbek 8・ 8 9 10 10 10,11 Alikhan Meldebekov ・西堀正英 ・揖斐隆之 ・辻 岳人 ・国枝哲夫

1 ホーチミン市ベトナム国家大学, アンジャン大学, ベトナム・2 パツアカリ科学技術大学, バングラデ

シュ・ 3 岡山理科大学・4 フエ大学, 農林大学, ベトナム・5 Zhangir Khan 西カザフスタン農業技術大学,

カザフスタン・6 M. Utemisov 西カザフスタン州立大学, カザフスタン・7 放射線生物学研究所 JSC

アスタナ医科大学, カザフスタン・8 動物学研究所, カザフスタン・9 広島大学大学院生物圏科学研究科・

10 岡山大学大学院環境生命科学研究科・11 岡山理科大学獣医学部

カザフスタン固有の品種であるクシュームウマについて 22 個体のサンプルを採取し，ミトコンド リア DNA D ループの 247bp の領域，Y 染色体上の雄特異的な真性クロマチン領域の 4SNPs (rAX, rW, rA, rD)，および毛色，体の構成，歩行特性に関する機能的遺伝子を用いてその遺伝的特性について 調べた。その結果，ウマミトコンドリア DNA の主要な 17 ハプログループの内の８ハプログループ に属する 10 ハプロタイプが検出され，クシュームウマのミトコンドリア DNA は高い遺伝的多様性 と固有のハプロタイプ構成を持つことが明らかとなった。さらに，クシュームウマには，トロッター 種とドン種に由来すると考えられる 2 つの Y 染色体ハプロタイプが存在することも明らかになった。 これらのミトコンドリア DNA と Y 染色体ハプロタイプに関する知見は，クシュームウマの母方およ び父方の祖先に関する記録と一致していた。毛色に関わる ASIP，MC1R，MATP 遺伝子の多型はク シュームウマの毛色の多様性と矛盾していなかった。持久力に関わる MSTN 遺伝子および歩様に関 わる DMRT3 遺伝子の対立遺伝子頻度は，これらの形質に関する選抜育種の結果と考えられた。以 上の知見はクシュームウマの由来と育種の歴史を理解する上で有用な情報となると考えられた。

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