New Primitive Marking (Bider) in Mongolian Native Horse and Equus Przewalskii

—ORIGINAL— New Primitive Marking (Bider) in Mongolian Native Horse and Equus przewalskii

Mioko MASUDA1, Junko TSUNODA2, Hiroko NOMURA2, Nami KIMURA2, Gombojav ALTANGEREL1, Bandi NAMKHAI3, Usukhjargal DOLJ3 and Michinari YOKOHAMA2* 1Mongolian State University of Agriculture, Zaisan-53, Ulaanbaatar-210153, Mongolia,2Faculty of Bioindustry, Tokyo University of Agriculture, Yasaka-196, Abashiri-shi, 099-2493 Hokkaido, Japan, 3Hustai National Park, P.O. Box 1160, Central Post Office, Ulaanbaatar, 13, Mongolia

The incidence of Bider (Bi marking) in Mongolian native horses was 0.010, and that J. Equine Sci. according to districts was the highest (0.032) in Tov Aimag. Bi marking was frequently Vol. 18, No. 4 observed in bay (0.482) and chestnut horses (0.351), and was also observed in gray and roan pp. 145–151, 2007 horses. The incidence of Bi marking was high (0.842) in horses with a dorsal strip (DS) as a primitive marking. In 164 Equus przewalskii, the incidence of Bi marking was 0.396, which was about 40 times the incidence in Mongolian native horses. Bi marking was observed in both males and females in the two species, showing autosomal inheritance. Analysis of the mode of inheritance based on the incidence of Bi marking in foals according to various crossing combinations showed the complete dominance of Bi marking in both species. Thus, it was concluded that Bi marking occurs in Bi/Bi and Bi/– genotypes. This Bi marking differs from DS, leg stripes (LS) and shoulder stripes (SS), and is considered to be a type of primitive marking peculiarly occurring in Mongolian native horse and Equus przewalskii. The high incidence of Bi marking in Equus przewalskii (0.396), and the results of crossing between Mongolian native horses and Equus przewalskii performed in Mongolia, suggest introgression of the Bi marking gene from Equus przewalskii to Mongolian native horses. Key words: Bider (Bi), coat color gene, Equus przewalskii, primitive markings, Mongolian native horse

The coat color of domestic horses shows variation, coat in Thoroughbreds is expressed by the Sabino- sometimes representing a species’ characteristic. At based white gene (S), and its homozygotes have a white present, 13 coat color-associated genes have been coat. identified [2, 6–8, 11], but new findings have also been In the basic coat, such as bay and chestnut in reported recently. Though homozygosity for the roan domestic horses, the loci of extension (E) and Agouti gene (R) as a coat color-associated gene was reported to (A) as coloring genes are closely involved, and the be lethal in the Belgian breed [7], Ueda [12, 13] and expression of coat color is controlled by melanocortin- Yokohama et al. [14, 15] showed the survival of 1 receptors (MC1R) and agouti-signaling protein Hokkaido native horses with R/R type. It was also (ASIP) genes [5, 10]. In these loci, point mutation of C reported that the white lethal gene (W) is involved in (E gene) at codon 901→T (e gene) in MC1R and the white coat, and that the homozygote white gene is deletion mutation (A gene→a gene) of 11 bp in 2174– also lethal [7], but Brooks and Bailey [1], and 2184 bp in the second exon of ASIP have been Yokohama and Ishida [16] confirmed that the white reported. The E locus is epistatic to the A locus, and chestnut color develops irrespective of the genotype of e e This article was accepted August 27, 2007 the A locus in recessive homozygosity (E /E ). On the a a *Corresponding author. other hand, when the A locus is A /A , a black coat e-mail: [email protected] develops in the presence of at least one dominant allele 146 M. MASUDA, J. TSUNODA, H. NOMURA ET AL.

The survey items were the basic color, incidences of primitive markings, and incidence of Bi marking in the parent-child relationship in each district. The number of investigated horses was 3,775 during 1997–2003 and 529 in 2006 (total 4,304). A survey of Equus przewalskii was performed in the Hustai National Park located in the suburbs of Ulaanbaatar, the capital of Mongolia, which is one of the three places designated as Equus przewalskii protection areas. Of a total of 194 horses in the park (June, 2006), 164, in which the presence or absence of Fig. 1. Survey areas (eight Aimags) of Mongolian native horses. Bi marking could be determined, were included in this Remarks: Number showed animal number in each area, and horse number with Bider showed in parentheses. study. Bi markings were confirmed by direct : Other Aimags; n=2,700 (2). observation using telescopes and photography. In horse that could not be observed, Bi markings were confirmed on the basis of literal data and photographs stored in the Hustai National Park archives. in both the A and E loci. In Equidae, DS, LS and SS as primitive markings are Investigation of coat colors and markings observed [9]. These are rare characters in improved In Mongolian native horses, four types of primitive domestic horses but are frequently observed in local marking (DS, LS, SS and Bi) were evaluated (Fig. 2). horse breeds and wild horses. These markings have All Equus przewalskii had DS and LS. linear patterns. However, in our survey in Mongolia in 1998, we observed Mongolian native horses with Bi Sex difference and mode of inheritance of Bi marking marking that had not been observed in domestic In 646 Mongolian native horses and 164 Equus horses. Unlike DS, LS and SS, the Bi marking has an przewalskii, the difference in the incidence of Bi irregular coat color/pattern. Subsequently, in 2003, we marking between males and females was analyzed by discovered in the literature the existence of Equus the χ2 test. Then, based on data on 296 Mongolian przewalskii which has the same marking and habitat of native foals and 164 Equus przewalskii foals, the the in Mongolian grassland [4]. Since the Bi marking theoretical incidences of foals with and those without is a very rare character and is also observed in Equus Bi markings were calculated from the probability of przewalskii, we considered that this marking might be a crossing combinations. The difference between the marker with which to clarify the genetic relationship theoretical and observed incidences was analyzed by between domestic and wild horses. Therefore, a the χ2 test, and the mode of inheritance was evaluated. statistical survey was performed to evaluate the incidence of Bi marking and the mode of its inheritance in Equus przewalskii, which are protected Results and proliferate in Mongolia, and Mongolian native horses. Development of Bider (Bi) markings Bi markings vary in size among individual horses, Materials and Methods exhibiting the small punctate type, the medium-sized belt-like type extending to the area around the shoulder, or the large type extending to the neck and Investigated horses costal region (Fig. 3). Unlike linear markings, the Bi Surveys of the basic coat color and Bi marking in marking has an irregular symmetrical pattern and is Mongolian native horses were performed during 1997– composed of skin tissue pigments, as is in tattoos (Fig. 2003 and 2006 (April–September) in eight Aimags, 4). These findings were similar in Mongolian native mainly in Tov, Hentii and Dundgovi Aimags (Fig. 1). horses and Equus przewalskii (Fig. 5). BIDER MARKING IN MONGOLIAN HORSE 147

Fig. 2. Primitive markings in Equus przewalskii. DS: Dorsal stripe, LS: Leg stripe, SS: Shoulder Stripe, Bi: Bider. Phot. 1 cited from Lekler B.(2003)6).

Fig. 3. Variation of Bider (Bi ) type in Mongolian native horses. 148 M. MASUDA, J. TSUNODA, H. NOMURA ET AL.

Fig. 4. Bider is an irregular symmetrical pattern. Remarks: Depicted horse has a bay coat color. L: Left side, R: Right side.

Fig. 5. Variation of Bider (Bi) patterns in Equus przewalskii in the Hustai National Park located in the suburbs of Ulaanbaatar.

The incidences of coat colors and primitive markings in Mon- marking was 0.149, but that of Bi markings golian native horses accompanied by DS was 0.842 (Table 1). These In Mongolian native horses with Bi markings, the findings suggest a close association between Bi and DS basic coat color was classified into 10 types (Table 1). markings. Table 1 shows that the Bi marking is not a character developing for specific coat colors. The incidence of Bi The incidence of Bi marking in Mongolian native horses marking was the highest for bay (0.482), followed by according to districts chestnut (0.351). In this study, the incidence of Bi The incidence of Bi marking in the Mongolian native marking not accompanied by any other primitive horses was 0.010, showing it to be a very rare character BIDER MARKING IN MONGOLIAN HORSE 149

Table 1. Frequency of appearance of coat colors and primitive markings in Mongolian native horses Coat colors Bay Brown Chestnut Dark Palomino Buckskin Gray Roan Bay Chestnut Total Primitive chestnut tabiano tabiano markings Bi 0.421 0.176 0.235 0.000 0.118 0.000 0.059 0.000 0.000 0.000 0.149 (7) (3) (4) (0) (2) (0) (1) (0) (0) (0) (17) Bi/LS 0.375 0.000 0.250 0.125 0.000 0.000 0.000 0.125 0.000 0.125 0.070 (3) (0) (2) (1) (0) (0) (0) (1) (0) (1) (8) Bi/DS 0.400 0.000 0.467 0.067 0.000 0.067 0.000 0.000 0.000 0.000 0.132 (6) (0) (7) (1) (0) (1) (0) (0) (0) (0) (15) Bi/LS/DS 0.521 0.00 0.37 0.00 0.00 0.041 0.041 0.00 0.014 0.014 0.640 (38) (0) (27) (0) (0) (3) (3) (0) (1) (1) (73) Bi/LS/SS 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.009 (1) (0) (0) (0) (0) (0) (0) (0) (0) (0) (1) Total 0.482 0.026 0.351 0.017 0.017 0.035 0.035 0.009 0.009 0.018 (114) (55) (3) (40) (2) (2) (4) (4) (1) (1) (2)

Bi: Bider, DS: Dorsal Stripe, LS: Leg Stripe, SS: Shoulder Stripe. ( ): Animal number.

Table 2. Frequency of appearance of primitive markings in Mongolian native horses Aimag Total animal No. Incidence and number of primitive markings Incidence in each Aimag [Studied number] Bi DS LS SS With primitive Without primitive marking marking Tov 1,150 0.032 0.120 0.109 0.024 0.285 0.715 [1,023] (37) (138) (125) (28) (328) (822) Dundgovi 188 0.011 0.149 0.128 0.043 0.330 0.670 [159] (2) (28) (24) (8) (62) (126) Hentiy 468 0.011 0.107 0.064 0.024 0.205 0.795 [422] (5) (50) (30) (11) (96) (372) Other 2,772 0.001 0.052 0.036 0.000 0.088 0.912 [2,700] (2) (144) (99) (0) (245) (2,527) Total 4,578 0.010 0.079 0.061 0.010 0.160 0.840 [4,304] ( 46 ) (360) (278) (47) (731) (3,847) Bi: Bider, DS: Dorsal Stripe, LS: Leg Stripe, SS: Shoulder Stripe. [ ]: Studied animal number, ( ): Total of animal number.

(Table 2). The incidence of Bi marking in Tov Aimags sex difference (p=0.2–0.1). These results confirm the was 0.032, which was about three times the incidence in autosomal inheritance of Bi marking. Dundgovi or Hentii Aimag (0.011). This suggests a The mode of inheritance of Bi marking was distribution of horses with Bi markings centered on Tov evaluated in Mongolian native horses (Table 3). Aimag. Theoretical values were obtained from the probability Among primitive markings, the incidence of DS was of crossing combinations. Tests assuming that Bi/Bi the highest (0.079), followed by that of LS (0.061), and and Bi/– types represent complete dominance for the the incidences of Bi marking and SS were 0.010. development of Bi marking showed consistency between theoretical and observed values (p=0.5–0.3), Genetic analysis of Bi marking showing that the mode of inheritance of Bi marking is The incidence of Bi marking in Mongolian native similar to that of the roan gene (R). Similar tests horses was 0.146 in males and 0.121 in females, with no performed in Equus przewalskii confirmed complete significant sex difference by the χ2 test (p=0.5–0.3). dominance as the mode of inheritance as shown in The incidence of Bi marking in Equus przewalskii was Table 4 (p=0.7–0.5). The parent-child relationship was 0.466 in males and 0.341 in females, with no significant surveyed in 164 Equus przewalskii protected in the 150 M. MASUDA, J. TSUNODA, H. NOMURA ET AL.

Table 3. Test of the inheritance mode of the Bider (Bi) gene using family data of Mongolian native horse Mating Animal No. Observed No.(O) Bi in foal χ2 test P Sire Dam Expected No. (C) Bi non-Bi Bi Bi 6 O 5 1 0.075 0.8–0.7 C 4.727 1.273 Bi non-Bi 52 O 28 24 1.225 0.3–0.2 non-Bi Bi 30 21 9 (Bi × non-Bi/non-Bi × Bi) 82 C 44.002 37.998 Total 88 O 54 34 0.573 0.5–0.3 C 50.489 37.511 Bi ? 3 / 1 2 / / ?Bi20/ 137 non-Bi ? 4 / 2 2 ? non-Bi 16 / 6* 10 non-Bi non-Bi 165 / 0 165 Total 296 76 220 Genotype of Bi: Bi/Bi and Bi/–. Non-Bi: –/–.

Table 4. Test of the inheritance mode of the Bider (Bi) gene using family data in Equus przewalskii Mating Animal No. Observed No. (O) Bi in foal χ2 test P Sire Dam Expected No. (C) Bi non-Bi Bi Bi 9 O 7 2 0.057 0.9–0.8 C 7.280 1.720 Bi Non-Bi 51 O 29 22 0.016 0.9 Non-Bi Bi 28 16 12 (Bi × non-Bi / non-Bi × Bi) 79 C 44.457 34.543 Total 88 O 52 36 0.401 0.7–0.5 C 54.88 33.12 Non-Bi Non-Bi 28 / 3* 25 / / ?Bi1 / 0 1 Non-Bi ? 3 / 1 2 ?Non-Bi6 / 0 6 ? ? 38 / 9 29 Total 164 / 65 99 / / Genotype of Bi: Bi/Bi and Bi/– (Frequency of appearance; 39.634%). Non-Bi: –/– (Frequency of appearance; 60.366%).

Hustai Nuruu Park. The incidence of Bi marking in were regarded as Bi marking, in the way nomads refer Equus przewalskii was 0.396, which was about 40 times to them. The findings of the survey were similar in the incidence in Mongolian native horses (Table 4). Mongolian native horses and Equus przewalskii. In general, Bi marking was darker than the coat color and could be readily distinguished, but was difficult to Discussion distinguish from coat colors such as black bay and gray. A previous study showed that the basic primitive Bider (Bi) has no special meaning in Mongolia, and marking is DS, and that LS or SS is observed in horses is used simply to express a conspicuous marking. with DS [9]. A close association between Bi and DS Unlike linear markings, the Bi marking has an irregular markings was also recognized in this study. symmetrical pattern and is composed of skin tissue In each country of the world, horse breeds with DS or pigments, as in tattoos. In this survey, all such markings LS have frequently been reported [9, 11]. Many BIDER MARKING IN MONGOLIAN HORSE 151

Mongolian native horses also have primitive markings. 3. Hintz, H.F., and Van Vleck, L.D. 1979. Lethal However, the incidence of Bi marking in Mongolian dominant roan in horses. J. Hered 70: 145–146. native horses is only about 1%. This marking has not 4. Lekeler, B. 2003. Loshadi:1000 fotografii (Horse: been confirmed in any other domestic horse breed and 1000 photographs), Astel’: p.7. is considered to be a rare primitive marking developing 5. Marklund, L., Johansson, M., Sandberg, K., and only in Mongolian native horses and Equus przewalskii. Andersson, L. 1996. A missense mutation in the Bi markings in Mongolian native horses and Equus gene for melanocyte-stimulating hormone przewalskii were autosomally inheritance, and the mode receptor (MC1R) is associated with the chestnut of inheritance in both species was similar to that of the coat color in horses. Mamm. Genome 7: 895–899. roan gene (R). Six horses with Bi marked with an 6. Marklund, S., Moller, M., Sandberg, K., and Andersson, L. 1999. Close association between asterisk mark in Table 3, were difficult to distinguish sequence polymorphism in the KIT gene and the because of coat colors such as black bay and gray. We roan coat color in horses. Mamm. Genome 10: 283– observed three contradictory cases in Table 4, but there 288. was a high possibility they were produced by crossing 7. Pulos, W.L., and Hutt, F.B. 1969. Lethal dominant with a young sire in Harlem. white in horses. J. Hered. 60: 59–63. The results of genetic analysis of Bi marking, such as 8. Sponenberg, D.P., and Weise, M.C. 1997. those of the present study, may become basic Dominant black in horses. Genet. Sel. Evol. 29: 403– information for the clarification of the relationship 408. between domestic horses and Equus przewalskii. 9. Stachurska, A.M. 1999. Inheritance of primitive In Mongolia, Mongolian native horses and Equus markings in horses. J. Anim. Breed. Genet. 116: 29– przewalskii have lived in the same areas since early times, 38. and the observation of their natural crossing has been 10. Rieder, S., Taourit, S., Mariat, D., Langlois, B., and orally passed on. In the 1950s, at the Racehorse Guerin, G. 2001. Mutations in the agouti (ASIP), Research Institute in Jargalant, Tov Aimag in Mongolia, the extension (MC1R), and the brown (TYRP) loci a crossbred (F1), between female Mongolian native and their association to coat color phenotypes in horses and male Equus przewalskii, was produced to horses (Equus caballus). Mamm. Genome 12: 450– obtain strong racehorses. This may be the reason for 455. the presence of horses with Bi markings among 11. Trommershausen-Smith, A. 1980. Coat color Mongolian native horses mainly in Tov Aimag. We genetics. Appaloosa News April: 11–18. speculate that Bi marking is a character that has been 12. Ueda, J. 1999. Molecular approaches to coat color transferred from Equus pzrewalskii to Mongolian native genetics of horses. Proceeding of the first joint horses. symposium between Hokkaido University and Yeugnam If Bi gene cloning can be realized by molecular University August: 20–21. biological methods in the future, the association 13. Ueda, J. 2000. Polymorohism of the KIT gene and roan coat color in Hokkaido native horses. between wild and domestic horses may be further Proceedings of the second joint symposium between clarified. In addition, in terms of genetic resources, the Hokkaido University and Yeugnam University August: clarification of genes associated with primitive 4–5. markings may allow the recognition of Mongolian 14. Yokohama, M., Nomura, H., Yasuhara, T., and native horses as a domestic horse breed with inherited Nozawa, K. 2002. Lethal dominant roan is not genes of Equus przewalskii. found in Hokkaido native horse. Jour. Agri. Sci. Tokyo Univ. of Agric. 47: 98–101. 15. Yokohama, M., and Nozawa, K. 2004. An References additional analysis on lethality of roan allele in Hokkaido native horses. Jour. Agri. Sci. Tokyo Univ. 1. Brooks, S.A., and Bailey, E. 2005. Exon skipping in of Agric. 49: 147–149. the KIT gene causes a Sabino spotting pattern in 16. Yokohama, M., and Ishida, I. 2007. On inheritance horses. Mamm. Genome 16: 893–902. of white thoroughbred in Japan. Jour. Agri. Sci. 2. Castle, W.E. 1954. Coat color inheritance in horses Tokyo Univ. of Agric. 52: 76–81. and in other mammals. Genetics 39: 35–44.