74 Medycyna Wet. 2012, 68 (2)

Artyku³ przegl¹dowy Review White markings in

ANNA STACHURSKA, ANNE PHAFF USSING*

Department of Breeding and Use, Faculty of Biology and Animal Breeding, University of Life Sciences, Akademicka 13, 20-950 Lublin, *The Royal Library, P.O. Box 2149, DK-1016 Copenhagen K, Denmark

Stachurska A., Phaff Ussing A. White markings in the horse Summary White markings in horses result from the lack of melanocytes in the skin and hairs. The trait is characteristic of most breeds of domestic horses. In the breeding of the Polish Konik horse, which should resemble its wild ancestors, the lack of white markings was accepted as a selection criterion. The heritability of the markings is high. MC1R and ASIP loci, as well as two other putative QTLs are involved in the appearance of white markings. A foal should inherit a number of genes from both parents to make it possible for the markings to appear. The ultimate extent of markings is caused by the genes, as well as by intrauterine factors. The latter consist of accidental and random events which disturb the survival, migration and clonal proliferation of melanoblasts. The recessive e allele from MC1R locus and the dominant A allele from ASIP locus cause a greater extent of the markings, behaving as major genes. The most desired genotype of the Polish Konik horse, aaEEDD, which in inter se matings produces solely blue dun foals, is also the most desired with regard to the low amount of markings. To increase the progress in the Polish Konik breeding with regard to the absence of the markings, marked individuals and identified carriers of e allele should be consistently eliminated from reproduction. Keywords: white markings, inheritance, horse

White markings are patches of white hairs growing melanocytes from the epidermis (24). The patches of on unpigmented skin on the horse’s peripheral areas: depigmentation usually appear around the anus and the head and limbs. Rarely, the markings appear in genital regions, as well as in the face (fig. 1). Vitiligo central areas, which in the majority of cases indicates is observed in many animal species: mostly mammals, that the horse carries a gene producing one of the pied but also in chickens. The studies performed in humans coat color patterns. The unpigmented skin and hairs show that besides genetic effects it may result from an result from a lack of melanocytes (17). The white autoimmune disorder (20). markings are present at the foal’s birth and persist In many horse breeds, white markings are assumed throughout the life of the horse. Besides the markings, to be flashy, highlighting the limb action which is vitiligo may develop in adult horses due to the loss of particularly important in shows but not desired in

Fig. 1. Vitiligo in Felin Ponies. D¹browica near Lublin Medycyna Wet. 2012, 68 (2) 75 dressage. Moreover, the markings enable the recogni- tion of the horse from a distance. Unpigmented hoof horn associated with the markings is equally hard and elastic as the pigmented one (9, 28). However, because of the unjustified opinion of the inferior quality of the unpigmented horn, horses with white markings are unfavorable in many breeds. Occurrence of white markings Archive sources on the (Equus caballus gmelini) living in Poland and Lithuania do not men- tion any white markings. It may be supposed that if the old chronicles name such details as the dorsal stripe, white markings would not be passed over, either. Gmelin (1745-1774) (quoted after 21) states that “the wild horses are similar to a mouse with regard to the coat color and their legs beneath the middle to the hooves are black”. The lack of white markings in the Fig. 2. Percentage of Polish Konik horses without markings registered in I to VIII volumes of the studbook (24) Przewalski horse (Equus caballus przewalskii), the last living wild representative of the species, and in the Paleolithic cave paintings illustrating the horse, as well minary stud book were then included in the main stud as the markings rarely occurring in primeval Asiatic book (26; fig. 2). The number of the horses without horses (12), indicate the trait is characteristic of markings is higher in the following volumes; however, domestication. Hence, in the breeding of the Polish it still only slightly exceeds 80%. These data show that Konik horse, which should resemble its wild ancestors, the constant occurrence of marked horses, despite the the absence of white markings was accepted as a se- selection, is a great problem in the breeding of Polish lection criterion. Similarly, in the Norwegian Fjord Koniks. horse which is bred for a yellow dun color, white mar- kings seldom occur and are undesired (29). A facial Inheritance of white markings star, up to 5 cm diameter, is solely accepted in brood For over 80 years geneticists have been studying , but not in , in Norway. The reason for the inheritance of white markings. In the beginning, the official acceptance is that such a marking was pre- the most extensive research performed in Germany sent in three famous Fjord ancestors in the nineteenth enabled the suggestion of the hypotheses of two pairs century. In Denmark, only white hairs on the forehead of genes controlling the trait (18, 30). Crew and in Fjords are accepted. Buchannan Smith (8) put forward a theory of a domi- Breeders of Polish Konik, Fjord and other horses nant M factor and modifiers. Afterwards, German stu- which should be without white markings always face dies assumed four genes responsible for the markings the dilemma if it is worth eliminating an otherwise (3-6). According to another hypothesis the markings valuable horse due to, for instance, a small white spot. were considered as the least developed pied pattern Hence, usually some exceptions are considered. In (2). Following that approach, Lauvergne et al. (15) Polish Konik breeding, two programs are in effect. suggested horse classification related to the extent of According to the Programme of Conservation Breeding white patches on the body. None of the hypotheses were accepted by the Minister of Agriculture in 1999, confirmed later. “Polish Koniks ( ) without white markings may be In the early 1980s, monozygotic horse twins produ- registered in the studbook ( ) temporary in mares ced by embryo micromanipulation attracted the gene- little markings on head (white hairs, snip) are allowed”. ticists’ attention (1). One colt had a marking on all The Breeding Programme of Genetic Resources four limbs and another colt had it solely on the left Conservation effective from 2000 is more strict, since limbs. In a pair of fillies one foal had a marking on the it allows only horses without any white markings. left forelimb and the other did not have any markings. Analyzing the stud book, it may be noticed that up to The result was not consistent with expectations, since Volume VI some individuals with a marking, includ- if the trait is genetically controlled in monozygotic ing the fetlock joint, occur. In Volume VII, mares with twins the genotype and consequently the markings a blaze, stripe or white pastern, as well as stallions should be identical (35). A similar riddle was noticed with a star are registered. There are still five stallions in the case of individual horses with asymmetrical with white hairs, one with a snip and two with a star in markings. The same genotype produces differently Volume IX, which is the latest volume. The percentage expressed traits on two sides of the body. The findings of horses without markings is lower in Volumes III indicated that additional non-genetic factors determine and IV due to the fact that individuals from the preli- the extension of the trait. 76 Medycyna Wet. 2012, 68 (2)

Extensive studies on the occurrence and amount of markings brought the solution to the problem. A high or medium correlation between the extension of the facial and limb markings (10, 19, 23, 30, 35) showed that they were due to one genetic mechanism. Results documenting a high sire-foal and dam-foal regression proved the mechanism could not be founded on one pair of genes which would cause the segregation in the offspring. It was also found that stallions were slightly more marked than mares, hence a gene producing the trait might be associa- ted with the horse’s gender (10, 23, 35). A hypothesis of the multifactorial mode of inheritance of white markings was put forward by Nebe (19) on the basis of data collected in over 8,000 foals of Hessian Fig. 3. Vertical black stripes in Polish Konik . The stripes are not horses. Broad investigations of Ara- present on the other side of the body. Roztocze National Park bian horses registered in the American stud- book, conducted by Woolf at the turn of the 1980s and (breed or identity marks) destroys the melanocytes, 1990s (31-36) explored the hypothesis on the multi- resulting in growth of white hairs in the involved area. factorial inheritance of the markings. Particular analy- Factors disturbing melanoblast migration and pro- ses concerned 2.5 thousand to 12 thousand horses. The liferation are not known. Although the effects should markings were scored from 0 to 5 depending on the similarly influence both sides of the organism, stocha- amount of whiteness at five areas on the head and stic events appear. It must be emphasized that the separately on each limb. According to those studies, migration and proliferation of the melanoblasts occur the heritability of the markings on the head is 0.69, on autonomously in each side of the embryo because of the limbs 0.68, and combining the facial score and four the mid-dorsal separation (33). Sponenberg (25) pre- limb score amounts to 0.77 (35). Other authors (19, sented a photograph of a horse with white narrow 22, 23) also reported high indices, but compared with vertical stripes on one side of the body. Such horses those mentioned above they were lower. MC1R and occur extremely rarely in the world and they do not ASIP loci, which control basic coat colors, show reproduce those stripes. Occasionally, vertical black a pleiotropic function affecting the white marking stripes on one side of the body occur (fig. 3). They expression. Recent studies on Swiss Franches-Mon- also may be caused by specific factors in the prenatal tagnes horses show that other two putative QTLs life. greatly influence the trait (14, 22). Woolf’s articles formed a view of how to explain A heritability of approximately 0.75 is very high. the inheritance of white markings (33, 35). A number That means the phenotypic variance is determined in of genes of equal and unequal effects on the markings ¾ by the genotype and in ¼ by environmental factors. is inherited from the parents by a foal. A parent with The latter finding was the most difficult to interpret. It markings may be heterozygous to some extent, hence has always been known that the markings were innate it produces gametes both with genes increasing the and constant, hence they did not seem to be influen- markings and with genes limiting the expression of ced by the environment. To explain the phenomenon, the trait. Heavily marked parents carry more genes pro- Mintz’es (17) and earlier hypotheses on the ordinary ducing markings and in some loci are homozygous, developmental noise in the intrauterine life were used hence the offspring will show a tendency towards the (33, 35). As is known, melanoblasts migrate from the presence of the markings. In contrast, parents less neural crest and enter distal ends of particular body marked or without any markings carry few genes for part buds. The melanoblasts proliferate in the presump- the markings and the progeny will show a tendency tive facial and limb tissues. After metamorphose, the not to be marked. According to Woolf (33, 35), a foal melanocytes that have settled in the skin, epidermis should inherit a threshold number of genes from both and follicles secrete melanines determining the color parents to make it possible that the markings appear. of the skin and hairs. During the prenatal development, If the threshold is not reached, the markings do not accidental and random events may affect the survival, appear. Horses without markings may differ with migration and clonal proliferation of the melanoblasts. respect to the number of potential genes producing the If the melanocytes are absent or did not develop markings. Ultimate extension of markings is influen- normally, melanogenesis does not occur and the skin ced by the genes, as well as by intrauterine factors. and hairs are unpigmented. Similarly, freeze-marking The polygenic inheritance is consistent with results of Medycyna Wet. 2012, 68 (2) 77

Tab. 1. Results of matings (%) of Polish Konik horses in 2002 research proved that chestnuts were more heavily mar- with regard to the presence of white markings (25) ked not only when compared to bays, but primarily in

Offspring regard to blacks (32, 33). On that basis, a hypothesis was suggested that the recessive e allele from the Parents Number Without With of foals markings a marking MC1R locus (mapped to ECA3p12) causes a greater size of the markings behaving as a major gene. The Without Without 209 88.5 11.5 markings markings dominant A allele from the ASIP locus (ECA22q15) shows a similar although weaker function. Hence, Without With 092 75.0 25.0 markings a marking a genotype producing the greatest amount of markings is AAee, whereas the fewest markings is produced by With With 013 69.2 30.8 a marking a marking aaEE. The strong MC1R locus effect causes that horses of matings of marked horses producing non-marked foals all homozygous ee colors have a greater amount of and, conversely, marked offspring originating from markings, i.e. besides chestnuts, those are red duns and non-marked parents. palominos. ASIP locus implies that homozygous aa As an example, data on matings of Polish Konik horses are relatively less heavily marked, i.e. besides horses are presented (27; tab. 1). For parents without blacks, this includes blue duns. Bays, yellow duns and markings, the progeny usually was not marked either buckskins are located at a medium position. Thus, the (88.5%). For parents with markings, the number of most desired genotype of the Polish Konik horse, progeny without markings was distinctly lower (75.0% aaEEDD, which produces solely blue dun foals, is also and 69.2%). The relation is visible in spite of select- the most desired with regard to the low amount of its ing the herd against the markings. markings. Blue dun Koniks of aaEeDD genotype car- The markings are more frequent on hind limbs than rying the e allele, with a similar parent, may produce on fore limbs and on left side limbs compared to right a red dun foal and in 50% of all kinds of matings will side limbs (10, 23, 35). Fig. 4 illustrates average transfer the tendency to the presence of the markings. white marking size scored on the 0-5 scale in over 16 MC1R locus is mapped in a group linked with KIT thousand Purebred Arabian and half-breed Arabian locus controlling tobiano, roan and other white pat- horses (33). The marking amount in forelimbs was terns (7, 13, 16); thus in the region important for the significantly lower than in hind limbs. In turn, the trait melanogenesis in horses. Recent studies by Rieder et extent on right side limbs was considerably lower than al. (22) show another putative major locus mapped to on left side limbs, which was found in both breeds, ECA3q at or near the KIT locus. A recessive single except for the hind right limb in Purebred gene could account for 20-80% of the total heritability Arabians. It turned out that half-breed Arabians were for the trait present at different parts of the body, parti- less marked compared to Purebred Arabians. It was cularly in pheomelanic (ee) horses. One more QTL also shown in the case of each limb that bay horses (MITF) responsible for the expression of white mar- were generally less heavily marked than chestnut horses. kings, mainly on eumelanic (E_) coat color, has been The fact that chestnut horses are more marked was found on ECA16q (14). These findings may indicate established a long time ago (3-6, 10, 19, 35). Further that not many genes (at least four) produce the trait. White markings versus breeding criteria The high heritability and QTLs involved mean that selection both towards and against the markings is effective. Simultaneously, the polygenic inheritance is associated with the impossibility of the total elimina- tion of markings. As shown, the genes affecting the markings may be recessive and masked by dominant genes, hence they are not easy to identify. Moreover, mutations are always possible. To increase the progress in the Polish Konik breeding with regard to the absence of markings, the marked individuals should be con- sequently eliminated. Moreover, identified carriers of e allele should not be considered in reproduction either. It seems that soon molecular studies can almost enti- rely explain the white markings inheritance. If the model includes only a few genes, it will allow the matings to be more directed. Fig. 4. Mean white limb marking size in bay and chestnut The high effectiveness of the selection resulted in Purebred Arabians and half-breed Arabians (32) great differentiation of horse breeds with regard to the 78 Medycyna Wet. 2012, 68 (2) extent of white markings. Besides the Polish Koniks associated with chestnut coat color in horses. Mamm. Genome 1996, 7, 895- and Fjords, marked horses are eliminated with various -899. 17.Mintz B.: Gene control of mammalian differentiation. Annu. Rev. 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J., Silvestrelli M., Langlois B., Renieri C., Poirel D., Gallizzi Arabian horse. J. Hered. 1989, 80, 173-178. Vecchiotti Antaldi G.: A new scheme for describing horse coat colour. Livest. Prod. Sci. 1991, 27, 219-229. Corresponding author: prof. Anna Stachurska, PhD, Department of 16.Marklund L. S., Moller M. J., Sandberg K., Andersson L.: A missense muta- and Use, University of Life Sciences, Akademicka 13, tion in the gene for melanocyte-stimulating hormone receptor (MC1R) is 20-950 Lublin, Poland; e-mail: [email protected]