Equine coat color genetics

This chestnut horse most likely has the e/e genotype at Extension.

Extension, also called MC1R, is in charge of deciding when a hair follicle should produce red pigment and when it should produce black. When the MC1R protein produced by this gene works properly, it is capable of making the hair either red or black. When it is broken, it can only tell the hair to be red. It has no effect on skin color. E symbolizes Extension, and the working version is dominant over the broken version. That means that an E/E or E/e horse will be capable of producing either red or black pigment in the hairs. Black pigment may be restricted to the points, as in a bay, or uniformly distributed in a black coat. Meanwhile, a horse with the genotype e/e will only be able to color the hairs red, such as in a chestnut horse.[7] Extension is also sometimes called "red factor" and can be identified through DNA testing.[8] Horses with the genotype E/E are sometimes called "homozygous black", however depending on the mate there is no guarantee that offspring will be black coated, only that no offspring will be "red".

This bay horse has a functional copy of MC1R (Extension) and has black hairs in the mane, tail, and lower legs.

There are two known mutations to the extension gene in horses, both resulting in a chestnut color. The first to be discovered is symbolized by e, and is a change of a single cytosine to thymine at base pair 901 which results in the serine in position 83 being changed to a phenylalanine. The other is symbolized by e^a, and is a change of a single guanine to adenine at base pair 903, resulting in aspartate being changed to asparagine at position 84 in the polypeptide. Visually there is no difference between the two, but some horses genetically tested before 2000 when the e^a allele was discovered may have gotten incorrect results. [9] [10]

The extension gene is found on equine chromosome 3 and codes for the melanocortin-1 receptor (MC1R), which straddles the membrane of pigment cells (melanocytes). MC1R picks up a chemical called alpha-melanocyte-stimulating hormone (α-MSH), which is produced by the body, from outside the cell. When MC1R comes into contact with α-MSH, a complex reaction is triggered inside the cell, and the melanocyte begins to produce black-brown pigment (eumelanin). Without the stimulation of α-MSH, the melanocyte produces red-yellow pigment (pheomelanin) by default.[11] Mutations that break protein function generally lead to recessively inherited lighter or redder coat colors in various mammals, while mutations that cause MC1R to be constantly active result in dominantly inherited black coats.[12][13] In horses, both known mutations break the protein and therefore result in red coats.

Various mutations in the human MC1R gene result in red hair, blond hair, fair skin, and susceptibility to sunburnt skin and melanoma.[11] Polymorphisms of MC1R also lead to light or red coats in mice,[14] cattle,[15] and dogs,[16] among others. The Extension locus was first suggested to have a role in horse coat color determination in 1974 by Stefan Adalsteinsson.[17] Researchers at Uppsala University, Sweden, identified a missense mutation in the MC1R gene that resulted in a loss-of-function of the MC1R protein. Without the ability to produce a functional MC1R protein, eumelanin production could not be initiated in the melanocyte, resulting in coats devoid of true black pigment. Since horses with only one copy of the defective gene were normal, the mutation was labeled e or sometimes E^e.[18] A single copy of the wildtype allele, which encodes a fully functional MC1R protein, is protective against the loss-of-function. The normal or wildtype allele is labeled E, or sometimes E⁺ or E^E.

Extension phenotypes

E/E and E/e both allow black hairs. Absent the dominant agouti allele (A) to restrict eumelanin to the points, this horse's coat is uniform black.

• E/E (+/+, E⁺/E⁺, E^E/E^E) wildtype, homozygous dominant. A horse with this genotype will be bay if it has the non-dun mutation and the wild-type version of every other allele. Otherwise, it could be black, seal brown, buckskin, perlino or smoky cream, bay dun or grullo, silver bay or silver black. They may also be gray or white. Horses that are E/E will always pass on a functional copy of the MC1R gene to its offspring, and will never produce offspring with the e/e genotype.
• E/e (+/e, E⁺/E^e, E^E/E^e) wildtype, heterozygous. These horses can be any of the same colors as with E/E, but only have half a chance to pass on a functional MC1R allele. In addition, a recent study that compared horse genotypes to their coat color phenotypes did find a statistically significant connection that suggested that lighter bay shades were heterozygous for the Extension mutation (E/e) and darker bay shades were homozygous.[19]
• e/e (E^e/E^e) homozygous recessive. A horse with this genotype will be chestnut if it has the non-dun mutation and the wild-type version of every other allele. Otherwise, it can be any of the red-based colors: red dun, palomino, cremello, gold champagne, and so on. Just like E/E horses, they can also be gray or white. Paired with an e/e mate, such horses will only ever produce red-family coat colors. At birth, the skin may be pink and the eyes blue, but these traits disappear after a few days and the eyes and skin of adult red coated horses are unaffected by this allele.[20] No health defects are associated with MC1R mutations in horses.

Agouti controls the restriction of true black pigment (eumelanin) in the coat. Horses with the normal agouti gene have the genotype A/A or A/a. Horses without a normal agouti gene have the genotype a/a, and if they are capable of producing black pigment, it is uniformly distributed throughout the coat.[21] A third option, A^t, restricts black pigment to a black-and-tan pattern called seal brown.[22] This allele is recessive to A and dominant to a, such that horses with the genotype A/A^t appear bay, while A^t/A^t and A^t/a horses are seal brown in the presence of a dominant Extension allele E.

The Agouti locus is occupied by the Agouti signalling peptide (ASIP) gene, which encodes the eponymous protein (ASIP). Agouti signalling peptide is a paracrine signaling molecule that competes with alpha-melanocyte stimulating hormone (α-MSH) for melanocortin 1 receptor proteins (MC1R). MC1R relies on α-MSH to halt production of red-yellow pheomelanin, and initiate production of black-brown eumelanin in its place.[23]

In many species, successive pulses of ASIP block contact between α-MSH and MC1R, resulting in alternating production of eumelanin and pheomelanin; hairs are banded light and dark as a result. In other species, ASIP is regulated such that it only occurs in certain parts of the body. The light undersides of most mammals are due to the carefully controlled action of ASIP. In mice, two mutations on Agouti are responsible for yellow coats and marked obesity, with other health defects. Additionally, the Agouti locus is the site of mutations in several species that result in black-and-tan pigmentations.[24][25] In normal horses, ASIP restricts the production of eumelanin to the "points": the legs, mane, tail, ear edges, etc. In 2001, researchers discovered a recessive mutation on ASIP that, when homozygous, left the horse without any ASIP. As a result, horses capable of producing true black pigment had uniformly black coats.[26] One genetics testing lab began offering a test for A^t,[22] but it was later found to be inaccurate and is no longer offered.

The mode of inheritance of the Agouti gene is theorized to be complicated by the presence of more than 2 alleles. The theorized A^t allele appears to be responsible for Dark Brown to near black color colors that have lighter brown/tan color on the soft parts of the horse (muzzle, withers and around the eyes), such as this one.

Agouti phenotypes

• A/A wildtype, homozygous. Visually, the horse may be bay, buckskin, bay dun, amber champagne, and so on, or gray, or any member of the red family. However, such a horse will never be black, grullo, and so on, nor will a homozygous A horse ever produce uniform-black offspring or seal brown offspring.
• A/A^t Heterozygous Agouti. Visually Indistinguishable from the homozygous A Bay colored horse. Such horses are unable to produce black colored foals.
• A/a wildtype, Heterozygous Agouti. Visually Indistinguishable from homozygous A. With the right partner, such horses have the ability to produce black colored foals.
• A^t/A^t The hypothetical A^t would modify the coat by restricting eumelanin in the coat to the points of the horse (Legs, ears, mane and tail). In the presence of at least one dominant E allele, this would result in a seal brown coat color. The body is dark brown to almost black with light brown to tan hairs on the muzzle, around the eyes and on the withers of the horse. Variations of the theorized brown/seal brown coat color include: dark buckskin (also referred to as smokey brown) - A brown based horse with 1 cream gene; Perlino - a brown horse with 2 cream genes; Brown Dun - A brown based horse with at least 1 dun gene; and Sable Champagne - A brown based coat with at least 1 Champagne gene.
• A^t/a Indistinguishable from the homozygous seal brown. Such horses have the ability to produce black and black based colored horses.
• a/a homozygous recessive. In the presence of a dominant E allele, the horse's coat will be black. With the presence of a single cream gene, the coat will be smoky black, and with the presence of 2 cream genes the coat will be smoky cream. The Presence of at least 1 Dun gene will result in a grullo colored coat (black dun). The presence of at least 1 Champagne gene will result in classic champagne coat, and the presence of at least 1 Silver gene will turn the black coat into a silver black coat, and so on.

The flat, earthy tone of the coat and vivid dorsal stripe are indicative of the D allele. Primitive markings are seldom visible on horses without the dominant, wildtype dun allele (D).

Dun is one of several genes that control the saturation or intensity of pigment in the coat. Dun is unique in that it is simple dominant, affects eumelanin and pheomelanin equally, and does not affect the eyes or skin.[27] Horses with the dominant D allele (D/D or D/d genotype) exhibit hypomelanism of the body coat, while d/d horses have otherwise intense, saturated coat colors. The mane, tail, head, legs, and primitive markings are not diluted. In some breeds, zygosity for Dun can be determined with an indirect DNA test.[27]

The Dun locus is TBX3 on equine chromosome 8.[2][28] The molecular cause behind the dun coat colors is not entirely understood, but the dilution effect comes from the placement of pigment in only part of the hair. The associated coat colors were assigned to the Dun locus in 1974 by Stefan Adalsteinsson, separate from Cream, with the presence of dun dilution indicated by the dominant D allele.[17] The dominant D allele is relatively rare compared to the alternative d allele, and for this reason, the dominant allele is often treated as a mutation. However, the pervasive coat color among wild equids is in fact dun, and researchers from Darwin to modern day consider dun to be the wildtype state.[29][30]

An older non-dun mutation was found in 2015 and named non-dun 1. It creates primitive markings but does not dilute the base color, and is co-dominant with the more common non-dun 2 but recessive to dun.[2]

Cream is another one of the genes that control the saturation or dilution of pigment in the coat. Cream differs from Dun in that it affects the coat, skin, and eyes, and unlike Dun, is dosage dependent rather than simple dominant. Furthermore, the effects on eumelanin and pheomelanin are not equal. Horses with the homozygous recessive genotype (C/C) are not affected by cream. Heterozygotes (C^Cr/C) have one cream allele and one wildtype non-cream allele. Such horses, sometimes called "single-dilutes", exhibit dilution red pigment in the coat, eyes, and skin to yellow or gold, while eumelanin is largely unaffected. Homozygotes (C^Cr/C^Cr) have two cream alleles, and are sometimes called "double-dilutes." Homozygous creams exhibit strong dilution of both red and black pigment in the coat, eyes, and skin to ivory or cream. The skin is rosy-pink and the eyes are pale blue. Cream is now identifiable by DNA test.[31]

The Cream locus is occupied by the Solute carrier family 45, member 2 (SLC45A2) gene, also called the Membrane associated transport protein or Matp gene.[32] The Matp gene encodes a protein illustrated to have roles in melanogenesis in humans, mice, and medaka, though the specific action is not known.[32]

Mutations in the human Matp gene result in several distinct forms of Oculocutaneous albinism, Type IV as well as normal variations in skin and hair color.[33] Mice affected by a condition homologous to cream, called underwhite, exhibit irregularly shaped melanosomes, which are the organelles within melanocytes that directly produce pigment.[34] The first descriptions of the dosage-dependent genetic control of the palomino coat color occurred early on in equine coat color inheritance research.[35] However, the distinction between Dun and Cream remained poorly understood until Stefan Adalsteinsson wrote Inheritance of the palomino color in Icelandic horses in 1974.[36] The mutation responsible, a single nucleotide polymorphism in Exon 2 resulting in an aspartic acid-to-asparagine substitution (N153D), was located and described in 2003 by a research team in France.[32]

Champagne is a gene that controls the saturation or dilution of pigment in the coat. Unlike Cream, Champagne is not strongly dosage-dependent, and affects both types of pigment equally.[37] Champagne differs from Dun in that it affects the color of the coat, skin, and eyes, and in that the unaffected condition is the wildtype. Horses with the dominant CH allele (CH/CH or CH/ch genotype) exhibit hypomelanism of the body coat, such that phaeomelanin is diluted to gold and eumelanin is diluted to tan. Affected horses are born with blue eyes which darken to amber, green, or light brown, and bright pink skin which acquires darker freckling with maturity.[38] The difference in phenotype between the homozygous (CH/CH) and heterozygous (CH/ch) horse may be subtle, in that the coat of the homozygote may be a shade lighter, with less mottling.[39] Horses with the homozygous recessive genotype (ch/ch) are not affected by champagne. Champagne is now identifiable by DNA test.[31]

The Champagne locus is occupied by the Solute carrier family 36, member 1 (SLC36A1) gene, which encodes the Proton-coupled amino acid transporter 1 (PAT1) protein.[40] This protein is one of many which is involved in active transport. The gene associated with the Cream coat colors is also a solute carrier, and orthologous genes in humans, mice, and other species are also linked to coat color phenotypes.[41] The single nucleotide polymorphism responsible for the champagne phenotype is a missense mutation in exon 2, in which a C is replaced with a G, such that a threonine is replaced with arginine.[42] This mutation was identified and described by an American research team in 2008.

• Equine coat color
• Tiger eye
• Horse genome
• Horse breeding
• Ann T. Bowling

Wikimedia Commons has media related to Horse coat colors.

• "Horse coat color tests" from the UC Davis Veterinary Genetics Lab
• "Introduction to Coat Color Genetics" from Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis. Web Site accessed January 12, 2008
• "Horse Color Calculator" From Animal Genetics Incorporated. This creates the possible coat coloring of the offspring from the imputed color of sire and dam.
• "Horse Genome Project" A quick summary of horse color genetics.