Equine coat color genetics

Equine coat color genetics determine a horse's coat color. Many colors are possible, but all variations are produced by changes in only a few genes. Extension and agouti are particularly well-known genes with dramatic effects. Differences at the agouti gene determine whether a horse is bay or black, and a change to the extension gene can make a horse chestnut instead. Most domestic horses have a variant of the dun gene which saturates the coat with color so that they are bay, black, or chestnut instead of dun, grullo, or red dun. A mutation called cream is responsible for palomino, buckskin, and cremello horses. Pearl, champagne and silver dapple also lighten the coat, and sometimes the skin and eyes as well. Genes that affect the distribution of melanocytes create patterns of white such as in roan, pinto, leopard,  white, and even white markings. Finally, the gray gene causes premature graying, slowly adding white hairs over the course of several years until the horse looks white. Some of these patterns have complex interactions.

Most wild equids are bay dun, and so were many horses before domestication, though at least some were non-dun with primitive markings. Non-dun 1 is one of the oldest coat color mutations, and has been found in remains from 42,700 years ago, along with dun. Non-dun 2, the version of the dun gene that most domestic horses have, is thought to be much more recent, possibly from after domestication. Leopard complex patterns are also very old, having been found in horse remains from 20,000 years ago. The mutation causing black or grullo also predates domestication, and was especially common in the Iberia. The mutations causing chestnut, sabino 1, and tobiano are all at least 5000 years old, and happened at about the same time as horse domestication. Pearl appeared at least 3,400-4,200 years ago, and silver and cream appeared at least 2,400 years ago. The gray mutation is thought to be thousands of years old as well.

Fundamental concepts

Terminology
Heritable characteristics are transmitted, encoded, and used through a substance called DNA, which is stored in almost every cell in an organism. Proteins are molecules that do a variety of different things in organisms. The DNA instructions for how to make a protein are called a gene. A change to the sequence of DNA is called a mutation. Mutations are not inherently bad; in fact, all genetic diversity ultimately comes from mutations. Mutations that happen within a gene create alternate forms of that gene, which are called alleles. Alleles of a gene are simply slightly different versions of the instructions on how to make that gene's protein. The term "allele" is sometimes replaced with the word "modifier", because different alleles tend to modify the horse's appearance in some way. DNA is organized into storage structures called chromosomes. A chromosome is simply a very long piece of DNA, and a gene is a much shorter piece of it. With some rare exceptions, a gene is always found at the same place within a chromosome, which is called its locus. For the most part, chromosomes come in pairs, one chromosome from each parent. When both chromosomes have the same allele for a certain gene, that individual is said to be homozygous for that gene.  When the two alleles are different, it is heterozygous. A horse homozygous for a certain allele will always pass it on to its offspring, while a horse that is heterozygous carries two different alleles and can pass on either one. A trait that is only seen when the gene is homozygous for its allele is called recessive, and a trait that has the same effect no matter whether there is one copy or two is called dominant.

Notation
Often, the dominant allele is represented by an uppercase letter and the recessive allele by a lowercase letter. For instance, in silver dapple, this is Z for the dominant silver trait and z for the recessive non-silver trait. However, sometimes the alleles are distinguished by which is the "normal" or wild type allele and which is a more recent mutation. In our example z (non-silver) would be wild type and Z would be a mutation. Wild type alleles can be represented as + or n, so Zz, Zz+, Z/+, and Z/n are all valid ways to describe a horse heterozygous for silver. Wild type notation is mainly useful when there is no clear dominant/recessive relationship, such as with cream and frame overo, or when there are many alleles on the same gene, such as with MITF, which has four known alleles. Using n is also common in the results of genetic tests, where a negative result usually means none of the known mutations were found, but does not rule out undiscovered mutations.

Melanin
Genes affecting coat color generally do so by changing the process of producing melanin. Melanin is the pigment that colors the hairs and skin of mammals. There are two chemically distinct types of melanin: pheomelanin, which is a red to yellow color, and eumelanin, which is brown to black. Melanin is not a protein and therefore there is no gene that changes its structure directly, but there are many proteins involved in the production of melanin or the formation of melanocytes during embryonic development. Mutations that change the structure of proteins with a role in melanin production can result in slightly different variations of melanin. Genes affecting melanocytes, the cells that produce the pigment melanin, do not alter the structure of melanin but instead affect where and whether it is produced.

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. Extension is also sometimes called "red factor" and can be identified through DNA testing. 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".

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 ea, 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 ea allele was discovered may have gotten incorrect results.

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. 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. 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. Polymorphisms of MC1R also lead to light or red coats in mice, cattle, and dogs, among others. The Extension locus was first suggested to have a role in horse coat color determination in 1974 by Stefan Adalsteinsson. 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 Ee. 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 EE.

Extension phenotypes

 E/E (+/+, E+/E+, EE/EE) 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+/Ee, EE/Ee) 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.
 e/e (Ee/Ee) 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. No health defects are associated with MC1R mutations in horses.

Agouti
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. A third option, At, restricts black pigment to a black-and-tan pattern called seal brown. This allele is recessive to A and dominant to a, such that horses with the genotype A/At appear bay, while At/At and At/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.

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. 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. One genetics testing lab began offering a test for At, but it was later found to be inaccurate and is no longer offered.

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/At 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.
 At/At The hypothetical At 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.
 At/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.

Dun

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. 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.

The Dun locus is TBX3 on equine chromosome 8. 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. 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.

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.

Dun phenotypes
 D/D (+/+, D+/D+) wildtype, homozygous dominant. Visually, the horse may be bay dun, grullo, red dun, palomino dun, amber dun, gray, and so on. Such a horse will always pass on the D allele and will therefore always have dun offspring.
 D/d (+/d, D+/Dd) wildtype, heterozygous. Visually indistinguishable from the homozygous D horse.
 d/d (Dd/Dd) non-dun, homozygous recessive. The entire coat, barring the influence of other alleles, is a rich, saturated color. The primitive markings are no longer visible. The horse may be chestnut, bay, black, gray, palomino, and so on.

Cream

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 (CCr/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 (CCr/CCr) 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.

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. The Matp gene encodes a protein illustrated to have roles in melanogenesis in humans, mice, and medaka, though the specific action is not known.

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. Mice affected by a condition homologous to cream, called underwhite, exhibit irregularly shaped melanosomes, which are the organelles within melanocytes that directly produce pigment. The first descriptions of the dosage-dependent genetic control of the palomino coat color occurred early on in equine coat color inheritance research. However, the distinction between Dun and Cream remained poorly understood until Stefan Adalsteinsson wrote Inheritance of the palomino color in Icelandic horses in 1974. 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.

Cream phenotypes
 C/C homozygous wildtype. Visually, the horse may be any color other than the cream dilute shades of palomino, buckskin, smoky black, cremello, perlino, smoky cream, and so on. 
 CCr/C heterozygous. The colors most commonly associated with this genotype are palomino, buckskin, and smoky black, though the phenotype may vary depending on other factors. Any pheomelanin in the coat is diluted to yellow or gold, and the eyes and skin are often slightly lighter than unaffected horses.
 CCr/CCr homozygous. The colors most commonly associated with this genotype are cremello, perlino, and smoky cream. Regardless, the coat will be cream- or ivory-colored, and the skin a rosy-pink. The eyes are pale blue.

Champagne

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. 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. 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. Horses with the homozygous recessive genotype (ch/ch) are not affected by champagne. Champagne is now identifiable by DNA test.

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. 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. 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. This mutation was identified and described by an American research team in 2008.

Champagne phenotypes
 ch/ch (N/N) wildtype, homozygous recessive. Visually, the horse may be any color other than the champagne shades.
 CH/ch (CH/N) heterozygous. The colors most commonly associated with this genotype are gold champagne, amber champagne, and classic champagne, though the exact phenotype depends on a variety of factors. At birth, the skin is bright pink and the eyes bright blue, darkening to freckled and light brown or green, respectively, with age. Both red and black pigment in the hair are also diluted.
 CH/CH homozygous champagne. Homozygotes, which will never produce non-champagne offspring, are indistinguishable from heterozygotes except that their freckling may be sparser, and their coats a shade lighter.

Alleles and effects

Notable color combinations

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

References

External links

 "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.

Genetics
Category:Equine genetics