Archive | Basic genetics

Where the wild things are


In response to the previous post about the Agouti locus, a reader questioned the importance of the fact that bay was the original color of wild horses—that it “came first”, before black or chestnut. Why should that matter?

This question touches on the reason why I have come believe that the way color gets explained matters so much. I have mentioned in previous posts that equine coat color has become far more complex since I first began writing about it. (At the risk of revealing my age, that was around 1990.) There was a time when everything could be explained in terms of the colors themselves, while the technical aspects of genetics could be skipped or at least minimized. In hindsight, while some of these explanations made certain concepts easier to grasp, they could also be misleading. Let me give an example of a common misunderstanding from fifteen years ago, the explanation that was used to clarify the situation, and how that same concept—so useful then!—is somewhat problematic now.

At the time in question, the concept of dominance was difficult for a lot of people. It was not uncommon to get questions like, “Which is more dominant, grey or bay?” Or, “I know bay is dominant to chestnut, so shouldn’t palomino be recessive to bay?” These questions arose because it was not clear that a color could not be dominant or recessive to an unrelated color. My approach was to point out that colors could only have this kind of relationship with their opposites. Tobiano, for example, could not be dominant or recessive to buckskin; tobiano could only be dominant or recessive to “not-tobiano”. The opposite of a color was not a different color, but the absence of the color. This was a very clear way to get the idea across that the genes for different colors were separate things, and that each presented an independent chance for inheritance.

Fifteen years ago, many did not understand that each aspect of this horse’s color—bay, cream and tobiano—involved a separate, unrelated gene.

It was a simple explanation, but behind it lurked some puzzling questions for anyone who cared to look a little closer. If tobiano was dominant to “not-tobiano”, what exactly was this “not-tobiano”? If tobiano was believed to have arisen after domestication, how on earth were those wild ancestors carrying around a gene for the absence of something that did not yet exist? The idea of “not-tobiano” worked when it came to predicting breeding outcomes, but looked at in this light it made no sense.

That is why something like the situation with bay as an ancestral color matters, because the key to understanding what is really going on with “not-tobiano” can be found there. As I mentioned in the previous post, bay (or bay dun) is the most likely ancestral, or wild, color for horses. The other two basic colors, chestnut and black, were later mutations to the two genes responsible for bay. Another word for those alleles that were already there is wild-type. The wild-type is the allele that is typical for a given population. Wild-type is the “normal” setting—the default—for a gene. “Not-tobiano” and all those other “not-colors” were really just that: the wild-type for their particular gene. In the case of things like dilutions and white patterns, the wild-type is usually just the instructions for normal pigmentation.

Shifting from a color-based approach to a gene-based approach

Looking at colors in terms of the wild-type eliminates the misunderstandings that come from thinking of the color itself as a gene. Because we often refer to colors this way—as the “tobiano gene” or the “cream gene”—it is easy to get the idea that something like the cream dilution is an additional gene that palomino, buckskins, and smoky creams have; one which non-diluted horses do not have. The cream dilution is actually a mutation that occurred to a gene, known as MATP, that all horses have. In the absence of the cream dilution, MATP is involved in the normal formation of pigment. So the wild-type for that gene gives a fully-pigmented horse.

Not knowing there is a wild-type makes it seem that the color (Cream) is the gene itself and therefor the starting point. That is why there is a tendency to assume later discoveries are “mutations of the color” rather than alleles for the same (non-mutated, wild-type) gene. So pearl, which is found in the same genetic location as cream, becomes a “mutation of cream” rather than a second, unrelated mutation of the MATP gene. But the starting point is not cream, but the wild-type at MATP. The cream mutation did not have to be present for pearl to occur; it is a mutation like cream, not necessarily a mutation to cream.

But perhaps more importantly, many colors were named before their relationships to other colors were understood. Things that once were assumed to be separate later proved to be alleles of the same gene. At one time we thought, and taught, that the opposite of tobiano was the absence of tobiano. But the “tobiano gene” is not a separate gene. Tobiano is a mutation to the KIT gene, which again is a gene that all horses have regardless of their color. Tobiano shares the KIT gene with a host of other alleles (like Sabino1, Roan and the White Spotting patterns) that have historically been thought of as unrelated. That complex situation is very difficult to explain, especially if someone’s basic understanding of the subject is color-based rather than gene-based, because the relationship between that group of colors is not visually obvious.

Tobiano and roan are both alleles of the KIT gene, which is why the combination does not breed true. The offspring can only get one or the other from the parent.

I know for many who have learned about horse color exclusively in terms of basic colors and their modifiers, focusing on the actual genes is a very different approach. It may seem like it adds a lot of unnecessary complexity to the subject. I certainly can appreciate that point of view, but genes and the importance of using their wild-type as a starting point is the missing piece of the puzzle for a lot of people. When that piece falls into place, color genetics—especially as it is currently understood—begins to make a lot more sense.

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Bay is not a modifier


When talking about a topic as complex as equine coat color, simplifying concepts is essential. This is particularly true when speaking to a non-technical audience. The trick is to avoid more detail than is necessary without reducing the topic to the point where the information is misleading or inaccurate. Ideally any simplified explanation is compatible with a more nuanced understanding, since it needs to provide a solid foundation for those listeners who want a more in-depth understanding of the subject.

One of the common conventions used to explain horse color is that of basic colors and modifiers. By structuring the explanation this way, it is easier to make sense of the wide variety of colors and patterns. When each color is understood to have three (or four) versions—chestnut, bay/brown, and black—it is easier to see the relationships between colors that are not visually similar. The other advantage to this system is that, because the basic colors are a given, you get to skip (or at least gloss over) the mechanics of basic coloration in horses. That is useful because the basic colors require a more complicated explanation than most of the dilutions and white patterns.

In an attempt to simplify basic colors, one approach that has become increasingly common in internet discussions is to move bay into the “modifier” category, and assert that horses are basically red and black. Bay, by this convention, becomes a modifier of black. In the “absence of agouti”, the explanation goes, a horse is black. This approach is problematic on a number of levels, not the least of which is that it obscures the fact that “agouti” (as it is used in horses) is a genetic locus. It is a place in the genetic code, and not the name of a specific color. (The term is used for specific colors in other species.) There is no “absence”, because all horses have Agouti (ASIP). Some of them have the allele at Agouti for bay (A).

A study of ancient remains showed that bay was the original color of horses. At the time it was not possible to test for dun, but based on the pervasiveness of dun in wild equids—like this Przewalski’s Horse—it is assumed that they were likely bay dun.

It also takes a concept that is really about pigment, and applies it to the horse. Pigment in mammals is understood to be basically red (or yellow) and black. At the animal level, though, animals are understood to have a wild color that is typically some combination of those two pigments. In horses, that wild color is not red (chestnut) or black, but bay. Bay—or more likely bay dun—was the original color for the species. Animals that are all-red, or all-black, are usually the result of mutations to (modifications of) the species’ original color. Presenting bay, the wild color for horses, as a modification of black gets this backwards. Black is not the default color, but a mutation to the Agouti (ASIP) locus that could have occurred as early as 5200 BCE. Samples from 9210 BCE and earlier were uniformly bay.

It requires more explanation than the “all horses are red or black” approach, but the basic colors are governed by a category of genes that control pigment-type switching. That is because pigment cells have the ability to produce both types of pigment (red or black), and these genes are what control the switch between the two possibilities. One of the clearest explanations of pigment-type switching can be found in The Colors of Mice: A Model Genetic Network:

Pigment-type switching describes the ability of pigment cells to switch between the production of eumelanin [black] and pheomelanin [red], under the control of the Agouti and Extension loci and modifying genes.

In horses, Extension is sometimes called “the black gene” because its dominant allele (E) is responsible for the colors often referred to as “black-based” (bay, brown and black). That term is somewhat misleading, however, because it does not mean the horses with that allele are “basically black”, but rather than the resulting colors have some portion of black in the coat. It is a category based on the presence of black, not on modification from an all-over black color. Despite its popular name, the dominant form of Extension (E) does not just produce black pigment, but rather black and red pigment. (Remember that pigment cells already have the ability to produce either type.)

For those horses that can have both red and black pigment (E), the alleles at Agouti control which parts of the horse will be black. Agouti does not “add red” or “dilute black to red”, which are the two common assumptions made when Extension is presented as giving either a black (E) or a red (e) horse. There is a recessive mutation to Agouti (a) that distributes black over the entire horse, effectively eliminating the red pigment, but Extension itself does not limit cells to producing only black pigment.

I understand the appeal of simplifying the situation with black and red pigment, but I do think that the distinction between basic pigment colors and basic horse colors is an important one. Because there are some unknowns in this area of color genetics, and because there have been surprises in other species, it is probably helpful to lay the foundation for pigment-type switches as a general category. That means being clear about the situation with Extension and Agouti, even if it takes a little more effort to explain.

This variant of bay, known as wild bay, involves a reduction of black pigment at the points, particularly the legs. It is presently assumed to be an allele at Agouti, but that has not yet been proven.

Note: I would like to talk about some of the pigment-type switching “surprises”, because there have been some fun discoveries in that area in recent years, but that will require wandering a little further afield so I’ll save that for a future post.

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Classical versus molecular genetics


As an artist, I always found variations in animal coloration fascinating, but I fell in love with coat color genetics in 1989 when I purchased a copy of Horse Color (1983) by Dr. Phillip Sponenberg and Bonnie Beaver. The book contained what seemed at the time to be countless color variations and a structure for categorizing them. I read the book cover to cover, and then set out to find as many of the referenced books and papers that appeared in the bibliography in the back. The whole system seemed so logical, and the basic framework fairly easy to understand.

One of the pages of color plates from my very worn copy of Horse Color. It is a good thing that eyeballs do not absorb printing ink, or this page would have ended up blank years ago!

Because it is in my nature to share information, it was not long before I was writing about horse color with the intention of teaching others that same framework. And that was how I usually presented the subject: “This is pretty simple.” I could explain the concept of base colors–black, chestnut and bay/brown–and then the modifiers that altered those colors, either by diluting the color itself, or adding white hairs, or covering them with a white pattern. All of those were governed by the rules of genetic inheritance most of us learned in high school biology, when we used Punnett squares to map out the ratios of green and yellow pea pods.  Those ratios would tell us if something was dominant or recessive, or incompletely dominant, or perhaps a homozygous lethal. All that was needed was to tie that knowledge to an eye for the nuances of shade and pattern, and you were set!

Using this approach, I could give an hour-long presentation without using scientific terms like allele or epistasis. Instead I used a system that relied on visual understanding, because I found that many non-scientists would shut down if the initial information was abstract or overly technical. Most of the questions I encountered had pretty straight-forward answers, so this tended to work well. It was easy enough to expand on the basic concepts with technical information once someone was more comfortable with the subject. But somewhere along the way, explaining horse color became a lot more complicated. Over time, I found that even the most basic questions required increasingly more technical answers.

Cycling through the same set of images, each showing the effect of a given modifier on each of the base colors, was one of the most effective ways to communicate the concept of modifiers. (It could be argued that my use of Comic Sans was perhaps less effective.)

So what changed? Why has color genetics become so complex and so much more technical?

The answer is that there has been a change in the field of genetics. Mendelian genetics, which is also known as classical genetics, predates the advent of molecular biology. The huge leaps in our understanding of coat color in mammals–horses included–come from advances in molecular biology. When horse color research relied on classical genetics, it was pretty easy to explain the subject in simple terms; after all, the original discoveries were made before even the most basic concepts about sexual reproduction were understood. But the science has advanced far past that point, making it not only possible to find the exact, physical mutation in the genetic code, but to understand why that particular change caused the final result that we can see. 

The albino Dobermans are a good example of this difference. Classical genetics could tell breeders that the trait was recessive, and the visual appearance of the dogs suggested that the dogs carried some form of albinism. Molecular genetics makes it possible to tell what kind of albinism (or dilution, if you prefer) is involved. That makes it possible to test for carriers, but it also allows comparisons with other dilutions to help determine what, if any, detrimental effects might occur because of the mutation. As more is known about the function of the different genes–and each mutation found adds to that body of understanding–it becomes easier to predict what might be directly caused by the change, what might be linked, and what might be unrelated. In horses, this kind of research determined that Multiple Congenital Ocular Anomalies (MCOA), formerly known as Anterior Segment Dysgenesis (ASD), was “tightly linked” to the silver dilution, and that homozygous silvers were at greater risk for eye defects regardless of their breed.

Another good contrast between classical genetics and molecular genetics is the prediction of lethality. In a comment from the last post, a reader asked about the belief that some forms of Dominant White were lethal in their homozygous form. In classical genetics, crosses that produced early lethals (that is, lost pregnancies rather than offspring that did not survive long) were determined by the absence of true-breeding individuals, and a ratio that indicated that one portion of the expected outcome was missing (2:1 instead of 3:1). While those factors are still considered, knowing what role each gene plays in the development of the organism allows researchers to predict outcomes in a way that just looking at production ratios cannot. If the gene is responsible for task A, B and C during development, and if shutting its function down at point X ends that process before reaching C, and if C is necessary for life, then it can be assumed that without a normal copy of the same gene (ie., if the animal is homozygous for that mutation) the resulting offspring is non-viable. That is why some recent studies have suggested that certain crosses might not be viable, even when the mutations themselves are rare enough that there are not statistically significant numbers to assess ratios, and where a lack of true-breeding animals might not be particularly informative.

This Punnet Square of Lethal Yellow in mice illustrates how homozygous lethals change the expected ratio from 3:1 to 2:1. Instead of the three yellow (two heterozygous and one homozygous) and one white, the result is two yellow to one white. 

For breeders, this level of understanding holds a lot of promise for the future, because it makes it possible to analyze the connection–or lack of connection–between a color and undesirable traits. In the past, defects have been tied to colors, often using nothing more than rumor or supposition. It was not unusual to see a superficial similarity, like white patches or lighter eyes, used to suggest that a given color was susceptible to the same defects. This is how a diluted dog becomes an “albino” carrying something that is “a defect in all species”. Molecular genetics offers the opportunity to examine the actual cause for the reduction in pigment, and a means of determining just exactly what other problems, if any, this alteration might create. Yes, this does mean that more often the answer to questions about color will be, “It is complicated,” rather than “It really is pretty simple.” But knowing the true nature of a given mutation, and being able to identify it, gives breeders more control when it comes to obtaining, or avoiding, certain colors. And instead of culling all suspect animals, and losing whatever else they might have to contribute to their breed, breeders can make more informed selections whatever their goals are in terms of color. Seen in that light, the increased complexity of modern genetics seems a small price to pay.

(Punnet Square graphics courtesy of Wikimedia Commons, with apologies to my rodent-loving readers. Molecular graphic courtesy of the U.S. National Library of Medicine.)

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