Anyone who reads this
magazine likely agrees that horses are amazing creatures. Valued
for their speed, their beauty, and their grace, not to mention
their generosity of spirit toward humans, horses are a
continuing marvel even to those of us who work with them every
day. And now, as researchers delve into the secrets of the DNA
strands that make horses what they are, we're discovering anew
just how miraculous they are--on a molecular level.
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ANNE M. EBERHARDT
We now
have at our disposal a great deal of information about
the genetics of coat color.
|
Every species of living
thing on the planet has a genetic code, which is a
characteristic number and array of chromosomes, hidden in every
cell, that supply the directions for the precise workings of the
organism's metabolic function, development, and reproduction. On
these chromosomes are genes, the term used to describe sections
of the DNA spiral that are responsible for creating individual
traits, some obvious to the eye, others invisible but no less
crucial. Genes can vary in size from just a couple of molecules
in the DNA strand, to large and complex sections with thousands
upon thousands of subunits.
Genes come in pairs, one
from each parent, and the various forms of each gene are called
alleles. An organism might be homozygous for a certain trait
(meaning both of the alleles are the same), or heterozygous
(meaning the two alleles are different). A dominant gene is one
that will create a certain physical characteristic whether it's
present in single or double form; a recessive only expresses
itself when it's present in homozygous form and not overruled by
the presence of a dominant gene. While this is the genetic
definition of dominant and recessive, unfortunately, in an
animal as genetically complicated as the horse, it isn't as
straightforward as it sounds.
Although observations of a
horse's physical characteristics (his phenotype) have provided
us with some information about his genetic make-up, and allowed
us to make some predictions about the heritability of certain
dominant and recessive traits, much of what we know about equine
genetics has been guesswork. Even when we've studied breeding
and registration records going back 20 generations, we've gotten
an incomplete picture--because horses with genetic defects that
affected their health usually weren't registered, because mares
and stallions tend to be registered more often than geldings,
because some individuals remained unregistered when they didn't
meet the standards of the breed registry, or because their
owners didn't consider them worth the economic outlay. But huge
advances in research, delving down to the molecular level, have
given us the hope of a far more complete picture.
Genetic Voyagers
The Equine Gene Mapping
Project, an on-going international effort involving universities
in more than a dozen countries, including Australia, England,
France, and the United States, has made remarkable progress
since it was begun in the early 1990s. Its goal: to identify at
least 300 "markers" (like pushpins on a map) that will give
researchers a reference plan for the location and character of
the approximately 70,000 genes located on a horse's 32 paired
chromosomes.
Once we can reliably locate
the genes that create a horse's various traits, it's a
straightforward step to being able to test for those traits, and
calculate the likelihood of a horse passing certain
characteristics on to his or her offspring. Heritability could
be just a matter of interest, for instance, when a mare owner
would like to know what the chances are of getting a palomino
foal from the pairing of her mare and a certain stallion. It
might be a matter of life and death, as when a breeder is trying
to determine whether an Arabian stallion is a carrier of the
gene for Combined Immunodeficiency Disease (CID), an
invariably fatal condition. Once, it would take several matings,
each one a roll of the dice, to determine an answer about a
horse's genetic design. These days, we're able to go straight to
the source, and decode the language of DNA itself.
Gene mapping for horses has
had a slow start, relatively speaking. Geneticists already have
established maps for several other species, including sheep,
pigs, chickens, cattle, and mice. Human gene mapping also is
nearing completion, with thousands of markers identified.
Researchers currently are able to map more than 4,000 genetic
abnormalities or diseases in humans, thanks to the
multi-billion-dollar Human Genome Project, which in many ways is
driving equine research.
One of the most interesting
discoveries of recent years is that the DNA record doesn't vary
that much from species to species, at least among vertebrates.
There is some basic genetic material that is common to all
species, and genes that "express" to produce similar traits tend
to be in roughly the same location relative to other sets of
genes when you compare one animal to another. For instance, the
gene that creates a certain coat color in mice will often have a
counterpart (called a homologue) that does the same thing in a
horse's DNA. This lucky coincidence has provided an invaluable
shortcut for researchers, who have been able to use species with
more complete DNA records as templates for the Equine Gene
Mapping Project. Mice, as it turns out, are an especially good
model for equine coat color genes (of which at least 10 are now
known), and many genetic diseases horses suffer, including CID,
HYPP, and even the overo "lethal white" syndrome, all have
parallels in humans or mice!
Gene mapping can provide
answers for a multitude of questions about heritability. For
instance, we might be able to identify a gene, or genes, that
would be a sure indicator of superior speed in a Standardbred,
or "gait" in a Tennessee Walker or Paso Fino. We might be able
to institute a high degree of predictability of conformation in
the offspring of a certain bloodline. Color breeders would be
able to choose breeding stock with the best possible chance of
producing a tobiano, a buckskin, or a blanketed Appaloosa. And
it's possible we might be able to identify a gene that
predisposes foals to Developmental Orthopedic Disease (DOD),
or older horses to heaves or navicular disease, or, at least,
determine whether genetics plays a role in those (or other)
conditions. On top of all this, gene mapping also can provide us
with insights into evolution on a chromosomal level, as we begin
to understand how some changes in DNA sequence (mutations) make
a breed or a species more, or less, successful than it was
before.
On a more prosaic level,
gene mapping will allow us to make a far more accurate
determination of the parentage of an individual horse, an
increasingly important requirement of many breed registries.
Blood typing, the current method used by most registries, is
reliable, but DNA testing can provide a far more definitive, and
informative, result, potentially telling us more than just the
likelihood that a foal is sired by a particular stallion. The
possibilities are, to say the least, tantalizing.
Gene Mapping Project
Gus Cothran, PhD, director
of the Equine Blood Typing Research Laboratory, Veterinary
Sciences Department, University of Kentucky, says that the
Equine Gene Mapping Project now has identified some 160 genetic
markers. While the project is by no means complete, he says, two
papers that provide the first fairly comprehensive maps of the
equine genome have been accepted by academic journals and are
awaiting publication. "Before the year is out, or very early in
1999, we should have the first good maps of the (DNA of
the) horse in the literature.
"The map is progressing
rather well," he adds. "We achieved our goal of 150 (markers)
pretty close to when we thought we would, and 300 looks well
within reach. Three hundred markers should give us adequate
coverage for almost anything we want to look for."
Genetic markers for the map
are called "microsatellites"--which Cothran describes as
subunits that are repeated a number of times. "Microsatellites
are not necessarily genes, although they may be incorporated in
a gene," he explains. "They're just repeating sequences which
are easy to spot and can be used as signposts to help us find
genes we're interested in. If you have a trait you'd like to
locate a gene for (in horses), you would find the coding genes
close to a marker, and compare that to the human gene map, which
is close to complete. From the comparison, you can pick out a
candidate gene for the trait."
The International Equine
Gene Mapping Project is involved in linkage mapping, which is
designed to provide researchers with a ready comparison of the
correlating sections of DNA between different species.
Meanwhile, at both the
University of California/Davis and the University of Kentucky,
another kind of mapping is simultaneously underway. Ann Bowling,
PhD, Executive Associate Director of the Veterinary Genetics
Laboratory at UC Davis, explains that her lab is working on
synteny mapping of the equine genome, a method that helps
researchers identify which genes are located together on a
single chromosome. Through an elaborate system which uses a
panel of cells created by fusing horse cells with those from
mice (a somatic hybrid), synteny mapping is making it easier to
locate specific genes on specific chromosomes.
Cothran mentions that there
is a third level of activity in gene mapping going on in an
independent project at the Animal Health Trust in Newmarket,
Great Britain. "It's fascinating work," he says. A group of
research mares there are being bred by artificial insemination,
with the embryos harvested soon after for their DNA material. In
this way, a "family" of genetic results of specific matings is
being built up very quickly, allowing researchers to examine the
results without the time and expense of raising the foals. All
the information is contained within the DNA of even the tiniest
embryo.
Bowling notes that although
the human genome likely will be completely sequenced within the
next five years (with the help of several billion dollars in
research funding), the Equine Gene Mapping Project will probably
never be absolutely complete. Research on horses, she points
out, tends to be under-funded, thanks both to the cost of
maintaining research animals and the perception that the horse
industry in North America has a relatively minor economic impact
when compared to livestock like cattle or swine. Still, on small
research budgets, equine geneticists already have uncovered some
fascinating information about the molecular makeup of horses.
We'll highlight just a few of them.
A Coat Of Many Colors
It's often been said that "a
good horse is never a bad color," but for many breeders, some
colors are better than others. Certain coat colors or patterns
have been prized and selected for over many centuries. Those who
breed paints and pintos would like a guarantee of spotted foals
every spring; Appaloosa breeders, too, are looking for "good
color," with no hint of pinto coloration or graying to dilute
the characteristic coat patterns. Those who breed palominos are
hoping for that 100% probability of a golden colt or filly. And
sometimes, the "wrong" color can be an indication of an outcross
somewhere in a horse's parentage, since many breed registries
forbid pinto coloration, for example, or limit the extent of a
horse's white markings. It should be kept in mind that the
molecular description of the genetics for coat colors for the
most part is in its infancy and much research will come from
Bowling's initial work in this field.
Sometimes an interest in
color heritability is more than academic or economic. In the
case of overo-patterned paints, there is a condition called
aganglionosis, more commonly called "lethal white,"
which sometimes crops up when breeders match two overo horses. A
foal with a double dose of the overo gene is born white and
appears normal, but it is unable to pass food through its
digestive tract due to a lack of nerve cells in its intestine
and soon dies. A similar condition in humans is called
Hirschsprung's disease and is associated with a white
"forelock." There are also at least three white spotting genes
in mice that can be lethal when they're homozygous. Because of
the possibility of lethal white, there has been a pressing need
to understand the genetics of the overo coat pattern, and to be
able to test for the gene for lethal white.
Fortunately, we now have at
our disposal a great deal of information about the genetics of
equine coat colors, much of it uncovered by Bowling and her team
at UC Davis. It turns out that all of the various colors horses
can be are created by the presence, or absence, of two
pigments--eumelanin (black/brown) and phaeomelanin (red/yellow).
These two pigments can be influenced by a number of genes that
can modify the basic coat color, diluting it or producing
patterns. There also is an allele for the absence of color.
Most color genes are
dominant, but one of the most common, the gene that controls red
(chestnut) coloration, is recessive. When a horse is homozygous
for the red gene (designated as ee), he will possess no black
hairs on his body or "points" (the mane, tail, ear tips, muzzle,
and lower legs). Depending on the modifier genes present, a
horse could be chestnut, sorrel, palomino, red dun, gray,
cremello, or even white, with the added possibility of roan,
Appaloosa, or paint patterning if those genes are present.
On the other hand, if a
horse's genetic code contains the "black factor," E, he'll have
black hairs in his points and possibly his body color as well.
He could be black, bay, or brown, or if there are other
modifiers present he could be buckskin, dun, grulla, perlino,
gray, or white (or any of the spotted-coat variations). A horse
which is homozygous EE doesn't have the red factor in his
genetic makeup and thus cannot produce red foals. This can be an
important detail in breeds where the color black is prized (as
it is in the Arabian). Because the "red factor" is a simple
recessive gene, it has been fairly straightforward to develop a
test for it. This test recently has become available, and with
this test, a breeder can discover whether or not a horse with
black points is homozygous (EE) or heterozygous (Ee). The best
chance of producing black horses will come when two EE horses
are mated. The red factor test also can be a tip-off as to
parentage--for example, two chestnut parents cannot produce a
horse with black points, such as a bay.
It's less simple to try and
detect whether black will breed true. Researchers originally
compared the black coat color in horses to a gene called
"agouti" in mice, but it's now known that black is controlled by
more than one gene. Being homozygous EE isn't enough to ensure
that a horse is black; he must also be free of body-color
modifiers that might make him a buckskin or grulla instead.
Another gene, designated as
A (for agouti), appears to control the distribution of black
hair. In combination with the presence of the E gene, it will
confine the black hair to the points, to produce a bay horse. If
the alternative gene, a, is present instead, it and the E gene
will produce a horse with a uniform black color. In most breeds,
the a gene is quite unusual, so true black horses are a rare
occurrence (except in breeds such as the Percheron, Friesian,
and Canadien, which select for it). As of yet, there is no
reliable test available to discover the "black factor."
Another interesting color
gene is G, which causes graying. Breeders have known for
centuries that a gray can only be produced in a mating with at
least one gray parent, making G a simple dominant. Horses with
the G allele, whether they are homo- or heterozygous (GG or Gg),
are born dark and gradually fade to gray, then to white, as they
age. Because these horses get lighter as they get older, they
can "lose" patterned markings such as tobiano or overo spotting,
or Appaloosa markings (which is the reason the Appaloosa
registry forbids gray horses).
Tobiano coloration also is
controlled by a simple dominant gene, designated TO or P (for
"piebald," the European term for a tobiano whose dark spots are
black). Tobiano spotting features large patches of color on a
basically white background. Horses marked this way usually have
white legs and dark faces (except for markings such as a blaze).
In order to achieve tobiano coloring, at least one parent must
be tobiano, and the chances are better if both parents have the
coat pattern. Fortunately, there seems to be no risk of lethal
white occurring as it does with overo spotting. Any lab
performing blood-typing can perform blood testing for the
tobiano gene, although Cothran notes that the procedure looks
for two linked genes associated with tobiano coloration, rather
than the tobiano gene itself, and the test is not 100% reliable.
Despite this, the lab receives two or three requests a week for
the test (which costs about $31 and takes a couple of weeks to
yield results).
The D gene is a dilution
factor or modifier, which can have an influence on the body
color of both red (ee) and black (Ee or EE) horses. When D is
present, a horse coded for chestnut body color becomes a red or
a claybank dun, while black coloration becomes grulla or mouse
dun. Horses with the D gene also have dark points, dorsal
stripes, and sometimes shoulder striping and zebra stripes on
the legs. If a horse is coded dd, however, his coat color will
be undiluted.
Likewise, the C gene has an
allele called Ccr which can dilute red pigment to
yellow on both the body and the points. When Ccr is
present, horses coded for chestnut (ee) become palomino, with
the mane and tail turning flaxen or white. A bay would become a
buckskin, the black color of his points unaffected. Ccr
does not affect genetically black horses when it's present in
heterozygous form. In homozygous form, however (CcrCcr),
just about any coat color becomes diluted to a very pale cream
with pink skin and blue eyes, usually called cremello or
perlino. It usually takes a mating between two "dilute" horses,
such as palominos or buckskins, to produce a homozygous
cremello. The alternative, CC (with no cr) is a fully pigmented
horse.
Researchers also have
identified a gene that codes for roaning, logically designated
R, and a Z gene, which codes for the rare color called "silver
dapple," found only in a few breeds (Shetlands, miniatures,
Rocky Mountain horses, Icelandics, and Dutch warmbloods, among
them). The Z gene is dominant, but only expresses itself when E
is present (in other words, the horse is not coded to be a
homozygous chestnut). Variations include a silver dapple black,
silver dapple bay, and a silver dapple buckskin, which is often
mistaken for a dappled palomino.
One of the more complicated
colors, from a genetic point of view, is the overo paint pattern
so popular among breeders of registered Paint horses. While
breeders have long known that when you cross two overos, you
generally have a 50% chance of overo-spotted offspring (along
with a 25% chance of a solid-colored foal, and a 25% chance of
homozygous lethal white), there have always been, in the
Quarter Horse breed, horses called crop-outs--those which arrive
with overo spotting despite no history of the pattern in their
parents or their ancestors. Furthermore, studbook investigations
have shown that some stallions, when bred to solid-colored
mares, buck the statistics by producing substantially more
overos than solids (although so far, no overo stallion has been
shown to sire 100% color).
Early on in her studies of
Paint coloration, Bowling speculated that overo spotting might
be controlled by several genes. Further investigation has
revealed that there might indeed be more than one kind of overo
and that blending of these ovaro patterns is common. The type
most often associated with the incidence of lethal white is what
breeders call a "frame overo," in which the white markings
usually are confined to the head and the sides of the body,
"framed" by color. A pure "frame" overo rarely has white on its
legs, ventral abdomen, or dorsal line. The type of overo
sometimes described as "sabino" (typified by the snowflake-edged
splotches of white, and significant roaning, often seen on
Clydesdales and Shires) now appears to be a separate type of
spotting with its own genetic coding. Unlike the frame overo
gene, sabino is fully dominant, so at least one parent must be
sabino in order to achieve a sabino foal.
Another spotting pattern
lumped by the registries under overo is a pattern called "splash
white," which features a continuous pattern of white on all four
legs, chest, and completely over the head. There also is a
relatively rare spotting pattern designated F, which produces a
"white head splash" pattern (occasionally accompanied by neck
and belly white as well). It's seen only in certain northern
European breeds (the F stands for Finland, where it was first
recognized), and it is a homozygous recessive.
Some spotted horses might in
fact possess several spotting genes all working together. Such
horses are called compound heterozygotes (also known as Paint
blends, as discussed above) by geneticists, but breeders might
describe them with names like tovero (a combination of overo and
tobiano characteristics). It's suspected that compound
heterozygous stallions are in fact the most successful producers
of color. They can sire a high percentage of spotted foals on
both spotted and solid-colored mares.
Fortunately for breeders,
who might find it difficult to define their horse's genetic
makeup by observation, there now is a test for the overo gene
that is known to produce lethal white. The test is useful to
breeders not only to help them avoid producing lethal white
foals, but also to help them in identify potential pedigree
sources of the overo pattern that could be useful in their
breeding programs. If you do have a frame overo, according to
the testing, it's safest to breed that horse to solid-colored
counterparts. However, in order to avoid a lethal white entirely
you should test both parents.
Appaloosa coat patterns are
an area not yet fully explored from a genetic point of view,
says Cothran, although an investigation by University of
Kentucky graduate student Rebecca Terry currently is underway.
Early indications are that all of the various Appaloosa
patterns, from blanket to leopard to varnish roan, are
controlled by a single dominant gene. Cothran elaborates, "We
have a strong suspicion as to the location of the Appaloosa
gene, as well...it's probably in the same area as the tobiano
and roan genes."
The silver dapple gene also
is being explored by undergraduate student Shawn Phillips, who
has an interest in Rocky Mountain horses (a breed in which the
color is prevalent).
Testing For Genetic Disease
One of the most important
applications of genetic testing is its use in detecting and
diagnosing genetically linked diseases and abnormalities.
Usually the result of a "typographical error" when DNA is
reproduced--an addition (the most common type), substitution,
deletion, or scrambling of the order of the individual codons--most
abnormalities, or mutations, are never seen because they create
an animal which isn't viable and is resorbed or perishes long
before birth.
Occasionally, though, a
mutation occurs that allows life to develop to adulthood. If the
animal possessing that mutation is used for breeding, there's
the chance that it could pass on its defect--or improvement--to
the population. New mutations are cropping up all the time;
researchers postulate that the process explains much about how
species change and adapt to their environment over the
millennia.
Bowling points out, "It's
not realistic to talk about 'wiping out' genetic disease in
horses, because mutations are always happening. No species of
animal is ever completely free of defects, and horsepeople need
to start thinking in those terms. We can't get rid of
everything. The trick is that you don't want to match up a sire
and dam who have the same deleterious genes."
Hyperkalemic periodic
paralysis, or HYPP, is the mutation that opened up a world
of possibilities in terms of the detection and diagnosis of
genetically linked diseases. HYPP is a disease that can be
traced back to a single Quarter Horse sire, Impressive. Due to a
"typo" the horse suffered at conception (although he never
demonstrated any disease symptoms himself), some of Impressive's
descendants have inherited a genetic defect that disrupts the
way sodium channels (tiny gateways in the membranes of muscle
cells) open and close. Because the gateways tend to get stuck on
"open," they can allow an uncontrolled flow of sodium ions into
the cells. This changes the voltage current of the cells,
causing weakness and/or twitching. High levels of potassium also
tend to build up in the blood when these disruptions occur.
Cases of HYPP can be mild or
severe, depending on how many sodium channels are affected. At
its worst, the condition can be fatal if respiratory failure or
cardiac arrest occurs. More often, an HYPP horse suffers attacks
of weakness and staggering, which can be exacerbated by stress.
He often is exercise-intolerant as well. The severity of the
disease is influenced by whether a horse has inherited a single
or double dose of the defective gene, called H. Homozygous
horses, as a rule, are far more severely affected than
heterozygous ones.
On a molecular level, the
HYPP mutation is the result of a single nucleotide error in the
DNA strand. Within the gene that controls sodium channels in
muscle cells, the amino acid leucine is substituted for the
correct one, phenylalanine. In other words, one single misplaced
base pair in the DNA sequence causes "all hell to break loose."
Because of Impressive's
popularity as a stud, HYPP now is widespread among registered
Quarter Horses and in other breeds that accept Quarter Horse
lineage (such as the Paint and Appaloosa). Fortunately, once the
initial shock of the disease's discovery wore off, the American
Quarter Horse Association became pro-active, vowing to wipe the
mutation from the breed's makeup by a program of vigorous
testing for any Impressive-descended horse. HYPP proved to be
the first equine disease traceable to a single bloodline by
genetic analysis, thanks to Eric Hoffman, PhD, a human
geneticist who had already worked on the human version of the
disease. He identified the HYPP gene in 1992 at the University
of Pittsburgh. It was also one of the first diseases for which a
reliable test was developed.
In the HYPP test, as in most
DNA tests, genetic material is extracted from a blood, hair, or
tissue sample. The gene coding for the muscle cell's sodium ion
channels is amplified (copied using PCR, polymerese chain
reaction), cut out using enzymes that cut specific DNA
sequences, separated by electrophoresis, then stained and
examined. The HYPP test is virtually 100% accurate. A horse
which has tested negative for the gene bears the notation HYPP
N/N on his registration papers; one which tests positive is N/H
or H/H and should not be used for breeding purposes. Although
HYPP was the first equine disease to be identified, beyond the
shadow of a doubt, as genetic, researchers expect it will not be
the last.
Close on the heels of the
development of the HYPP test in the early 1990s was a test for
combined immunodeficiency, a devastating condition in
Arabians that breeders long had suspected of being heritable. In
CID, foals are born without a functioning immune system.
They appear normal at first, but once their colostral immunity
wears off, they succumb to the first bacterial or viral
infection to invade their systems. For decades, the only way to
identify a carrier of the CID gene was for the horse to produce
a CID foal.
Twenty years of painstaking
research, and a lot of blind alleys and dead ends, finally
yielded the exact location of the CID mutation in 1996, when a
team led by Dr. Katheryn Meek, at the University of Texas
Southwestern Medical Center, found a five base-pair deletion in
the DNA of CID-affected horses and known CID carriers. It soon
was confirmed that CID was an autosomal (not sex-linked)
recessive defect. From there, it was a simple process to develop
a CID test for breeders, a test that when launched in the summer
of 1997, was expected to revolutionize the Arabian industry, or
destroy it, depending on who you talked to.
John Duffendack, president
and CEO of VetGen, the Michigan-based company that made
available the commercial CID test, notes that a year later, "the
CID test is doing quite well. It's becoming more accepted now
that the initial scare is over, and we've tested several
thousand Arabians in the past 12 months. The carrier rate is
working out to be about 16-18%. Testing is still voluntary--it's
not legislated by the breed registry--but I think most
conscientious breeders want to know, and they want to eliminate
the defect from the breed."
The CID test is particularly
simple for owners or veterinarians to access; the VetGen labs
can analyze either leukocytes, isolated from a blood sample, or
a swab taken from the inside of a horse's cheek, by the same
basic techniques described earlier. Generally speaking, tissue
samples are better for DNA analysis than blood samples. Blood
must be carefully handled and analyzed quickly, but tissue
samples, properly harvested and stored (under refrigeration),
are more durable. Once extracted, a DNA sample will "keep
virtually forever," says Cochran.
Bowling notes that the lab
at UC Davis recently developed a method of extracting DNA from
the bulb at the root of a strand of horse hair, "because we
recognized the problem of shipping blood or tissue long
distances in the United States. The distances became a major
concern when you're dealing with a sample which must be kept
under refrigeration. Hair is clean and neat and doesn't degrade,
and it's easy for the owner to obtain without having to call a
veterinarian out. Furthermore, it can be easily stored
indefinitely. The HYPP test is now being performed using hair,
and the CID test would work this way as well."
Other Genetic Defects
Several other genetically
linked conditions are currently the focus of research to
identify their locations on the DNA strand, and to develop a
reliable testing procedure. For example, the University of
Kentucky genetics lab has been collecting tissue samples from
Miniature horses exhibiting signs of dwarfism "for a
couple of years now," says Cochran.
"There are lots of types of
dwarfism in humans, so we need to narrow down the candidates
that would be most likely to parallel what goes on in horses. So
far, our investigation has shown that the (gene) which causes
about 80% of dwarfism in humans is not the one which causes the
same condition in horses or cattle. We have to keep looking. But
we do know that in horses, dwarfism seems to be a recessive
trait."
Genetic research also might
yield some answers for an alarming condition called
epitheliogenesis imperfecta, or EI, which occurs in
Belgians, Quarter Horses, and Saddlebreds (some researchers
suspect that up to 80% of Belgians carry the gene). EI foals are
born with large areas of their skin, oral membranes, and
sometimes their hooves, missing. They usually die within hours
or days, starving because their delicate mouth parts hurt too
much to allow nursing, or suffering massive infections as a
result of the large swathes of unprotected tissue. A similar
condition in humans is called epidermolysis bullosa, but
Cochran says that initial studies have indicated that EI
probably isn't controlled by the single gene originally
suspected. That's a mixed blessing, he says, "because it was a
large and complex gene, and would have been hard to work with."
The studies are continuing.
Yet another genetically
linked condition for which there likely will be a reliable test
for soon is anterior segment dysgenesis, or ASD,
which has a high frequency among Rocky Mountain Horses. Horses
which are heterozygous for the ASD defect tend to develop cysts
in their eyes, while homozygotes can range anywhere from almost
unaffected, to having no eyes at all (this, fortunately, is
rare). In any case, the horse's vision is definitely
compromised. A candidate gene has been identified at Michigan
State University, and it's expected that we might soon know a
lot more about this unusual condition.
Sex-Linked Defects
Some genetic defects are
sex-linked--that is, they are associated with the X chromosome.
Two examples of these occasionally crop up as an explanation for
infertility in broodmares--XO Gonadal Dysgenesis, in
which a mare is missing one X chromosome (in other words, she
has a total of 63 chromosomes, not 64), and XY Sex Reversal,
sometimes called testicular feminization, in which a
horse has the outward appearance of a mare, but is genetically
male. Both have parallels in humans. The XO defect is known as
Turner's Syndrome in women. It's also possible for a horse to
have extra chromosomes (65 instead of 64; this has been
demonstrated in a few cases of small, unthrifty foals and, like
Downs syndrome in people, is associated with foals born to older
mares. The first extra-chromosome foal to be identified with
certainty happened, by sheer coincidence, to be born to a
24-year-old mare belonging to Bowling. That filly, now entering
middle age, remains small and suffers from a number of angular
limb deformities and stiff gaits. Since then, a couple of other
foals with an extra chromosome have been identified.
Question Of Genetics
Unfortunately, it's not
always clear whether a disease or defect has a genetic basis.
Witness a condition called degenerative suspensory ligament
desmitis, or DSLD, in which a healing error forces
the body to repair torn or strained suspensory ligament tissue
with non-stretchable cartilage. A horse with DSLD ends up with
ankles that sink toward the ground. This horse often tries to
dig holes in the ground to stand with his toes down to relieve
his discomfort. Although the condition has been found in many
breeds, it appears to be particularly common in Peruvian Pasos,
and many researchers strongly suspect the existence of a DSLD
gene (especially since there is a candidate gene in the human
code). But not all breeders admit there is an increased
incidence, and some actively resist the idea of further
exploration, perhaps for fear the results will devalue their
breeding stock.
Conditions that are
suspected to have a genetic link, and probably will be testable
in the future, include developmental orthopedic disease (DOD),
heaves, cerebellar hypoplasia, cryptorchidism
(undescended testicles), parrot mouth, and club foot.
Even sweet itch, a hypersensitivity to a type of gnat, and
cataracts, might end up having a genetic element.
Testing In The Future
Like computer technology,
which seems to change so fast that your current home system is
always a little out of date, advances in genetic testing are
proceeding faster than they can be implemented on a practical
level. At the moment, says Cochran, the University of Kentucky
labs are involved in parentage testing for approximately 30
different breed registries in North, Central, and South America,
and the labs at UC/Davis "are doing at least that many"...not to
mention many other university-based and privately operated
laboratory companies. That parentage testing is based largely on
blood-typing, a technology that pre-dates true DNA testing and
that categorizes factors on the red blood cell surfaces, much as
blood typing in humans designates us as type O, A, B, or AB, and
as positive or negative.
Blood-typing is perfectly
adequate for some purposes; not only can it provide a fairly
reliable determination of parentage (based on the idea that a
foal's blood type must be inherited from the sire and dam), but
it's also useful for detecting problems like neonatal
isoerytholysis (NI, also called hemolytic disease or
foal jaundice). NI is similar to Rh incompatibility in expectant
mothers and babies--if a foal's blood group is incompatible with
the mare's, he will become weak, anemic, and jaundiced within
six hours to five days after drinking his first colostrum, and
might require blood transfusions of thoroughly washed red blood
cells from his dam in order to survive. A mare which has had one
NI foal is likely to produce another, so most veterinarians
recommend testing her blood three weeks prior to foaling, with a
blood grouping test that can indicate whether she is carrying an
incompatible foal. (Disaster can be quickly averted if the
answer is yes, by having an alternate colostrum source on hand
to feed the newborn.)
Blood-typing also can
provide an indirect indicator of a color gene. The tobiano
spotting pattern is strongly associated with the presence of
certain protein variants in the blood--so analyzing the blood
for those factors can help you determine whether your tobiano
horse is homozygous and likely to pass on his coloration. The
current tobiano test is based on an analysis of these two linked
genes.
However, blood typing
doesn't provide nearly as much information, or do it as
accurately, as DNA testing. The procedure, which uses a process
called allele-specific polymerase chain reaction, or ASPCR, can
be performed on any source of nuclear DNA extracted from a
cellular source. A few registries already have made the switch
from blood to DNA testing, most notably the AQHA. It moved from
blood typing to DNA testing for all registered Quarter Horses
two years ago. Other registries are considering the move, but
just as you might hesitate to buy a new piece of electronics
equipment for fear something better is just on the horizon, some
are holding back, waiting for new developments that might make
DNA testing simpler and easier.
"The question," says
Cochran, "is whether the DNA testing technology we have now is
the best we can do, and I suspect not. And if we switch (a breed
registry) to another technology, we have to re-type all the
parents from scratch, because different markers are used. It's
debatable whether that's worth doing.
"The upcoming technology is
going to be better within five years, no question. And it has
the potential to be quite inexpensive," he adds.
Shortly after the turn of
the century, we can expect to take a hair sample from a horse,
extract DNA from the hair bulb, float it in a solution and flood
a microchip with hundreds of tiny "wells" on it, Cochran
explains. On each chip will be DNA that has been denatured to a
single strand--so when it encounters a match from the hair
sample, it will bind to it. Through a simple chemical reaction,
a match in a single well will produce a visible color change,
making the chip simple and economical to read.
The possibilities of this
new system are virtually limitless, helping to identify hundreds
of traits and/or abnormalities in a single test.
"The microchip we envision
will hold all of the parentage information for a horse, plus
diagnostic data, all from a single hair sample," says Cothran.
The face of things to come.
DNA Testing Helps Resurrect the
Quagga
Once upon a time, horses had
a strange-looking relative on the south African veldt--a
pony-sized animal with striping over part of his body, like a
zebra, but with pale brown hindquarters designed to help him
camouflage on the dusty plains of the Karoo (the southern part
of the present Orange Free State of South Africa). European
immigrants designated him Equus quagga, then merrily
hunted him to extinction, as they did many other species in the
18th and 19th Centuries. They used his carcass for food and his
hide for harness, ropes, and even grain sacks. On Aug. 12, 1883,
the last quagga on earth, a lonely old mare, died at the Artis
Magistra Zoo in Amsterdam. It would be several years before
anyone even realized that she had been the final representative
of her kind.
The quagga was assumed to be
lost forever--until German taxidermist Reinhold Rau, working for
the South Africa Museum in Cape Town, found his curiosity piqued
in the late 1960s when he was charged with restuffing and
restoring several preserved quagga skins (of the 23 left in the
world). The more he examined the quagga's characteristics, the
more he became convinced that the beast was not a separate
species to the zebra, but a sub-species of the Burchell's, or
plains zebra, which survives in southern Africa and shows a
great deal of variation in its stripes from region to region.
The Burchell's (unlike its cousin, the Grevy's zebra) often has
brown "shadow" striping between the main black stripes on its
coat, especially on the hindquarters. As you go south toward the
southernmost tip of the African continent, its hindquarter
striping becomes less distinct.
Only DNA would tell the tale
for sure. Miraculously, Rau was able to extract some tiny bits
of blood and tissue found clinging to one of the preserved
quagga skins. He sent them to California's San Diego Zoo, where
there is a depository of rare animal tissues, for analysis.
Although it is almost impossible for genetic material from a
sample more than 100 years old to survive, the tissue fragments
did contain enough mitochondrial DNA to allow the San Diego
geneticists to amplify it and compare it with that of the
Burchell's zebra. To everyone's excitement, it was a match. The
quagga had not been a separate species, after all--it had just
been the southernmost sub-species of zebra, living in a
different habitat, but genetically one and the same.
Suddenly there was the
possibility of bringing the quagga back from extinction, by
selective breeding of Burchell's zebras who demonstrated "quagga-like"
characteristics. A team of researchers ranging from taxonomists
and veterinarians to conservationists and geneticists founded
the Quagga Project in 1987, capturing nine Burchell's zebras
with brown shadow striping and transporting them from the Etosha
Game Reserve to the Nature Conservation Station near Cape Town,
where they were separated into breeding groups. In December
1988, the first generation foal of the founding herd was born.
The team estimated that it would take at least two, and possibly
three to four generations before they would see if it was
possible to resurrect the quagga through selective breeding.
There was much excitement when several years later, the first of
the second-generation foals arrived and quagga characteristics
were clearly emerging.
Today, there are 53 animals
in the breeding program; some are grazing on the slopes of Cape
Town's Table Mountain, and a herd of 11, with striping on their
shoulders and light brown hindquarters, was released in the
spring of 1998 into their ancestral territory in the Karoo
National Park. Future generations are expected to exhibit, more
and more, the patterns of the century-old skins of their lost
brethren.
The saga of the quagga is by
no means a cure-all for extinction, for few other species are
likely to be resurrected in this manner. But it certainly is a
heartening application of genetic research, and the righting of
a huge wrong inflicted over a century ago on an odd-looking
equine who used to roam the arid plains.
