Construction and Validation of Parentage Testing for Thoroughbred Horses by 53
Single Nucleotide Polymorphisms
Christine Noonan
Biol 302
11/04/10
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According to a 2005 study conducted by the American Horse Council, the horse industry
at that time contributed about $100 billion annually to the US economy. $26 billion of that came
from the racing industry, and of that $26 billion, about $21 billion came from Thoroughbred
racing specifically. At the time of the study, thoroughbreds made up 14% of the total horse
population in the US, which is an astonishing figure when one considers that there are hundreds
of horse breeds in the world. Of this 14%, approximately 43% were being used for racing, which
means that they would have to be registered with the Jockey Club, which is a very intensive
process requiring DNA parentage verification. Another 30% of the horses were Quarter Horses,
but only 4% of them were being used for racing, and therefore the statistic may be skewed by
breed reporting errors. In summary, this study shows just how much of an impact thoroughbred
racing has on the horse industry. Racing thoroughbreds only made up 6% of the horse
population, but they still brought in 20% of the total amount of money that the horse industry
generated annually at the time of the study (The American Horse Council, 2005). Because the
thoroughbred racing industry is so large and so tightly regulated, thoroughbreds are often used in
equine research.
The thoroughbred horse originated about 300 hundred years ago in England when three
stallions (The Godolphin Arabian, the Darley Arabian, and the Byerly Turk) were imported from
the Middle East and bred to local mares. Combining the “hot,” an equine term for a high strung,
fine boned horse, with the calmer, larger English mares resulted in the modern thoroughbred, a
breed that is known for being the best at gallop races over medium distances, but that is also used
in almost every kind of equine competition because of its incredible versatility. The Jockey Club
has been the breed registry for thoroughbreds in the US since 1894 (The Jockey Club, 2010).
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Currently, in order for a thoroughbred to be registered with the Jockey Club, it must
follow many guidelines. The three that are most relevant to parentage research are listed below.
“1. Foals must be genetically typed and qualified by parentage verification
by a laboratory approved and authorized by The Jockey Club.
2. To be eligible for registration, a foal must be the result of a stallion’s
Breeding with a broodmare (which is the physical mounting of a broodmare by a
stallion with intromission of the penis and ejaculation of semen into the
reproductive tract). As an aid to the Breeding, a portion of the ejaculate produced
by the stallion during such mating may immediately be placed in the uterus of the
broodmare being bred. A natural gestation must take place in, and delivery must
be from, the body of the same broodmare in which the foal was conceived.
Without limiting the above, any foal resulting from or produced by the processes
of Artificial Insemination, Embryo Transfer or Transplant, Cloning or any other
form of genetic manipulation not herein specified, shall not be eligible for
registration.
3. The following colors are recognized by The Jockey Club: Bay, Black,
Chestnut, Dark Bay/Brown, Gray/Roan, Gray, Roan, Palomino, White.” (The
American Stud Book, 2008)
These guidelines were put in place to protect potential buyers and/or breeders from false
advertising about a foal’s parents, but are rapidly becoming outdated due to technology. In 2001,
the Jockey Club began using DNA genotyping for parentage verification (The American Stud
Book, 2008). Currently, short tandem repeats (STRs) are used by breed registries (including the
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Jockey Club) worldwide to verify parentage. STRs, however, are prone to slippage mutation
errors. These can lead to incorrect parentage reports. For that reason, two different STR assays
are usually run to verify parentage. The study performed by Hirota et al. looked at using SNPs
instead of STRs to verify parentage in 95 randomly chosen thoroughbreds. It was hypothesized
that SNPs would be better than STRS for use in parentage verification because they have lower
mutation rates, and SNP assays are cost effective, able to be analyzed with automated
technology, can be detected via multiple methods, and are more useful than microsatellites in
other research fields (Hirota et al., 2010).
DNA was collected from a random sample of 95 thoroughbreds and the various
experiments were run. Three different final tests were performed: an iPLEX SNP assay, an
assay using STRs reported by Kakoi, and the Microsatellite 15 TKY System. The latter two
assays are currently used in parentage verification and were therefore the controls of the
experiment. The assay using STRs reported by Kakoi is for basic parentage verification, and the
Microsatellite 15 TKY System is then used to support the data obtained from the first STR assay
(Hirota et al., 2010).
The study started out with 120 SNPs (randomly chosen from known equine SNPs) which
were then directly sequenced in 8 thoroughbreds in order to obtain the allelic frequencies. 60 of
the 120 SNPs were chosen for designing the assays due to their high heterozygousities. These 60
SNPs were then analyzed with MassARRAY software and the final assay excluded 7 more SNPs
because of instabilities in amplification and/or primer extension. The final iPLEX assay
consisted of two panels, JPN_01 (27 SNPs) and JPN_02 (26 SNPs), because the machine was
only able to run up to 40 SNPs at once (Hirota et al., 2010).
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The International Stud Book committee requires the average exclusion probability (PE01)
for parentage results to be above 99.95%. The SNP assay initially resulted in a PE01 of
99.996%. When the Hardy-Weinberg equation was applied to each SNP allelic frequency,
however, three SNPs deviated from the equilibrium. There are evolutionarily biased genomic
regions in thoroughbreds, so it could be that those regions are what caused the deviations. When
those three SNPs were excluded from the statistical analysis, the PE01 was 99.994%. The STR
assays resulted in PE01s of 99.994% (STRs reported by Kakoi) and 99.998% (Microsatellite 15
TKY System) (Hirota et al., 2010).
The above results indicate that in terms of PE01, SNP assays and STR assays are equally
effective. An individual SNP was not as discriminatory as an STR, but a combination of
multiple SNPs was highly discriminatory, indicating that as long as multiple SNPs were run, the
resulting parentage results would be accurate. Because the two methods are equally effective,
other factors must be taken into consideration (Hirota et al., 2010).
One main reason for using SNPs over microsatellites is the fact that SNPs have more
potential research applications in the fields of equine coat color genetics and equine diseases
(Hirota et al., 2010). Coat color genetics may seem unimportant, but actually understanding the
genetics of coat color can sometimes help diagnose characteristics that future generations of
foals might inherit. Coat color genetics also relate back to the research described above because
of the Jockey Clubs rules regarding which colors are recognized in the registry (The American
Stud Book, 2008).
The Jockey Club currently does not recognize “colored” thoroughbreds (The American
Stud Book, 2008). A “colored” horse is simply any horse that has white markings other than on
the face and legs. Some examples include tobiano, overo, sabino, and leopard spotted. Tobiano
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is a spotting pattern characterized by white patches, usually rounded or oval with clearly defined
edges, that generally extend over the spine, white legs (usually), and tails that can be a mixture of
two colors. Tobiano is a dominant gene (UC Davis, 2009). Overo is a spotting pattern
characterized by white irregular, scattered white patches that do not cross the spine and dark legs
(usually) (American Paint Horse Association, 2010). Sabino is a spotting pattern characterized
by flecks or roan patches (places where dark and light hair grow intermixed), and it is one of the
most varied spotting patterns (Sabino horses). Leopard spotting is a pattern characterized by
small dark spots on a white background over the entire body (Lloyd). Below are examples of
each of the patterns.
Tobiano
Overo
Sabino
Leopard
Figure 1. Examples of spotting patterns in horses.
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As mentioned above, the Jockey Club currently will not register horses with the above
patterns or any of the other spotting patterns (The American Stud Book, 2008). If accurate
parentage verification is available on a horse that shows up with a spotting pattern, why wouldn’t
they allow that horse in the registry? If color genetics progress further, they may be able to help
solve this problem.
Currently, there is a lot of information available about color genetics in horses. After all,
horses are generally speaking similar in body shape, if not size. Breeders want excellent
conformation, of course, but having a striking color pattern can often distract the eye. Some
breeders like blue eyes, some don’t, it’s all personal preference, but the fact remains that over
time these patterns have clearly been selected for by breeders. All of the patterns, however, trace
back to genetic mutations.
White coat coloring in horses is dominant, and a 2009 paper by Haase et al. looked into
how white patterns are related in horses (Haase et al., 2009). Haase et al. looked at the KIT gene
and its effect on white patterns, but there is an earlier paper that first discovered that KIT had to
do with the tobiano spotting pattern by Brooks et al (Brooks et al., 2007).
The paper by Brooks et al. specifically studied the tobiano pattern. As mentioned above,
the tobiano trait is dominant. The researchers had seen in a previous study on mice that similar
spotting patterns in mice were caused by chromosomal rearrangements near the Kit gene, and
therefore they decided to look there in horses for anomalies. In 2002 and 2006 Brooks looked at
linkage of Tobiano and KIT, but in this 2007 study they specifically looked for chromosomal
rearrangements. The study found that a chromosome 3 inversion near KIT caused the tobiano
phenotype. In cases of homozygous tobiano horses, both copies of chromosome 3 were inverted,
while in cases of heterozygous tobiano horses (no phenotype difference from homozygous
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tobiano horses), there was one normal chromosome and one inverted. This chromosome
inversion does not cause any problems in the coding sequence of any genes, so it is still unknown
exactly how it causes the tobiano phenotype. The inversion may simply disrupt other regions of
the KIT gene. Tobiano horses are healthy, but on an interesting note, heterozygous tobiano
horses may be less fertile (20 – 50%, according to the study). The study goes on to say that this
infertility may not be noticed by horse breeders, but in the racing world, where live cover is
mandatory, so you can’t alter semen or place it inside the uterus, decreased fertility very well
could have been one of the reasons why early thoroughbred breeders stayed away from colored
horses (Brooks et al., 2007). At the same time, this paper demonstrates that a harmless KIT
mutation could cause an unusual color phenotype in a thoroughbred that had Jockey Club
registered parents.
The 2009 paper by Haase took the above research even further and looked at KIT
mutations in horses with white coat patterns. In 2007, Haase et al. identified four KIT mutations
in dominant white horses and named them W1 – W4. This study was a more comprehensive
investigation, using nine horse families from five different breeds segregating for white or
partially white coats. The researchers sequenced the KIT exons and nearby sequences in 192
horses, 35 of which had a white or partially white coats. They then tested the 35 horses for the
absence of the known KIT mutations. The researchers found 30 new polymorphisms in the KIT
gene, seven of which were mutations affecting the coding sequence. These mutations were then
named W5 – W11. Three different thoroughbred families were studied, and they contained W5,
W6, and W7 (Haase et al., 2009). Therefore it is quite clear that even pure thoroughbreds can
develop these mutations.
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Coat color genotyping may actually prove useful in breed differentiation, which would
help the Jockey Club keep the registry pure without excluding colored thoroughbreds. In a 2009
study by Kakoi, a method was developed for genotyping coat color loci simultaneously in order
to differentiate between Japanese thoroughbreds and a native breed, the Misaki horse. The
researchers developed a method for simultaneous genotyping of multiple color loci using SNPs.
The Misaki breed is a very small population, but it is historically important to the Japanese, and
so they wish to keep it as close to the ancestral population as possible. It is currently thought that
in 1913 a foreign stallion was introduced to the Misaki population and since then phenotypic
variations not previously seen have been cropping up in the population. This study hoped to help
separate the “impure” Misaki horses from the ancestral population by developing an alternate
method coat color genotyping. The study did achieve this, and in combination with DNA
parentage verification, it is probable that future efforts to conserve the Misaki horse will be
successful (Kakoi et al., 2009).
The above study indicates that coat color genotyping could be used to separate different
horse breeds based on which color genotypes are present in each breed. There are currently
methods for testing the genotypes of the basic color genes in horses, but in the past this
information has mostly been used by breeders hoping for a foal of a certain color.
DNA parentage verification has greatly enhanced the horse industry. In the past, when
someone said a foal was by a certain stallion, there was no way of telling if that was true.
Nowadays, the truth is a simple assay away. The Jockey Club still, however, refuses to
recognize some thoroughbreds simply because they are not the correct color (The American Stud
Book, 2008). This concern would be understandable if DNA testing was not readily available,
but since the Jockey Club actually requires genetic parentage verification and it requires both
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parents to have been registered with the Jockey Club, this restriction is outdated (The American
Stud Book, 2008). White coat patterns in horses are caused by KIT mutations that can occur at
any time, no matter how many generations of solid colored horses there are in a foal’s past
(Haase et al., 2009). Some breeders in the past have taken these mutations and based whole new
breeds off of them, as with the American Paint Horse Association. According to the American
Paint Horse Association, a Paint Horse must have a combination of white and any other color
found in horses (American Paint Horse Association, 2010). Other times, these mutations have
been unintentionally used to create new breeds, as with the Gypsy Vanner. The Gypsy Vanner
was originally just a plain cart horse in England, but according to Deborah Noonan, at one point
the British government began conscripting horses for the army, but it would only take solid
colored horses, because they don’t stand out on a battlefield, so the Travelers started breeding for
color, and while today there are solid colored Gypsy Vanners, the vast majority are black and
white tobianos. With thoroughbreds, there’s so much inbreeding already that it would be better
for the genetic structure of the population to allow colored thoroughbreds, if only for genetic
diversity. As for future research, studies could be done to see why KIT mutations occur, how
often they occur, and how they actually affect coat color phenotype, and coat color genotyping
could be combined with SNP parentage assays to look at relatedness of breeds.
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References
UC Davis. (2009). Tobiano. Veterinary Genetics Lab.
http://www.vgl.ucdavis.edu/services/tobiano.php
American Paint Horse Association. (2010). Overo Pattern.
http://www.apha.com/breed/overo.html
Brooks, S., Lear, T., Adelson, D., & Bailey, E. (2007). A chromosome inversion near the KIT
gene and the Tobiano spotting pattern in horses. Cytogenetic And Genome
Research, 119(3-4), 225-230.
Haase, B., Brooks, S., Tozaki, T., Burger, D., Poncet, P., Rieder, S., et al. (2009). Seven novel
KIT mutations in horses with white coat colour phenotypes. Animal Genetics, 40(5), 623629.
Hirota, K., Kakoi, H., Gawahara, H., Hasegawa, T., & Tozaki, T. (2010). Construction and
validation of parentage testing for thoroughbred horses by 53 single nucleotide
polymorphisms. The Journal Of Veterinary Medical Science / The Japanese Society Of
Veterinary Science, 72(6), 719-726.
Kakoi, H., Tozaki, T., Nagata, S., Gawahara, H., & Kijima-Suda, I. (2009). Development of a
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investigation of basic colour variation in Thoroughbred and Misaki horses in
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Leopard spotted photo, http://www.petsfoto.com/wp-content/uploads/2010/04/Appaloosa3.jpg
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