Eureka! The Appaloosa Gene (LP) Has Been "Spotted"!
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Eureka! The Appaloosa Gene (LP) Has Been “Spotted”! By Sheila Archer and Rebecca Terry, PhD Note: This article was first published in Vol.12, No.1 of “The Appaloosa”, the magazine of the Appaloosa Horse Club of Canada, and is reprinted here with permission from the editor. Most Appaloosa breeders anxiously await the birth of a new foal, wondering how it will appear when it arrives. Will it be a leopard or fewspot, will it have a spotted blanket or a snowcap blanket? Perhaps it will only have characteristics and roan later, or maybe it will be a “true solid”? For many years researchers have tried to gain an understanding of what genes play a role in the inheritance of appaloosa patterning in order to help limit the “guessing game” for breeders. Past research has demonstrated that there is one dominant gene responsible for appaloosa patterning, namely, LP. For several years researchers have been trying to identify the LP gene. Through recent advances made in the mapping of the horse genome, molecular biologists can determine what genes play a role in many important heritable traits. By using this current gene map of the horse we were able to identify the location of the LP gene. Background: What is the LP gene? LP stands for “leopard complex”, and is the symbol used to describe the gene responsible for appaloosa patterning. It was given this name early in the history of Appaloosa genetic research for several reasons. First the name “leopard” was attached, to denote the spotted coat associated with many Appaloosas (Figure 1). Those who know and breed Appaloosas are well aware that they also come in a number of other coat patterns, and that “leopards” are only one of these. Therefore the term “complex” was added to the name to include all of the other types of appaloosa patterning (Bowling 1996; Sponenberg et al.1990; Sponenberg 2003). LP is believed to function as the gene that must be present in its dominant form for Appaloosas to display “characteristics” and any of the various appaloosa coat patterns. Appaloosa characteristics consist of white sclera, vertically striped hooves and mottled skin most commonly found around the eyes, nose and genital region (Figure 7). The various coat patterns include, but are not limited to leopard, fewspot, spotted blanket, snowcap blanket, varnish roan and snowflake (Figures 1 - 6). Fig. 1 - Leopard Fig. 2 - Fewspot leopard Fig. 3. Spotted blanket Fig. 4. Snowcap blanket
Fig. 5 – Varnish roan Fig. 6 – Snowflake roan It is believed that the interaction of LP with other genes, known as modifying genes, determine what pattern/patterns the horse will express. In other words, the LP gene is the “on-off” switch for appaloosa patterning and the modifying genes are the “dimmer switches” that control the type and appearance of the pattern produced. What this means is that LP is essential for characteristics and coat patterning to appear, the LP gene controls for the presence or absence of appaloosa patterns. Other genes play a role in determining which patterns are produced and where these patterns are located on the coat of the animal. Fig. 7 – Traits associated with the presence of LP – striped hooves, white sclera and mottled skin. The LP “on-off switch” has two forms, known as alleles: the dominant allele (LP) and the recessive allele (lp). A horse will inherit two copies of the LP gene; one allele from its sire and one from its dam. If a horse inherits one copy of the dominant form (allele) of the LP gene (LP) from either parent, the switch is in the “on” position and the horse will display characteristics and some type of appaloosa pattern. Furthermore, if the horse inherits two copies of the dominant allele of LP (LP/LP), one from the sire and one from the dam, the horse will display a relatively “spot-free” pattern ranging from a snowcap blanket to fewspot, suggesting an incomplete dominance effect (Sponenberg 2003). If the horse inherits the recessive allele of this gene from both of its parents (lp/lp) then the switch is in the “off” position and this horse will display neither characteristics nor a coat pattern. To help demonstrate this concept of incomplete dominance, see Figure 8. For more on the action of LP please refer to Dr. Phillip Sponenberg’s book, “Equine Color Genetics” (second edition, 2003).
Fig. 8 - A
Physical appearance of horses that are homozygous for LP: Note that the amount of white patterning is
not the critical factor. Rather, the doubled effect of LP’s “command to be white” is the key to recognizing
these horses – only a “few spots” break through.
Fig. 8 – B
Physical appearance of horses that are heterozygous for LP: Again, the amount of white patterning is
not the key. A moderate to large amount of spotting occurs in dense white pattern areas, since a single
copy of LP gives only a partial “command to be white”.
Note to Diagram 8: In the case of roan-only LP-carriers, it is not possible to be sure from physical
appearance whether the horse in question is heterozygous or homozygous for LP.
So How Does a Gene Get Discovered?
Phenotype research examining pedigree information and photographs (Miller 1965;
Sponenberg et al. 1990; Lapp and Carr 1996) demonstrated the basis for the single
dominant gene theory of LP as previously discussed. This initial phenotype-based
research was the first step in understanding the genetics behind appaloosa patterning.
This initial step must then be followed by molecular research to determine the biological
mechanism of LP, in other words what gene encodes for LP.
Earlier molecular research performed by Rebecca Terry at the University of Kentucky
Veterinary Science Department ruled out the obvious candidate genes that cause similar
coat color patterns in other species (Terry et al. 2001, Terry et al. 2002). In order to
identify other candidate genes for investigation it was necessary to determine the
chromosome location of LP.
To identify which chromosome a gene is located on, molecular biologists perform an
experiment termed a “whole genome scan” (WGS). This type of research is based on the
concept of genetic linkage and recombination. Two genes located close together on a
chromosome are inherited together and are thus linked. Two genes located on different
chromosomes will be inherited independently from one another.
Performing a WGS involves looking at genetic markers on every chromosome to
determine if one of these markers is linked to the gene of interest. Researchers look for
an allele (remember that means form) of a marker that is being inherited or “traveling”
consistently with the physical feature being studied. For example, let’s assume we know
that a marker X has two alleles (A and B), and we know the two alleles of the LP gene
are (LP) and (lp). If form A always segregates with (LP) and form B always with (lp)
then these two markers are said to be linked and thus on the same chromosome.
However, if form A segregates with (LP) in some offspring and with (lp) in other
offspring and vice versa than these two markers are not linked. In order to detect linkage
the marker and the gene of interest must be fairly close together on the same
chromosome.
Through collaborative efforts involving a team of researchers we conducted a WGS for
LP during May and June of 2003. Two stallions were selected for the study, both of
which had large numbers of foals by non-Appaloosa mares (lp/lp). This type of mating
was important because solid, non-Appaloosas dams could only contribute the “switch
off” allele of LP (lp) therefore we could follow the inheritance of the LP alleles
transmitted by the sires. Both stallions were heterozygous for LP, meaning they had one
copy of the dominant allele (LP) and one copy of the recessive allele (lp). Thus the sire
transmitted the dominant allele (LP) to all appaloosa patterned offspring (LP/lp) and the
recessive allele (lp) to all solid offspring (lp/lp). For a visual demonstration of these
matings see Figure 9. Foals to be sampled were carefully inspected, and then divided into
two study groups - appaloosa patterned offspring with characteristics (inherited LP allele)
and those which were solid in color and did not have any of the appaloosa characteristics
discussed earlier (indicating they inherited the lp allele).
Fig. 9: Study Design of the Genome Scan to Locate LP
Heterozygous Appaloosa stallions were crossed to non-
Appaloosa mares. DNA samples were collected and
analyzed from the stallions, the Appaloosa-patterned
offspring, and the solid offspring. Since the dams were
solid (lplp), it was possible to follow the inheritance of
LP alleles from the sire to the resulting offspring.
DNA (deoxyribose nucleic acid) was extracted from the over 50 blood samples that were
graciously collected by owners and sent in for this study. This DNA was used to
examine the inheritance of more than one hundred markers on the 32 horse
chromosomes. Two of these markers located on horse chromosome 1 showed very strong
linkage with appaloosa patterning, while the others did not. In other words, for those two
markers that showed strong linkage, one allele was inherited with the LP allele (patterned
horses) and not (or vary rarely) with the lp allele (solid horses). For visual images of this
data see Figure 10 and Figure 11. After the initial placement of LP on chromosome 1
was established, other markers in this same chromosome region were tested to further
refine the location of the LP gene (Figure 12). This research is described in detail in
volume 35 of Animal Genetics (Terry et al. 2004).
Fig. 10 – Demonstration Showing Linkage of LP to
the Genetic Marker ASB08 on Horse Chromosome 1:
LP and lp denote the two forms of LP. Alleles A
and B denote two forms of ASB08. Allele A of
ASB08 was inherited with the dominant (LP) allele,
while allele B was inherited with the recessive (lp)
allele.
Fig. 11 – Demonstration that LP is Not Linked to
Genetic Marker ASB23 on Horse Chromosome 3:
LP proved to be linked to genetic markers on chromosome
1, but not to markers on other chromosomes. The diagram
here shows the type of scenario that is observed when two
markers are not linked. The marker exemplified here is
ASB23, which is on horse chromosome 3. LP and lp
denote the two forms of LP. Alleles A and B denote the
two forms of ASB23. Allele A was inherited with the
dominant (LP) allele and with the recessive (lp) allele.
Likewise, allele B was inherited with both the dominant
(LP) and recessive (lp) alleles.
What does this finding mean for the Appaloosa Breeder?
Now that we know that LP is located on horse chromosome 1, we are one step closer to
one of the goals of the LP research, to develop a genetic test for this gene so we can
definitively classify horses as homozygotes, heterozygotes or non-carriers for LP.
Practically speaking, breeders will be able to determine whether an Appaloosa carries
two, one or no copies of the critical allele for this gene. Testing for LP will allow
breeders to know which Appaloosas are homozygous for LP (LP/LP), without having to
rely on the physical appearance of the horse. As well, the difficulty of correctly
identifying horses that do not show physical signs that they carry LP will be solved – the
“true solid” will be distinguishable from a suppressed LP-carrier. No more expensive
and time-consuming test breeding, and no more painstaking physical inspections.
However, in order to reach this goal several more steps must be taken. Through our
research this summer we have been able to place LP on the map in a bracketed region on
chromosome 1 (Figure 12). To develop a DNA test we must have DNA sequence
information for the two forms of the LP gene, the dominant or “on” version, and the
recessive or “off” version. Biologically speaking, that means we have to know what gene
encodes for LP. To do this we can refer to what is already known in other animals. We
know that the region on chromosome 1 that we have LP bracketed within is homologous,
or “equivalent” if you will, to a region of human chromosome 15 and mouse
chromosome 7. Within these regions there are several genes that cause pigmentation
abnormalities in humans and mice. We have chosen several genes as candidates for LP
and are currently investigating them. Ultimately we hope to find the mutation that causes
appaloosa patterning and thus have a genetic test for breeders.
Fig. 12: LP is Located on Equine Chromosome 1
The genome scan was successful in identifying the chromosome region where LP is located.
Further testing led to the refinement of the location of LP, which lies between two genetic
markers. Shown here is a diagram of equine chromosome 1 with the location of LP indicated
with respect to the bracketing markers.
Fig. 12: LP is Located on Equine
Chromosome 1
The genome scan was successful in
identifying the chromosome region where LP
is located. Further testing led to the
refinement of the location of LP, which lies
between two genetic markers. Shown here is
a diagram of equine chromosome 1 with the
location of LP indicated with respect to the
bracketing markers.
Furthermore, we are working on identifying the modifying genes. Recent phenotypic
research by Sheila Archer (unpublished 2002) suggests one of these modifying genes,
that we have termed the white pattern gene, PATN, is on a different chromosome than LP.
Thus it is possible for a solid horse to inherit a form of PATN that it can pass on to its
descendents that inherit the LP allele from their other parent. Put in other terms, this
solid horse has the potential to contribute a pattern-enhancing gene to a horse that inherits
the “on” allele of LP from the other parent. Future investigations will focus on trying to
answer the ever-present question, “Why can a solid horse of Appaloosa ancestry bred to a
minimally marked Appaloosa produces a loudly marked foal?
Conclusion We are excited to announce the news of our discovery that the LP gene is located on horse chromosome 1. Our research team will continue to investigate further into the DNA identity of LP, and continue to pursue answers to pressing questions related to Appaloosa physical traits with the hope that one day we might fully understand the genetics of appaloosa patterning. Thank You! Discoveries of this magnitude don’t just happen without the help of many people. We would like to take this time to thank the individuals and organizations that supported the efforts of our research. Thank you to the scientists that gave up countless hours of their time to help in this endeavor: Dr. Ernest Bailey, Dr. Meco Bernoco, Samantha Brooks, Dr. Terri Lear, and Dr. Gus Cothran, Dr. Cheryl Harrison and Dr. Phillip Sponenberg. Thanks also to the students who helped in the lab: Mindy Bateman, Naz Gandikal and Erin Flaherty. Our sincerest thanks goes out to the Appaloosa Horse Club of Canada (ApHCC) for providing access to registry photographic records and for providing funding to carry out molecular research. Thanks also to the Colorado Ranger Horse Association (CRHA) for providing photographic data courtesy of Sherry Byrd. Funding was also provided by a Dana Grant at the University of Tampa and a Lorraine Clay Fellowship at the University of Kentucky Department Of Veterinary Science. Furthermore, this research would not have been possible with out the help of all the breeders (forgive us for not naming all of you!) who contributed time and funds to send in blood samples. Special thanks to Cheri Moats and Mellanie Burkhart, the owners of the two stallions used for the LP research. They not only sent in the stallion samples but also facilitated in the collection of the samples for all of the necessary offspring. References Bowling, Ann. T (1996) Horse Genetics. CAB International. New York, New York. Miller, R.W. (1965) Appaloosa Coat Color Inheritance. Unpublished PhD Dissertation, Animal Science Department, Montana State University. Bozeman, Montana. Lapp, R.A., & Carr, G. (1998) Applied Appaloosa color genetics. Appaloosa Journal March:113-115. Sponenberg, D.P., Carr, G., Simak, E., & Schwink, K. (1990) The inheritance of the leopard complex of spotting patterns in horses. The Journal of Heredity 81(4):323-331. Sponenberg, D.P. (2003) Equine Color Genetics. (2nd ed.) Iowa State University Press, Ames, Iowa.
Terry, R.R., Bailey, E., Bernoco, D., & Cothran, E.G. (2001) Linked markers exclude KIT as the gene responsible for appaloosa coat colour spotting patterns in horses. Animal Genetics 32:98-101. Terry, R.B., Bailey, E., Lear, T.L., & Cothran, E.G. (2002) Rejection of MITF and evidence against MGF as the genes responsible for appaloosa coat colour spotting patterns in horses. Animal Genetics. 33:82-83. Terry, R.B. Archer, S., Brooks, S., Bernoco, D., & Bailey E. (2004) Assignment of the appaloosa coat color gene (LP) to Equine Chromosome 1. Animal Genetics 35 (in press).
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