Identifying Equine Roan Coloration Inheritance through Linkage Analysis
Samantha Kubeck
Mentors: Cecilia Penudo and Stephanie Jones
Veterinary Genetics Lab, UC Davis
May 26, 2009
BIT 188
Samantha Kubeck, BIT 188, 2009
Abstract
Equine breeding is a 25 billion dollar industry where the foals that are bred gain most of
their value through their coat color and pedigree [9]. Roan coloration is a dominant phenotype
characterized by the appearance of gray hairs interspersed over a base color on the torso of the
horse. It is a prized color for breeding, but the exact mode of inheritance is still unknown. Roan
coloration occurs from polymorphisms in genes linked to the Roan locus, but such researched
polymorphisms have not yet shown a direct correlation to the Roan phenotype. Previous studies
indicate that the mutation for Roan coloration may be located in the 3’ and 5’ regions of the KIT
gene. In light of this the KIT gene will be analyzed, but also other genes closely linked to Rn will
be analyzed to either support or reject the hypothesis that the Roan mutation is caused solely by
KIT. The Rn locus is vital to veterinary and medical science because it lies in a region that is
homologous to chromosome regions in mice, pigs, and humans. This region contains LG II
(where Rn is located) and the KIT gene, and they are important to understand because they play
key roles in cell communication and regulation. Thus, by revealing the mechanisms of Roan
inheritance, more knowledge is gained about important biological processes in horses and other
mammals. Roan inheritance can be revealed through RT-PCR and RT-RTFL methods where
polymorphisms are identified and subsequently analyzed using Southern blots and genome
comparisons of different Roan horses. In this experiment the KIT gene will either be supported as
the single cause for Roan coloration, or polymorphisms in other linked genes will prove to be
correlated with Roan coloration, indicating that Roan expression is dependent on multiple genes.
Samantha Kubeck, BIT 188, 2009
Table of Contents
Background……………………………………………………………………… 1
Specific Aims……………………………………………………………………. 3
Rationale and Significance………………………………………………………..3
Materials and Methods…………………………………………………………... 4
References………………………………………………………………………...7
Biographical Sketch of Mentors…………………………………………………..8
Resume……………………………………………………………………………9
Samantha Kubeck, BIT 188, 2009
Background
Roan is a horse coat color characterized by white hairs interspersed among pigmented
hairs only around the torso of the horse [3]. It is a dominant trait controlled by the locus Rn,
where homozygous dominant individuals are inviable [4]. In a study by Heintz and Van Vleck,
Heterozygous roan horses were bred with one another and the resulting offspring produced a 2:1
ratio instead of the expected 3:1 ratio for an autosomal dominant cross. A 2:1 ratio is also
observed in “White Lethal Allelsim” where homozygote dominant offspring are inviable [4].
Thus, it can be assumed that the offspring from the roan cross follow the same trend and are
either heterozygous (Rn/rn) or homozygous recessive (rn/rn). Researchers believe that the
homozygous dominant embryos possess a tag that causes them to be reabsorbed into the uterus
[4]. Current breeders claim that there are many homozygous roans that are available on the
market, but there is currently no way to test if an individual is homozygous dominant or
heterozygous for the roan trait [4]. The geneotype is important because it determines whether a
horse will be viable or not, but gene linkage is vital in revealing the mechanism for Roan
inheritance.
Thus far, four linkage groups have been identified in the horse: LG I, LG II, and LG III
[1]. LG II historically has four loci (Al, TO, Gc, and Es) where Al codes for albumin serum, TO
codes for the Tobiano coat color, Gc codes for the Vitamin D binding protein, and Es codes for
Esterase serum [1]. Andersson and Sandberg wanted to test if the recessive gene on the extension
loci encoding chestnut (e) and roan coat color (Rn) are associated with LG II, so they bred
Swedish Trotter horses because those breeds have variants of black, gray, chestnut, and roan coat
color [1]. Albumin and esterase were analyzed by starch gel electrophoresis during
recombination, and the results were evaluated with the chi-square method [1]. The results
showed close linkage between Al and Rn, and loose linkage between Es and Rn [1]. In addition, a
close linkage between Al and e, and a loose linkage between Es and e was observed [1]. Thus, Rn
and e are considered a part of LG II. In total, three coat color genes (TO, e, and Rn) are linked
with three serum protein loci (Al, Gc, and Es), making it the largest autosomal linkage group [1].
The recombination estimates for LG II provide the following tentative order for the genes [1]:
Al
Gc
Rn
TO
e
Es
Approximately one year later, mitochondrial glutamate oxaloacetate transminase (GOTm)
was assigned to linkage group II. GOTm was mapped against serum esterase (Es), and the
recombination frequency between them was very low [5]. This indicates that GOTm is linked to
Es. Since Es is in LG II, GOTm can also be categorized into LG II. Recombination frequencies
indicate that the order of the genes in LG II is as follows [5]:
Al
Gc
Rn
TO
e
Es
GOTm
In addition, the homology in equine LG II and mouse chromosome 8 is strongly supported by the
linkage of Es to GOTm [5]. The linkage of equine Es to GOTm also indicates that the mouse
esterase locus on chromosome 8 is homologous to the equine esterase locus in LG II [5]. The
assumptions extend to include rabbit LG V and human chromosome 16 in the homology pattern
[5]. Identifying the homologies between the different organisms around LG II is vital so we can
better understand the inheritance patterns of the Rn locus in equines. After discovering GOTm
Samantha Kubeck, BIT 188, 2009
researchers completed the list of genes within LG. Unfortunately, no data was produced to
conclude that the extension locus, which determines the base coat color, is linked to the Roan
locus.
Sponenberg et. al recognized this gap in knowledge and conducted an experiment to
identify the relationship between the two genes. In this experiment, a bay roan Barabant Belgian
stallion with genotype E,Rn/e,rn was bred to 8 chestnut American Belgian mares with genotype
e,rn/e,rn (where E indicates the dominant gene of extension locus coding for Black body, e
indicates the recessive gene of extension locus coding for brown/chestnut body, Rn indicates the
dominant roan allele, and rn indicates the recessive roan allele) [2]. 57 foals were produced: 30
were bay roan (E,e/Rn,rn or E,E/Rn,rn), and 25 were chestnut (e,e/rn,rn) [2]. Chi-square analysis
was preformed and the recombination frequencies indicate that the extension locus E encoding
for the black body is closely linked to Roan locus Rn [2]. Recombination mapping was
performed, and 2 possible loci orders resulted [2]:
Es
E
Al
Al
E
Rn
Rn
After LG II was analyzed in detail, researchers focused on finding genes that were linked to the
Roan locus. Those genes would then be examined for any polymorphisms that could contribute
to Roan inheritance.
One such gene is the KIT gene, which encodes for the mast cell growth factor (MGF)
receptor, a tyrosine kinase receptor [3]. The KIT gene, with its MGF, plays a key role in growth
and differentiation of melanocytes, hematopoietic cells, and germ cells [3]. Thus, mutations that
disrupt the function of the KIT gene are associated with pigmentation disorders, anemia, and/or
sterility (often leading to lethality) [3]. Recombination studies indicate a close linkage between
KIT and Rn (no recombinant observed) and strong linkage disequilibrium between KIT sequence
polymorphism and the Rn allele [3]. In exon 19 of the KIT sequence there is a TaqI restriction
enzyme site that is spliced in 2 different ways in a heterozygote roan (Rn/rn) [3]. This shows that
Rn does not regulate coloration by abolishing the KIT expression. Unfortunately, no sequence
polymorphism was found in the equine genome that would cause roan phenotypes to occur
among all horses [3]. This indicates that the mutation in the KIT gene might occur at the end of
exon 21 (which has not been translated yet) or at the 3’ or 5’ ends because they possess
regulatory genes [3]. The KIT gene must be further analyzed because the mutation for Roan
coloration is still believed to be in the gene. In addition, other closely linked genes should be
simultaneously analyzed to either support or reject the idea that the Roan mutation is caused by
the KIT gene alone.
Samantha Kubeck, BIT 188, 2009
Specific Aims
Objective #1:
Identify all genes that are genetically linked to the locus Rn, which encodes equine Roan
coloration.
Objective #2:
Find the locations of any polymorphisms in the 3’ and 5’ regions of the KIT gene and in the
genes that are closely linked to Rn.
Objective #3:
Determine whether the polymorphisms in the KIT gene and the polymorphisms in the genes
linked to Rn are directly associated with the Roan phenotype.
Significance and Rationale
According to an Iowa State study, the United States equine industry has a GDP of $112
billion, where equine breeding accounts for $25 billion [9]. A highly prized characteristic of a
foal is its color, which is determined through parental lineage. By revealing the inheritance of
Roan coloration, horse breeders and equine aficionados can accurately predict the coat color of a
potential Roan foal. In addition, homozygous dominant Roans are inviable, so by revealing the
polymorphisms involved in inheritance, researchers can determine why this prenatal death is
occurring. This will save breeders money, considering the stud fee for a thoroughbred mating is
between $2,500 and $250,000 [10].
Roan coloration is also important to the scientific realm because the chromosome region
that contains Rn in horses is homologous to parts of the human chromosome 4 (HSA4), the
mouse chromosome 5 (MMU5), and the pig chromosome 8 (SSC8) [3]. Humans, mice, and pigs
do not exhibit Roan coloration because they lack the Rn locus, but the homologous region
contains many of the genes in LG II, where Rn is located, and the KIT gene. The KIT gene
encodes the mast cell growth factor receptor (MGF), and the alpha subunit of the platelet-derived
growth factor receptor (PDGFRA) [3]. Both of these receptors are part of the tyrosine kinase
receptor family, and they are trans-membrane proteins that bind to extracellular ligands to trigger
a signal transduction into the cell. Thus, they are vital in the control of cell proliferation,
survival, motility, and differentiation [3]. They are also vital to embyogenesis, where mutations
in the genes could lead to prenatal lethality [3]. Since the mutation for Roan coloration is
believed to be in the KIT gene, our study will focus on KIT and its interactions with the Roan
locus. Through this we can reveal the control mechanisms for Roan coloration and also
understand more about KIT and the tyrosine kinase receptors associated with it. Since these
genes are present in horses, mice, pigs, and humans, gaining knowledge about them is vital to
veterinary and human medicine alike.
Samantha Kubeck, BIT 188, 2009
Materials and Methods:
Objective 1: Identify all genes that are genetically linked to the locus Rn, which encodes equine
Roan coloration.
The goal of this research project is to determine how the Roan phenotype is inherited.
The KIT gene is believed to contain the mutation for Roan coloration, but other linked genes
must be identified so their polymorphisms can be compared to those in KIT. These comparisons
will either support or refute the hypothesis that the Roan mutation is located in KIT, and that KIT
is the only gene responsible for Roan coloration. To complete the first objective, we will identify
the genes that are linked to the Rn locus in both Roan and non-Roan horses. Once the linked
genes from the two different samples are identified, they can be compared to each other to
identify polymorphisms that potentially contribute to the Roan phenotype.
The first step in identifying the linked genes is to obtain DNA samples from 100 Roan
and non-Roan horses. Blood samples from Roan horses all over the country are archived at the
UC Davis Veterinary Genetics Lab, so Roan blood samples and recessive non-Roan blood
samples will be collected from quarter horses that display the Roan phenotype. To isolate the
genes responsible for Roan coloration, we must first isolate the mRNA in each sample of blood.
This is accomplished using an RNeasy Mini Kit® manufactured by Qiagen®. This kit is able to
purify 100µg of high quality RNA from cells and stabilize it with a stabilizing reagent [6]. The
blood cells are first lysed and homogenized in the presence of a GITC-buffer, and then
homogenate is run through a mini affinity column [6]. The RNA attaches to silica beads in the
column, and the purified RNA is eluted out with water [6]. The sample then undergoes further
purification with centrifugation to separate large RNA molecules (mRNA) from small RNA
molecules (tRNA and rRNA).
RT-PCR is then run on the purified mRNA, where the primers are targeted to gene
regions near to the Rn locus. We will perform 20 RT-PCR cycles to achieve an abundant number
of clones with the amplified region. The RT-PCR products will then be purified with a QIAEX II
Gel Extraction Kit® where silica particles bind to DNA and are centrifuged, washed, and eluted
out [6]. The purified DNA from RT-PCR is then transformed into a bacterial vector, and the
vector is genetically sequenced with Sequencher 4.9® software [7]. The data from the
sequencing is aligned in contigs and provides accurate DNA sequence assembly. The data from
the sequencing will give the recombination frequencies between locus Rn and nearby genes.
The genes that produce a recombination frequency less than 5% will be considered
tightly linked, and will be used in the subsequent experiment. Since we had a sample size of 100
Roans and non-Roans, we use 5% as the cut-off because it accounts for genes that are closely
linked and it allows for 0.5cM resolution [8]. The genes that produce a recombination frequency
less than 5% will be reanalyzed with the Sequencher 4.9® software to create LOD scores. If the
LOD score is greater than 3.0 (the minimum conventional score for linkage) we will consider the
genes to be closely linked and they will be subject to polymorphism analysis in the subsequent
experiment.
Samantha Kubeck, BIT 188, 2009
Objective 2: Find the locations of any polymorphisms in the 3’ and 5’ regions of the KIT gene
and in the genes that are closely linked to Rn.
It is assumed that the KIT gene contains the mutation for the Roan phenotype, but it is
important to define the other polymorphisms in the genes closely linked to Rn to determine if the
polymorphisms are directly correlated with the Roan phenotype. To identify these
polymorphisms, the non-Roan linked genes are used as a control against the Roan linked genes.
The polymorphisms, mutations, or alternative splice sites that result in each of the samples will
be compared to one another to determine any differences. If there are any polymorphisms in the
Roan linked genes that are not present in the non-Roan linked genes, there is a possibility that
those polymorphisms contribute to the Roan phenotype.
To accomplish this, the genes that are identified as linked to locus Rn will be analyzed
through PCR-RFLP techniques. RT-PCR is used to amplify the section of DNA containing locus
Rn and its linked genes. We will conduct 20 cycles of PCR to achieve sufficient PCR products.
Those products are then digested with RFLP enzymes, and the resulting fragments are visualized
with Sequencher 4.9® software. The sequencer will produce various peaks that represent DNA
fragments, and by comparing the peaks from the Roan horse to peaks from the non-Roan horse,
we will determine the specific sites where polymorphisms, mutations, or alternative splices occur
[2]. The sequence changes may be responsible for the expression of the Roan phenotype, or they
may be caused by other genetic factors. Hence, we must conduct further screening to isolate the
polymorphisms that specifically contribute to the Roan phenotype.
Objective 3: Determine whether the polymorphisms in the KIT gene and the polymorphisms in
the genes linked to Rn are directly associated with the Roan phenotype.
The polymorphisms that were identified in the KIT gene may contribute to Roan
expression, or they may contribute to an unrelated trait. The same applies for the polymorphisms
in the genes that are closely linked to Rn. Each of the polymorphisms must be screened and
analyzed to be sure they are associated with Roan coloration.
First, we will screen additional clones to be sure the identified polymorphisms in KIT and
the other linked genes were not random mutations. To accomplish this we will use the identified
polymorphic sites as a control to compare similar sites in clones. PCR screening will be
conducted and we will create 50 clones of both the Roan genes and the non-Roan genes. Once
the clones are prepared, we will amplify the genes linked to Rn with RT-PCR, and then identify
the polymorphisms in the linked genes with PCR-RTFL (as seen above). If the polymorphisms
are the same as those in the 50 clones, we can conclude that the polymorphisms identified in the
previous experiment were not random mutations affecting only a single clone.
These polymorphisms will then be subject to Southern blotting techniques to determine if
they are common throughout the entire Roan genome. They are first tagged with a radioactive
label so the polymorphisms will light up when the whole genome is analyzed. If the
polymorphism is fairly common throughout the genome, we can assume that it is not specialized
to control the Roan phenotype. If it is rare, though, it can be considered a candidate in Roan
coloration control.
Samantha Kubeck, BIT 188, 2009
Lastly, we will determine if the polymorphism is common throughout different breeds of
horses. To do this, we will obtain a random blood sample from a chestnut horse and a Belgian
horse because they have significantly different coloration patterns than that of Roan. We will use
RT-PCR techniques to amplify the genes that contain the polymorphisms in the Roan, and then
conduct PCR-RTFL to establish if the same polymorphisms in Roans exist at the same sites in
the two other breeds. If they are present, we can assume that the polymorphism in the Roan does
not control expression of the Roan phenotype because it is not specific to Roans alone.
By conducting these screening procedures, we can hopefully narrow down the
polymorphisms to a single gene. If only the polymorphisms in the KIT gene are found to be
correlated with Roan coloration, we can assume that the trait is caused by mutations in KIT. If
the polymorphisms in the other linked genes show a correlation, though, the mode of inheritance
cannot be attributed to KIT alone. In that case, Roan expression would be attained through
mutations in multiple genes, and further research would reveal the important medical benefits
associated with those genes.
Problems/Alternate Approaches
We may run into problems in detecting polymorphisms in closely linked genes. Most of
the time, linked genes will not be fully sequenced. The terminal ends are often left un-sequenced
because it is assumed they are only signals for transport. If a polymorphism is not detected in a
closely linked gene (0% recombination) we will sequence the entire linked gene to search if
polymorphisms occur in rarer locations on the gene. If there is still no polymorphism, the gene is
not responsible for the Roan coloration and can be discarded from other potentials.
Timeline
The determination of gene linkage should take approximately 2 months to conduct. The
detection of polymorphisms should take approximately 3 months, but determining if the
polymorphisms are relevant will take approximately 5-6 months due to the numerous factors that
can cause polymorphisms.
Samantha Kubeck, BIT 188, 2009
References:
1. Andersson, L., Sandberg, K. (1982). A linkage group composed of three coat color genes
and three serum protein loci in horses. The Journal of Heredity. 73:91-94.
2. Sponenberg, D.P., et al. (1984). Direct evidence for linkage of roan and extension loci in
Belgian horses. The Journal of Heredity. 75:413-414.
3. Marklund, S., et al. (1998). Close association between sequence polymorphism in the
KIT gene and the roan coat color in horses. Mammalian Genome. 10:283-288.
4. Hintz H.F., Van Vleck, L.D. (1979). Lethal dominant roan in horses. The Journal of
Heredity. 70:145-146.
5. Andersson, L., et al. (1983). Linkage of the equine serum esterase (Es) and mitochondrial
glutamate oxaloacetate transaminase (GOTm) loci. The Journal of Heredity. 74:361-364.
6. Qiagen Website www.qiagen.com
7. Gene Codes Website www.genecodes.com
8. Bradley W, et al. 2004. Neurology in Clinical Practice: Principles of Diagnosis and
Management. Taylor and Francis Publishing. 799.
9. Miller-Auwerda P. 2004. Equine Program. Iowa State University Animal Industry
Report. Leaflet R1928.
10. Blood Horse Magazine. 2008. 2008 Leading Sires. Blood Horse Website
www.bloodhorse.com
Samantha Kubeck, BIT 188, 2009
Biographical Sketches of Mentors
Stephanie Jones VGL Livestock Department, stephiej414@gmail.com
Stephanie Jones is a UC Davis researcher with her Master’s degree in Animal Biology.
She was drawn to UC Davis over the other UC campuses because it has a renowned Animal
Science program and Veterinary School. At first, she was most interested in attending the
university because she wanted to become a Veterinarian. During her senior year of her B.S. in
Animal Science, though, she interned at the Veterinary Genetics Lab and gained an appreciation
for research. She enjoyed working in the field of genetics because she believed the subject to be
not only very interesting, but incredibly versatile since it can be used to study a variety of animal
characteristics. After her B.S. she went directly into the UC Davis Master’s program for Animal
Biology to obtain more research experience.
Stephanie’s research in her Master’s program focused on the Roan coloration project,
where she studied the underlying mechanisms of Roan inheritance. This project was very
significant because coat color is a prized trait for breeding, and the genes linked to coat color
may be responsible for the expression of certain diseases in horses and similar organisms as well.
Roan coloration is found in horses, dogs, and cattle, and it is thought that diseases associated
with genes linked to the Roan locus can reveal similarities in diseases between these organisms.
The KIT gene is closely linked to the Roan locus, and Stephanie spent two years isolating and
analyzing the polymorphisms that could contribute to the coloration. Through this research, she
did not obtain significant data to associate any single polymorphism to Roan coloration, but she
mapped many of the genes closely linked to the Roan locus. Stephanie has not had any formal
publications, but she drew a comprehensive Master’s thesis from the Roan project.
Cecilia Penudo VGL Livestock Department, mctorrespenedo@ucdavis.edu
Cecilia Penudo runs the department of the Veterinary Genetics Lab concerning genetic
testing of larger livestock. She began her education in Brazil, where she received her B.S. in
Biological Sciences from the University of Sao Paulo. After she graduated, she left Brazil to
pursue a PhD in genetics at UC Davis. She focused her studies on genetics because she found the
subject very enjoyable, and she liked the prospect of discovering new things. Science involves
many questions, and the act of coming up with a scientific answer to daily questions appealed to
Cecilia. After she received her PhD she returned to Brazil to work for a genetics research lab, but
in 1982 she was invited to run the portion of the Veterinary Genetics Lab that dealt with genetic
testing for cattle and other livestock. She had previously worked at the lab from 1976-1978 on
Immunogenetics for her dissertation, so she understood how the lab ran and was eager to be a
part of it again.
Cecilia has remained at the Veterinary Genetics Lab since 1982 and has published over
60 scientific articles. The topics for the articles include the Horse Linkage Map, the genetic
diversity of cattle and the variation of the Y chromosome, and the genetic diversity between
horses and cattle. The current project Cecilia is working on involves the inheritance of cerebellar
abiotrophy in horses, a disease that damages neurons in the cerebellum to disrupt balance and
coordination. She is attempting to isolate the gene responsible for the disease so breeders can
eliminate the trait from their horse herds. Most of the equine genome has already been mapped,
so now it is a matter of isolating which genes are responsible for a phenotype. One such
phenotype in question is the Roan coat color. The Veterinary Genetics Lab already provides
testing for coat color because breeders find value in horse coloration, so she wants to expand on
the topic. Thus, she promoted the Roan project and has been at the head of it since 2002.
Samantha Kubeck, BIT 188, 2009
206 Tercero Hall Circle
Davis, CA 95616
Phone: 209.327.0655
E-mail: sakubeck@ucdavis.edu
Samantha Kubeck
Objective
To find a research position in animal genetics that will augment my education in
the Biotechnology program at UC Davis. I plan on obtaining my Ph.D in genetics,
thus research experience is vital to be well-prepared for graduate school.
Education
2006-Current
U.C. Davis
Davis, Ca
 College of Agriculture and Environmental Sciences
 B.S. in Biotechnology (with animal emphasis)
 Awarded Dean’s List Fall 2007, Winter 2007, Spring 2007, Spring 2008
 Breadth courses include: chemistry (organic and general), biology, physics,
genetics, calculus, physiology, and statistics.
 Projected graduation date: June 2010
Employment 2007-Current
Experience Resident Assistant
U.C. Davis Student Housing
Davis, Ca
 Responsible for the residential experience of 51 students which includes
educational, recreational, social, and cultural programming, as well as policy
enforcement, conflict resolution, and diversity issues.
2008-Current
A+ Grades Up Tutoring Center
Wallace, Ca
Tutor
 Test K-12th grade students to assess their tutoring needs, and contact parents
to inform them of the tutoring services. Assist in tutoring students in math and
reading during the afterschool program.
2008-Current
UC Davis Student Housing
Davis, Ca
Academic Assistant
 Responsible for checking in advisors and tutors into the Tercero Academic
Advising Center. Advise students to on-campus resources and create flyers to
inform residents of the services provided in the Academic Advising Center.
Additional
Information
 Proficient in electrophoresis analysis and chromatography.
 Many hours of experience using distillation techniques to isolate compounds such
as acetaminophen.
 Able to organize large quantities of data either with physical files, or on a
computer database.
 Enrollment in the National Society of Collegiate Scholars.
Samantha Kubeck, BIT 188, 2009