Genetics and the Shape of Dogs

The "dog genome project" was launched in the early 1990s, motivated by
scientists' desire to find the genes that contributed to many of the ills suffered by
purebred dogs. Most dog breeds have only been in existence for a few hundred
years. Many exhibit limited genetic diversity, as dog breeds are typically
descended from a small number of founders, created by crossing closely related
individuals. Further, breeds often experience population bottlenecks as the
popularity of the breed waxes and wanes. As a result of this population structure,
genetic diseases are more common in purebred dogs than in mixed-breed dogs.
Scientists have been motivated to use dog populations to find genes for diseases
that affect both humans and dogs, including cancer, deafness, epilepsy, diabetes,
cataracts and heart disease. In doing so we can simultaneously help man and
man's best friend.
The initial stages of the dog genome project involved the building of maps that
allowed scientists to navigate the dog genome. Quick to follow were the
production of resources that facilitated the manipulation of large pieces of dog
genome DNA and a numbering of the dogs' 38 pairs of autosomes (non-sex
chromosomes) as well as the X and Y chromosomes. Finally, in 2003, a partial
sequence of a standard poodle was produced that spanned nearly 80 percent of
the 2.8 billion base pairs that make up the dog genome. This was followed quickly
by a concerted effort to fully sequence the boxer genome, producing what is today
the reference sequence for the dog.
How is this information being used by geneticists today? The availability of a high-
quality draft sequence of the dog genome has quite literally changed the way
geneticists do their work. Previously scientists used so-called "candidate gene"
approaches to try and guess which genes were responsible for a particular
disease or trait of interest. By knowing something about what a gene does or
what family it belongs too, we can sometimes, but not always, develop excellent
hypotheses as to what happens when a specific gene goes awry. However,
candidate gene approaches are often characterized by frustration and great
expense. Hence, companion-animal geneticists are turning increasingly to the
more sophisticated genomic approaches made possible by the success of the
dog genome project.
Central to our ability to use the newly available resources is an understanding of
breed structure, the strengths and limitations of the current molecular resources,
and consideration of the traits which are likely to lend themselves to mapping
using available resources. In this article I highlight first our current understanding
of what a dog breed really is and summarize the status of the canine genome
sequencing project. I review some early work made possible by this project:
studies of the Portuguese water dog, which have been critical to our
understanding of how to map genes controlling body shape and size, along with
studies aimed at understanding the genetics of muscle mass.
Dog Breeds
The domestic dog is believed to be the most recently evolved species from the
family Canidae. Within the Canidae there are three distinct phylogenetic groups,
or clades; the domestic dog shares a clade with the wolflike canids such as the
gray wolf, coyote and jackals. Dogs are thought to have arisen perhaps as
recently as 40,000 years ago, with initial domestication events occurring in
eastern Asia. Most domestic breeds that we recognize today, however, likely are
the product of human breeding over the last 200-300 years. Many of the most
common modern breeds were developed in Europe in the 1800s. Some of the
breeds represented in antiquity, including the greyhound and the pharaoh hound,
are particularly interesting to study, as it is unclear whether dogs from these
breeds are re-creations of ancient breeds or whether dogs alive today can truly
trace their lineage to founders from thousands of years ago.
Adapted by Barbara Aulicino and Linda Huff from Lindblad-Toh et al. 2005.
The American Kennel Club (AKC) currently recognizes about 155 breeds of dog,
but new breeds are created and given breed-recognition status frequently. What
defines a dog breed? Although a dog's parentage can be recognized by its
physical attributes—coat color, body shape and size, leg length and head shape,
among others—the concept of a breed has been formally defined by both dog
fanciers and geneticists.
Dog regulatory bodies such as the AKC define an individual's breed by its
parentage. For a dog to become a registered member of a breed (say, a golden
retriever), both of its parents must have been registered members of the same
breed, and their parents in turn must be registered golden retrievers. As a result,
dog breeds in the United States today are generally closed breeding populations
with little opportunity for introduction of new alleles (variations in the genome). At
a genomic level, purebred dogs are usually characterized by reduced levels of
genetic heterogeneity compared to mixed-breed dogs. Breeds that derive from
small numbers of founders, have experienced population bottlenecks or have
experienced popular-sire effects—that is, the effect on the breed of a dog who
does well in shows producing a disproportionate number of litters—display further
reductions in genetic heterogeneity.
Recently, my laboratory group and others have begun to use genetic tools such
as markers to define the concept of a dog breed. A genetic marker is a position
in the genome where there is variability in the sequence that is inherited in a
Mendelian fashion (that is, following the rules of classical genetics). Two common
kinds of markers are microsatellite markers, where the variation comes from the
number of times a repeat element is reiterated at a given position on a
chromosome, and single-nucleotide polymorphisms (SNPs, pronounced
"snips"), in which the DNA sequence varies when a single nucleotide (denoted A,
C, T or G) in a sequence differs between the paired chromosomes of an
individual.
These alterations are proving invaluable for understanding the role of genetic
modifications both within and between breeds. Because the alleles of markers
are inherited from parent to child in a Mendelian fashion, they can be used to
track the inheritance of adjacent pieces of DNA through the multiple generations
in a family. There are thousands of microsatellite markers and millions of SNPs
distributed randomly throughout the canine genome.
In order to determine the degree to which dogs could be assigned correctly to
their breed group, my lab utilized data from 96 microsatellite markers spanning
all the dog's 38 autosomes in a set of 414 dogs representing 85 breeds. We
found, first, that nearly all individual dogs were assigned correctly into their breed
group when we used a set of statistical tools called clustering algorithms, which
look for similarities in the frequency and distribution of alleles between individuals.
The exceptions largely included six sets of closely related breed pairs (for
example whippet-greyhound and mastiff-bullmastiff) that could only be assigned
to their respective breeds when considered in isolation from other breeds.
We also showed that the genetic variation between dog breeds is much greater
than the variation within breeds. Between-breed variation is estimated at 27.5
percent. By comparison, genetic variation between human populations is only 5.4
percent. Thus the concept of a dog breed is very real and can be defined not only
by the dog's appearance but genetically as well.
A second part of the study used an assignment test to determine whether we
could correctly identify each dog's breed by its genetic profile alone. In a blinded
study, where the computer program did not know what data set came from which
breed, 99 percent of dogs were correctly assigned to their breed based on their
DNA profile alone.
To determine the ancestral relationship between breeds, Heidi Parker from my
lab used data from the same set of dogs and sought to determine, ideally, which
dog breeds were most closely related to one another. To do this we utilized a
computer program called structure, which was developed by Jonathan Pritchard
at the University of Chicago and his colleagues. The program identifies
genetically distinct subpopulations within a group based on patterns of allele
frequencies, presumably from a shared ancestral pool.
The structure analysis initially ordered the 85 breeds into four clusters,
generating a new canine classification system. Cluster 1 comprised dogs of Asian
and African origin—thought to be older lineages—as well as gray wolves. Cluster
2 included largely mastiff-type dogs with big, boxy heads and large, sturdy bodies.
The third and fourth clusters split a group of herding dogs and sight hounds away
from the general population of modern hunting dogs, the latter of which includes
terriers, hounds and gun dogs. As more dog breeds have been added to the
study, additional groupings have emerged.
These data are extremely useful for disease-gene mapping studies. In some
cases, dogs from breeds that are members of the same cluster can be analyzed
simultaneously to increase the statistical power of the study. This will not only aid
in the identification of genomic regions in which the disease gene lies, but will
also assist in "fine mapping" studies which aim to reduce the region of DNA
linkage to a manageable size of about 1 million bases. Once a region is well
defined, we can begin to select candidate genes for mutation testing.
Sequencing the Dog Genome
The first published sequence of the dog genome was completed in 2003 in an
effort lead by Ewen Kirkness at The Institute for Genome Research. Genomes
are typically sequenced in many thousands of overlapping segments, and to
ensure that the whole genome is recorded at least once, it is estimated that there
have to be seven or eight iterations, or "reads," across the entire genome. The
2003 genome, from a standard poodle, was a so-called survey sequence. The
genome was sequenced just 1.5 times, so about 80 percent of the genome was
present in the final data set. This work was followed shortly thereafter by the
release of the draft assembly of the boxer genome, led by Kerstin Lindblad-Toh
and colleagues at the Broad Institute, which was done at 7.5x density. With
millions of reads successfully completed, nearly 99 percent of the genome is
present in the final data set.

Barbara Aulicino
Both resources have proved to be extremely useful. The 1.5x sequence provided
the first glimpse into the organization of the dog genome, number of genes and
organization of repeat elements. One surprise was the discovery of a large
number of short interspersed nuclear elements (SINEs) littered throughout the
dog genome that were occasionally located at positions with the potential to affect
gene expression. For example, the insertion of a SINE element into the gene
encoding the hypocretin receptor, a neuropeptide hormone found in the
hypothalamus of the brain, results in the disease narcolepsy in the Doberman
pinscher. Similarly, a SINE element inserted into the SILV gene (known to be
related to pigmentation) is responsible for merle, the mottled patterning of a dog's
coat.
The 7.5x female boxer sequence spans most of the dog's 2.4 billion bases in a
sum total of 31.5 million sequence reads. The sequence is estimated to cover
over 99 percent of the eukaryotic genome and provides data for the existence of
about 19,000 genes. For about 75 percent of the genes, the homology (amount
of similarity arising from shared ancestry) between the dog, human and mouse
genome is very high. The majority of genes contain no sequence gaps, which is
a great aid to scientists seeking to test particular genes as candidates for
diseases.
Over the course of its evolution, the canine genome acquired more than two
million SNPs, which are proving invaluable for understanding the role of genetic
variation both within and between breeds. Such SNPs, analyzed using DNA chips
or bead arrays, will be important for scientists conducting whole-genome
association studies aimed at identifying genes that underlie complex traits in the
dog. A dog chip with about 127,000 SNPs is currently available, allowing
scientists to interrogate the dog genome at several thousand positions
simultaneously. When the data from dogs with a given disease, for instance
lymphoma, are compared to those from dogs without the disease, we can quickly
pinpoint regions of the genome where disease genes are likely to lie.
The Shape of Things
Our research group, along with others, has been interested for several years in
identifying genes that define the differences in body size, shape and appearance
between breeds. Dog breeds vary not only in overall body size, but also in leg
length, head shape and many other body features, all of which are controlled at
least in part at the genetic level. The amount of morphologic variation observed
in the dog is reported to surpass that of all living land mammals.
Adapted by Barbara Aulicino and Linda Huff from Parker et al. 2004.
The first important molecular study aimed at understanding the genetics of canine
morphology was done at the University of Utah and led by Gordon Lark and Kevin
Chase. The project, termed the Georgie Project in memory of a favored dog,
focused on the Portuguese water dog, which is ideal for this type of study
because it derives from a small number of founders, largely from two kennels,
that came to the United States in the early 1950s. The breed standard permits a
significant amount of variation in body size compared with other breeds. The
community supporting the project is composed of highly motivated owners and
breeders who have sought to improve the health of the breed through
collaboration with scientists.
To date, the project has collected DNA from more than 1,000 dogs and has
completed a genome-wide scan using more than 500 microsatellite markers on
nearly 500 dogs. In addition to family history and medical data, more than 90
measurements have been collected for nearly 500 animals. These were derived
from a set of five x-rays taken at the time of initial sample collection. Analysis of
these metrics led to the development of four primary principal components (PCs),
sets of correlated traits that define Portuguese water dog morphology. It is
important to keep in mind that PCs are not genes but traits, and as such, they are
susceptible to genetic analysis.
Analysis of the genome scan data and four PCs initially highlighted 44
putative quantitative trait loci (QTLs) on 22 chromosomes that are important for
heritable skeletal phenotypes in the Portuguese water dog. QTLs derive from
complicated statistical analysis and indicate locations in the genome that
contribute coordinately to a particular trait. Of particular interest to us was a locus
on canine chromosome 15 (CFA15) that showed a strong association with overall
body size. Although this was only one of seven loci hypothesized to play a role in
body size in the dog, we chose it as an initial focus because of the strength of the
effect and the proximity to a compelling candidate gene.
To find the gene on CFA15, we searched for SNPs in a 15 million-base-pair
region and then genotyped the resulting set of markers on all the Portuguese
water dogs for which size information was available. The distribution of these
markers displayed a single peak close to the insulin-like growth factor-1 gene
(IGF1), which is known to influence body size in humans and mice. We
investigated IGF1 in detail and showed that 96 percent of Portuguese water dog
chromosomes carry one of just two patterns of alleles, which are
termed haplotypes. The haplotype associated with small dogs was termed "B"
and the one associated with large dogs "I." Portuguese water dogs homozygous
for haplotype B—that is, dogs that have the B pattern on both chromosomes—
have the smallest median skeletal size, whereas dogs homozygous for I are
largest. Dogs that are heterozygous—that is, those with a different pattern on
each chromosome—fall between.
Adapted by Barbara Aulicino and Linda Huff from Parker and Ostrander
2005.
To study the presumably more general role of IGF1 in size differentiation among
breeds, we surveyed genetic variation associated with 122 SNPs, spanning the
relevant 34 million- to 49 million-base-pair interval of chromosome 15 in 353 dogs
representing 14 small breeds and 9 giant breeds. Several lines of evidence
pointed to IGF1 as the gene likely to account for small body size in the dogs.
Most notably, we observed a dramatic reduction in heterozygosity in small breeds
over the IGF1 gene. These results demonstrate the presence of a selective
sweep in this region, showing that IGF1 has been under tight selection by
breeders seeking to create ever smaller dogs. In addition, the dominance of a
single unique haplotype in our panel of many unrelated small dog breeds,
together with its near absence in giant breeds, suggests that the mutation is
ancient and likely evolved early in the history of domestic dogs.
Sexual Dimorphism
The Georgie Project is remarkable for the number of putative loci that have been
discovered by the initial analysis. In addition to loci for head shape, body size, leg
length and a host of other traits, loci have also been described that reportedly
control differences in size between the sexes, so-called sexual dimorphism.
Sexual dimorphism is observed in almost all mammals including, of course, dogs.
The mechanisms for maintaining sexual dimorphism are not well understood. It
has been shown that the Sry locus on the Y chromosome plays an important role
in sex determination and dimorphism, but this is clearly only a small part of the
story.
Photographs courtesy of Kevin Chase.
The study of the Portuguese water dog has filled in some additional pieces of this
interesting puzzle. This vignette has its roots in the original observation that a
locus on chromosome 15, which may or may not be IGF1, interacts with other
genes to make males larger and females smaller.
On average, female Portuguese water dogs are 15 percent smaller then males.
Chase, Lark and their colleagues observed that in females, a particular haplotype
is dominant for small body size. In males, a different set of variants (another
unique haplotype) associated with large overall body size is dominant. The locus
on CFA15 interacts with another locus on the X chromosome that is known to
escape inactivation, meaning that both copies of the genes in this region are
turned on (in most locations on the X, only one copy is active).

Data courtesy of Kevin Chase. Adapted by Barbara Aulicino from Chase et

al. 2005.
Females who are homozygous at the X-chromosome locus and who are also
homozygous for the large-size CFA15 haplotype are, on average, as large as
large males. However, all females that are heterozygous at the X-chromosome
marker are small, regardless of their CFA15 genotype. This result suggests
several scenarios for how genes interact to affect major complex traits, such as
body size, and suggests a mechanism for the evolution of sexual dimorphism.
Two observations from the study must be accounted for in the development of
any model to explain canine sexual dimorphism. The explanation must include a
discussion of the reversal of dominant haplotypes between males and females
associated with CFA15 locus as well as an explanation for the interaction
between the CFA15 and X-chromosome loci.
To address the first question, Chase and his colleagues propose the existence of
another sex-specific factor. For example, the CFA15 locus might contain two
distinct genes associated with two haplotypes; the so-called Ahaplotype acts in
both males and females to upregulate size, while the B haplotype and its
associated allelesdo not upregulate size but rather contain another gene that
suppresses the up-regulator.
The second phenomenon, heterozygote-specific interaction, could be explained
by arguing that the activation of haplotype A's critical upregulator gene requires
interaction with a protein produced by the X chromosome.
The data of Chase, Lark and their colleagues are consistent with predictions
made in the early 1980s that sexual dimorphism evolves because females
secondarily become smaller than males as a result of naturalselection for optimal
size. Reduction of female size relative to that of males takes place, according to
this hypothesis, through an inhibition of major genes that enhance growth, such
as the locus on CFA15.
A Faster Dog
Studies such as those described above are well designed for understanding
complex or multigenic traits. But there remains some "low-hanging fruit" to be
harvested in the study of canine morphology—other cases where apparently
single genes contribute to major traits of interest. An example is provided by my
research group's study of the whippet and a mutation in the gene coding for
myostatin, a growth factor that limits the buildup of muscle tissue. In this study
we found a new mutation in the myostatin gene, MSTN, and observed that it
results in a double-muscled phenotype known as the "bully" whippet.

Stuart Isett/Polaris
The typical whippet, a medium-sized sight hound, is similar in appearance to dogs
of the greyhound breed and weighs about nine kilograms. Whippets are
characterized by a slim build, long neck, small head and pointed snout. Bully
whippets, however, have broad chests and an unusually well-developed leg and
neck musculature that makes them unattractive to fanciers of the breed.
Using a candidate gene approach, we showed that individuals with the bully
phenotype carry two copies of a two-base-pair deletion in the third exon (a gene
region that is transcribed to make portions of proteins) of MSTN, with the result
that a truncated or mutant protein is produced. These findings were somewhat
expected, as the double-muscle phenotype observed in the whippet is
reminiscent of what has been reported in mice, cattle and sheep and in a single
case in humans, each of which was caused by a mutation in the myostatin gene.
The specifics for dogs, however, were useful to the whippet dog community,
which is seeking to develop a genetic test that will reduce the number of dogs
produced with the bully phenotype.
Interestingly, we also found that individuals carrying only one copy of the
mutation are, on average, more muscular than wild-type individuals, as measured
by their neck and chest girth as well as mass-to-height ratio. Indeed, we
estimated that mutations in myostatin explain approximately 60 percent of the
variation in both the ratio of height to weight and neck girth, and 31 percent of the
variation in chest size. In addition to the statistically significant differences
between dogs that were bully and wild types, dogs who carried one copy of the
variant allele were more heavily muscled then their wild-type counterparts,
although not nearly as heavily muscled as the bully dogs.
This observation caused us to ask whether dogs that carried one copy of the
mutation were faster racers—a success that would likely lead them to be bred
more, which in turn could produce bully dogs if two recessive-gene individuals
were paired. Careful analysis revealed an association between individuals
carrying one copy of the MSTN mutation and racing speed. Dogs that were the
faster racers (class A) were more likely to carry the mutation then were dogs that
were slower racers (classes B, C and D). Least likely to carry the mutation were
dogs that had never raced and were primarily show dogs.
We considered the possibility that the result could be explained solely by the fact
that A racers tended to be mated more often to A racers as opposed to B, C, D
or nonracing dogs. This tendency would predict a significant amount of population
substructure among A racing dogs. Although we demonstrated that some
population substructure exists, we were able to show that it did not fully account
for the observation that an excess of A racing dogs carried the myostatin mutation
compared to dogs that either did not race or were class B, C or D. Indeed, 50
percent of the A racers tested carried the mutation. We did not find the variant in
greyhounds or any of the heavily muscled mastiff breeds such as the bulldog.
Remaining Selective
The advances of the past three years in canine genetics have been enormous.
The dog genome has been mapped and sequenced. A host of disease loci have
been mapped, and in many cases the underlying mutations identified. Our
understanding of how dog breeds relate to one another is beginning to develop,
and we have a fundamental understanding of the organization of the canine
genome. The issue of complex traits is no longer off-limits. We have begun to
understand the genetic portfolio that leads to variation in body size and shape,
and even some performance-associated behaviors.
Certainly the next few years will bring an explosion of disease-gene mapping.
The genetics of canine cancer, heart disease, hip dysplasia, vision and hearing
anomalies have all been areas of intense study, and investigators working on
these problems are poised to take advantage of the recent advances described
here. Whole-genome association studies are likely to replace family-based
linkage studies as a way of finding genes associated with not only disease
susceptibility and progression, but morphology and behavior as well.
What will the companion-animal and scientific communities do with this new
information? It is certainly hoped that the disease-gene mapping will lead to the
production of genetic tests and more thoughtful breeding programs associated
with healthier, more long-lived dogs. It will be easier to select for particular
physical traits such as body size or coat color, not only because we understand
the underlying genetic pathways, but because genetic tests are likely to be made
available as quickly as results are published. Finally, canine geneticists will finally
have a chance to develop an understanding of the genes that cause both breed-
specific behaviors (why do pointers point and herders herd?).
What is far less clear is whether we will come to understand what makes the
domestic dog unique to us among all the animals in the mammalian world. We
have domesticated dogs to the point that they display loyalty, friendship and
companionship. We seek their company and approval and bring them into our
homes, often as equal members of our family. We rejoice in their victories and
mourn their deaths, often as we celebrate or mourn our own children. Is the
genetics that defines this relationship within the dog, within ourselves, or both?
None of the studies proposed are likely to answer that question, and perhaps that
is okay. The comparative-genome projects of humans and dogs were designed
to bring about an understanding of our similarities and differences. Perhaps
scientists will have to be satisfied to understand that much, and leave as a
mystery the genetic basis of approval, adoration and loyalty. At least for me and
my dog, it's enough.