Collie or Pug? Study Finds the Genetic Code
By MARK DERR
Published: May 21, 2004
In a study that alters conventional wisdom and paves the way for a better understanding of canine behavior and evolution, scientists say they have found genetic variations that allow them to distinguish among 85 dog breeds and to identify an individual dog's breed with 99 percent accuracy.
Traditionally, appearance and a written pedigree have been used to define a dog's breed. But scientists had not been able to identify breed from DNA alone in more than a few cases until now.
"I was surprised that you could assign dogs to their breed with 99 percent accuracy," said Dr. Robert K. Wayne, an expert in canine evolution at the University of California, Los Angeles, who was not involved in the study. "That's pretty astounding."
More surprising to dog lovers might be some of the relationships among breeds that the research revealed. The German shepherd, for example, is closer genetically to mastiffs, boxers and other "guarding" dogs than to herding dogs. The fleet greyhound, Irish wolfhound, borzoi, or Russian wolfhound, and lumbering Saint Bernard count herding dogs among their closest kin. And the pharaoh hound and Ibizan hound, often called the oldest of breeds, are really recent constructions, as is the Norwegian elkhound.
The researchers emphasized that they had not yet found genes that account for differences in behavior and appearance among breeds - the mesmerizing stare, or "eye," of a border collie or the spotted coat of a Dalmatian but they now have a tool for studying genetic relationships among breeds that should help in that search.
"We can assign a dog to a breed, but we can't tell what behavior it will have," said Dr. Elaine Ostrander, a geneticist at the Fred Hutchinson Cancer Research Center and University of Washington. "There is huge variation in behavior between dogs within breeds."
Dr. Ostrander directed the study with a colleague, Dr. Leonid Kruglyak. The results were published in today's issue of Science magazine.
Dr. Ostrander said the research would help in the study of canine disease as well as human disease, because certain breeds of dogs are prone to some of the same genetic diseases as humans.
Dr. Wayne suggested that the research could someday be used to create a genetic gold standard for any given breed, or allow a veterinarian to identify a dog as purebred even without written papers.
The researchers sorted the 85 breeds into four major groups, based on genetic similarities. Three groups turned out to share physical characteristics, geographic origins or uses: guarding, herding and hunting. The fourth group consisted of ancient breeds that showed close genetic relationship to wolves. By contrast, the American Kennel Club sorts dogs primarily by use into sporting, working, herding, terrier, hound, toy and nonsporting groups.
Most of the 85 breeds fell into the hunting, herding or guarding groups and were created primarily in Europe or North America in the past 200 years to conform with the concept of purebred dogs, defined by appearance, behavior and closed gene pools, the researchers said. The oldest breeds tend to be most distinct, while the more recent creations, like retrievers, setters, pointers and hounds in the hunting group, are less well-defined genetically.
Because most breeds come from mixed ancestral stock, the differences among them result mainly from reproductive isolation, reliance on a limited number of "founders" and inbreeding to fix desired traits, Dr. Ostrander said.
The breeding practices have also left many purebred dogs susceptible to one or more of 350 genetic diseases.
The "ancient" group includes 14 geographically diverse breeds that are not usually grouped together, including the Asian chow chow, Shar-Pei, Shih Tzu, Pekingese, Tibetan terrier, Akita and Shiba Inu; the African basenji; the Middle Eastern Saluki; and the Siberian husky and Alaskan malamute.
The group's geographic and physical diversity surprised a number of geneticists not involved in the study and even the researchers themselves.
Dr. Kruglyak speculated that these breeds were directly descended from the first dogs and then spread out with their nomadic owners.
When and where dogs first separated from wolves is hotly disputed, with time estimates based on mitochondrial DNA evidence ranging from 15,000 to 135,000 years ago.
While generally praising the research, Dr. Wayne, who has proposed that dogs and wolves split 135,000 years ago, questioned the assignment of dogs to the ancient breed group, saying that any recent crossbreeding with wolves, as has happened with malamutes and Siberian huskies, could make a breed look primitive.
The researchers based their conclusions on DNA samples from 414 dogs representing 85 of the 152 breeds recognized by the American Kennel Club.
The research team drew genetic samples from 96 locations on the dog genome. Using variations in the DNA sequences from those locations, they were able to assign all but four of 414 dogs to their proper breed.
In one case they misidentified a beagle as a perro presa de Canario - a large, mastiff-like dog often used for guarding and fighting.
The study indirectly confirms and greatly expands on work published in 2003 by Dr. Mikko T. Koskinen, a Finnish geneticist, reporting success in using DNA to distinguish among five breeds.
(Note, these studies have been supported by the DCA Health & Welfare Trust Fund)
Genome Resources to Boost
Canines’ Role in Gene Hunts
The strict breeding practices that produce champion purebred dogs are proving a boon to geneticists. New results suggest that our pedigreed canine companions may be a major help in finding the genetic keys to common human afflictions such as cancer, diabetes, and mental disorders.
In one development, described on page 1160, Elaine Ostrander, a geneticist at the Fred Hutchinson Cancer Research Center in Seattle, Washington, and her colleagues show that purebred dogs should be more useful to genetic researchers than previously thought.
The team found that each breed has a unique genetic signature that will help geneticists decide which breeds will be most useful in revealing information about a disease.
Purebred dogs also may offer advantages in efficiency. At a genome meeting held last week at Cold Spring Harbor Laboratory in New York, Ostrander’s postdoc Nathan Sutter reported that dogs within a breed have less sequence variation than do humans. These findings should speed progress in understanding canine diseases, and that in turn “is likely to simplify the study of the corresponding diseases in humans,” says Jaime Modiano, a geneticist at the University of Colorado Health Sciences Center in Denver.
Another milestone reported at the meeting should also assist in the search for causes of disease. Kerstin Lindblad-Toh of the MIT Broad Institute in Cambridge, Massachusetts, and her colleagues outlined the initial analysis of the 2.4 billion bases of the dog genome. “A lot of researchers are primed to take that sequence and leap forward” to use dogs to study genetic diseases, says Francis Collins, director of the National Human Genome Research Institute, which supported the project.
Geneticists have been focusing on the dog as a possible model for gene searches because the species can help them circumvent a frequent problem with studies in humans.
When seeking the genes at fault in a disease, they find all too often that the trail goes cold because there aren’t enough members of affected families or isolated groups of people to pin down the genetic risk factors.
In contrast, dogs are “ideally suited because we have many breeds that are the equivalent of small populations,” says Gregory Acland, a geneticist who studies eye diseases at Cornell University in Ithaca, New York.
But to get the most out of canine gene hunts, Ostrander realized that she needed to know the degree of genetic differences among different breeds. She and graduate student Heidi Parker set out to determine these differences by identifying variations in 96 microsatellites—short pieces of unique sequence that serve as landmarks for gene seekers—in 414 dogs from 85 breeds. The work was far more extensive than previous studies and “has been needed for a long time,” says Carles Vilà, an evolutionary biologist at Uppsala University in Sweden.
The results are clear-cut. The researchers identified a set of microsatellites specific to each breed. Using the microsatellites, Parker, Ostrander, and the Hutch’s Leonid Kruglyak demonstrated that, with very few exceptions, they could group boxers with boxers, elkhounds with elkhounds, and so on. “It suggests a [DNA] gold standard that certifies a dog as [belonging to] a specific breed,” comments Robert Wayne, an evolutionary biologist at the University of California, Los Angeles. The work has also shed light on dog evolution, showing among other things that Asian spitzes are the most genetically distinct and thus the oldest breeds.
This confirms earlier evidence about the history of man’s best friend.
The findings should aid in tracking down disease genes, says Ostrander. She can now expand the search for a gene in one breed to other breeds shown to be related by their microsatellite compositions. Having a larger sample will make it easier to detect the mutation at fault. “This is what I see as the most powerful use of the data,” she notes.
The dog offers other advantages over humans for gene hunts, says Sutter. To find the mutated genes underlying complex diseases such as cancer, geneticists look for base changes along the DNA where the implicated gene seems to be. Initial analyses suggest that geneticists will need to gather about 400,000 base differences—called single nucleotide polymorphisms—in the human genome to begin to pin down a problematic gene implicated in a disease.
But as Sutter reported at the Cold Spring Harbor meeting, such gene tracking should be much easier in dogs. By incorporating genomic information from 20 dogs from each
of five breeds and the previously published poodle sequence (Science, 26 September 2003, p. 1898), he calculated that the job can be accomplished with just 30,000 SNPs.
At the same meeting, Lindblad-Toh described her progress sequencing the genome of a boxer named Tasha, chosen because the breed has very little genetic variation. Working with Ostrander and more than two dozen collaborators, Lindblad-Toh has sequenced enough DNA to cover the genome more than seven times over and expects that the consortium will put these data together into a high quality draft. Once that goes public, which should occur in the next few weeks, finding disease genes in dogs will be even easier.
Dog breeders should be proud.
Genetic Structure of the
Purebred Domestic Dog
Heidi G. Parker,1,2,3 Lisa V. Kim,1,2,4 Nathan B. Sutter,1,2
Scott Carlson,1 Travis D. Lorentzen,1,2 Tiffany B. Malek,1,3
Gary S. Johnson,5 Hawkins B. DeFrance,1,2
Elaine A. Ostrander,1,2,3,4* Leonid Kruglyak1,3,4,6
We used molecular markers to study genetic relationships in a diverse collection of 85 domestic dog breeds. Differences among breeds accounted for 30% of genetic variation. Microsatellite genotypes were used to correctly assign 99% of individual dogs to breeds. Phylogenetic analysis separated several breeds with ancient origins from the remaining breeds with modern European origins.
We identified four genetic clusters, which predominantly contained breeds with similar geographic origin, morphology, or role in human activities. These results provide a genetic classification of dog breeds and will aid studies of the genetics of phenotypic breed differences.
The domestic dog is a genetic enterprise unique in human history. No other mammal has enjoyed such a close association with humans over so many centuries, nor been so substantially shaped as a result. A variety of dog morphologies have existed for millennia, and reproductive isolation between them was formalized with the advent of breed clubs and breed standards in the mid–19th century. Since that time, the promulgation of the “breed barrier” rule—no dog may become a registered member of a breed unless both its dam and sire are registered members—has ensured a relatively closed genetic pool among dogs of each breed. At present, there are more than 400 described breeds, 152 of which are recognized by the American Kennel Club (AKC) in the United States (1). Over 350 inherited disorders have been described in the purebred dog population (2). Many of these mimic common human disorders and are restricted to particular breeds or groups of breeds as a result of aggressive in-breeding programs used to generate specific morphologies. We have previously argued that mapping genes associated with common diseases, including cancer, heart disease, epilepsy, blindness, and deafness, as well as genes underlying the striking diversity among breeds in morphology and behavior, will best be accomplished through elucidating and taking advantage of the population structure of modern breeds (3). Understanding the genetic relationships among breeds will also provide insight into the directed evolution of our closest animal companions.
Mitochondrial DNA analyses have been used to elucidate the relationship between the domestic dog and the wolf (4–6), but the evolution of mitochondrial DNA is too slow to allow inferences about relationships among modern dog breeds, most of which have existed for fewer than 400 years (1, 7, 8). One previous study showed that nuclear microsatellite loci could be used to assign dogs from five breeds to their breed of origin, demonstrating large genetic distances among these breeds (9). Another study used microsatellites to detect the relatedness of two breed pairs in a collection of 28 breeds but could not establish broader phylogenetic relationships among the breeds (10). The failure to find such relationships could reflect the properties of microsatellite loci (10), the limited number of breeds examined, or the analytical methods used in the study. Alternatively, it may reflect the complex structure in purebred dog populations, resulting from the recent origin of most breeds and the mixing of ancestral types in their creation. Here, we show that microsatellite typing of a diverse collection of 85 breeds, combined with phylogenetic analysis and modern genetic clustering methods (11, 12), allows the definition of related groups of breeds and that genetic relatedness among breeds often correlates with morphological similarity and shared geographic origin.
To assess the amount of sequence variation in purebred dogs, we first resequenced 19,867 base pairs of noncontiguous genomic sequence in 120 dogs representing 60 breeds.
We identified 75 single nucleotide polymorphisms (SNPs), with minor allele frequencies ranging from 0.4 to 48% (table S1). Fourteen of the SNPs were breed specific. When all dogs were considered as a single population, the observed nucleotide heterozygosity (13) was 8 104, essentially the same as that found for the human population (14, 15).
To further characterize genetic variation within and among breeds, we genotyped 96 microsatellite loci in 414 purebred dogs representing 85 breeds (five unrelated dogs that lacked any common grandparents were sampled from most breeds; table S2). We predicted that, because of the existence of breed barriers, dogs from the same breed would be more similar genetically than dogs from different breeds. To test this prediction, we estimated the proportion of genetic variation among individual dogs that could be attributed to breed membership. An analysis of molecular variance (16) in the microsatellite data showed that variation among breeds accounts for more than 27% of total genetic variation. Similarly, the average genetic distance between breeds calculated from the SNP data is FST= 0.33. These observations are consistent with previous reports that analyzed fewer dog breeds (9, 10), confirming the prediction that breed barriers have led to strong genetic isolation among breeds, and are in marked contrast to the much lower genetic differentiation (typically in the range of 5 to 10%) found among human populations (17, 18). Variation among breeds in dogs is on the high end of the range reported for domestic livestock populations (19, 20). Strong genetic differentiation among dog breeds suggests that breed membership could be determined from individual dog genotypes (9). To test this hypothesis, we first applied a Bayesian model–based clustering algorithm, implemented in the program structure (11, 12, 21), to the microsatellite data. The algorithm attempts to identify genetically distinct subpopulations on the basis of patterns of allele frequencies. We applied structure to overlapping subsets of 20 to 22 breeds at a time (22) and observed that most breeds formed distinct clusters consisting solely of all the dogs from that breed (Fig. 1A). Dogs in only four breeds failed to consistently cluster with others of the same breed: Perro de Presa Canario, German Shorthaired Pointer, Australian Shepherd, and Chihuahua. In addition, six pairs of breeds clustered together in the majority of runs. These pairings—Alaskan Malamute and Siberian Husky, Belgian Sheepdog and Belgian Tervuren, Collie and Shetland Sheepdog, Greyhound and Whippet, Bernese Mountain Dog and Greater Swiss Mountain Dog, and Bullmastiff and Mastiff—are all expected on the basis of known breed history. To test whether these closely related breed pairs were nonetheless genetically distinct, we applied structure to each of these clusters. In all but one case, the clusters separated into two populations corresponding to the individual breeds (Fig. 1B). The single exception was the cluster containing Belgian Sheepdogs and Belgian Tervurens. The European and Japanese Kennel Clubs classify these as coat color and length varieties of a single breed (23, 24), and although the AKC recognizes them as distinct breeds, the breed barrier is apparently too recent or insufficiently strict to have resulted in genetic differentiation.
We next examined whether a dog could be assigned to its breed on the basis of genotype data alone. Using the direct assignment method (25) with a leave-one-out analysis, we were able to assign 99% of individual dogs to the correct breed. Only 4 dogs out of 414 were assigned incorrectly: one Beagle as a Perro de Presa Canario, one Chihuahua as a Cairn Terrier, and two German Shorthaired Pointers as a Kuvasz and a Standard Poodle. All four errors involved breeds that did not form single-breed clusters in the structure analysis.
Having demonstrated that modern dog breeds are distinct genetic units, we next sought to define broader genetic relationships among the breeds. We first used standard neighbor-joining methods to build a majority rule consensus tree of breeds (Fig. 2), with distances calculated using the chord distance measure (26), which does not assume a particular mutation model and is thought to perform well for closely related taxa (27). The tree was rooted using wolf samples. The deepest split in the tree separated four Asian spitz-type breeds, and within this branch the Shar-Pei split first, followed by the Shiba Inu, with the Akita and Chow Chow grouping together. The second split separated the Basenji, an ancient African breed. The third split separated two Arctic spitz-type breeds, the Alaskan Malamute and Siberian Husky, and the fourth split separated two Middle Eastern sight hounds, the Afghan and Saluki, from the remaining breeds.
The first four splits exceeded the majority rule criterion, appearing in more than half of the bootstrap replicates. In contrast, the remaining breeds showed few consistent phylogenetic relationships, except for close groupings of five breed pairs that also clustered together in the structure analysis, one new pairing of the closely related West Highland White Terrier and Cairn Terrier, and the significant grouping of three Asian companion breeds of similar appearance, the Lhasa Apso, Shih Tzu, and Pekingese (fig. S1). A close relationship among these three breeds was also observed in the structure analysis, with at least two of the three clustering together in a majority of runs. The flat topology of the tree likely reflects a largely common founder stock and occurrence of extensive gene flow between phenotypically dissimilar dogs before the advent of breed clubs and breed barrier rules. In addition, it probably reflects the fact that some historically older breeds that died out during the famines, depressions, and wars of the 19th and 20th centuries have been recreated with the use of stock from phenotypically similar or historically related dogs.
Whereas the phylogenetic analysis showed separation of several breeds with ancient origins from a large group of breeds with presumed modern European origins, additional subgroups may be present within the latter group that are not detected by this approach for at least two reasons (28). First, the true evolutionary history of dog breeds is not well represented by the bifurcating tree model assumed by the method because existing breeds were mixed to create new breeds (a process that continues today). Second, methods based on genetic distance matrices lose information by collapsing all genotype data for pairs of breeds into a single number. The clustering algorithm implemented in structure was explicitly designed to overcome these limitations (11, 12, 28) and has been applied to infer the genetic structure of several species (17, 28, 29). We therefore ran structure on the entire data set using increasing values of K (the number of subpopulations the program attempts to find) to identify ancestral source populations. In this analysis, a modern breed could closely mirror a single ancestral population or represent a mixture of two or more ancestral types.
At K=2, one cluster was anchored by the first seven breeds to split in the phylogenetic analysis, whereas the other cluster contained the large number of breeds with a flat phylogenetic topology (Fig. 3A). Five runs of the program produced nearly identical results, with a similarity coefficient (17) of 0.99 across runs. Seven other breeds share a sizeable fraction of their ancestry with the first cluster. These fourteen breeds all date to antiquity and trace their ancestry to Asia or Africa. When a diverse set of wolves from eight different countries was included in the analysis, they fell entirely within this cluster (Fig. 3B). The branch leading to the wolf outgroup also fell within this group of breeds in the phylogenetic analysis (Fig. 2).
At K= 3, additional structure was detected that was not readily apparent from the phylogenetic tree. The new third cluster consisted primarily of breeds related in heritage and appearance to the Mastiff and is anchored by the Mastiff, Bulldog, and Boxer, along with their close relatives, the Bullmastiff, French Bulldog, Miniature Bull Terrier, and Perro de Presa Canario.
Also included in the cluster are the Rottweiler, Newfoundland, and Bernese Mountain Dog, large breeds that are reported to have gained their size from ancient Mastiff-type ancestors. Less expected is the inclusion of the German Shepherd Dog. The exact origins of this breed are unknown, but our results suggest that the years spent as a military and police dog in the presence of working dog types, such as the Boxer, are responsible for shaping the genetic background of this popular breed. Three other breeds showed partial and inconsistent membership in this cluster across structure runs (fig. S2), which lowered the similarity coefficient to 0.84.
At K= 4, a fourth cluster was observed, which included several breeds used as herding dogs: Belgian Sheepdog, Belgian Tervuren, Collie, and Shetland Sheepdog. The Irish Wolfhound, Greyhound, Borzoi, and Saint Bernard were also frequently assigned to this cluster. Although historical records do not suggest that these dogs were ever used to herd livestock, our results suggest that these breeds are either progenitors to or descendants of herding types. The breeds in the remaining cluster are primarily of relatively recent European origins and are mainly different types of hunting dogs: scent hounds, terriers, spaniels, pointers, and retrievers. Clustering at K 4 showed a similarity coefficient of 0.61, reflecting similar cluster membership assignments for most breeds but variable assignments for other breeds across runs (fig. S2).
At K= 5, the similarity coefficient dropped to 0.26 and no additional consistent subpopulations were inferred, suggesting a lack of additional high-level substructure in the sampled purebred dog population.
click for tables
Our results paint the following picture of the relationships among domestic dog breeds. Different breeds are genetically distinct, and individuals can be readily assigned to breeds on the basis of their genotypes. This level of divergence is surprising given the short time since the origin of most breeds from mixed ancestral stocks and supports strong reproductive isolation within each breed as a result of the breed barrier rule.
Our results support at least four distinct breed groupings representing separate “adaptive radiations.”
A subset of breeds with ancient Asian and African origins splits off from the rest of the breeds and shows shared patterns of allele frequencies.
At first glance, it is surprising that a single genetic cluster includes breeds from Central Africa (Basenji), the Middle East (Saluki and Afghan), Tibet (Tibetan Terrier and Lhasa Apso), China (Chow Chow, Pekingese, Shar-Pei, and Shi Tzu), Japan (Akita and Shiba Inu), and the Arctic (Alaskan Malamute, Siberian Husky, and Samoyed). However, several researchers have hypothesized that early pariah dogs originated in Asia and migrated with nomadic human groups both south to Africa and north to the Arctic, with subsequent migrations occurring throughout Asia (5, 6, 30). This cluster includes Nordic breeds that phenotypically resemble the wolf, such as the Alaskan Malamute and Siberian Husky, and shows the closest genetic relationship to the wolf, which is the direct ancestor of the domestic dog.
Thus, dogs from these breeds may be the best living representatives of the ancestral dog gene pool. It is notable that several breeds commonly believed to be of ancient origin, such as the Pharaoh Hound and Ibizan Hound, are not included in this group.
These are often thought to be the oldest of all dog breeds, descending directly from the ancient Egyptian dogs drawn on tomb walls more than 5000 years ago. Our results indicate, however, that these two breeds have been recreated in more recent times from combinations of other breeds. Thus, although their appearance matches the ancient Egyptian sight hounds, their genomes do not. Similar conclusions apply to the Norwegian Elkhound, which clusters with modern European breeds rather than with the other Arctic dogs, despite reports of direct descent from Scandinavian origins more than 5000 years ago (1, 24).
The large majority of breeds appears to represent a more recent radiation from shared European stock. Although the individual breeds are genetically differentiated, they appear to have diverged at essentially the same time. This radiation probably reflects the proliferation of distinct breeds from less codified phenotypic varieties after the introduction of the breed concept and the creation of breed clubs in Europe in the 1800s. A more sensitive cluster analysis was able to discern additional genetic structure of three subpopulations within this group. One contains Mastiff-like breeds and appears to reflect shared morphology derived from a common ancestor.
Another includes Shetland Sheepdog, the two Belgian Sheepdogs, and Collie, and may reflect shared ancestral herding behavior. The remaining population is dominated by a proliferation of breeds dedicated to various aspects of the hunt. For these breeds, historical and breed club records suggest highly intertwined bloodlines, consistent with our results.
Dog breeds have traditionally been grouped on the basis of their roles in human activities, physical phenotypes, and historical records. Here, we defined an independent classification based on patterns of genetic variation. This classification supports a subset of traditional groupings and also reveals previously unrecognized connections among breeds. An accurate understanding of the genetic relationships among breeds lays the foundation for studies aimed at uncovering the complex genetic basis of breed differences in morphology, behavior, and disease susceptibility.
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31. Supported by the Burroughs Wellcome Innovation
Award (E.A.O. and L.K.), grants from the AKC–Canine
Health Foundation (G.S.J.), NIH training grant T32
HG00035 (H.G.P.), and a postdoctoral fellowship
from the Waltham Foundation (N.B.S.). E.A.O. also
acknowledges support from K05 CA90754. L.K. is a
James S. McDonnell Centennial Fellow. We thank the
many dog owners, researchers, and breeders who
provided DNA samples for this work, especially G.
Brewer, C. Gaiser, and K. Murphy; D. Lynch, A. Ziska,
C. Ramirez, M. Langlois, and D. Akey for assistance
with sample collection; M. Stephens for advice and
assistance with the program structure; K. Markianos
and J. Akey for helpful discussions; M. Eberle for
computing assistance; H. Coller, E. Giniger, R. Wayne,
and three anonymous reviewers for comments on the
manuscript; and the AKC and C. Jierski for use of the
canine artwork included in Fig. 3.
Supporting Online Material
Materials and Methods
Figs. S1 and S2
Tables S1 to S5
2 March 2004; accepted 21 April 2004