Genome sequence, comparative analysis and haplotype structure of the domestic dog

Abstract

Here we report a high-quality draft genome sequence of the domestic dog (Canis familiaris), together with a dense map of single nucleotide polymorphisms (SNPs) across breeds. The dog is of particular interest because it provides important evolutionary information and because existing breeds show great phenotypic diversity for morphological, physiological and behavioural traits. We use sequence comparison with the primate and rodent lineages to shed light on the structure and evolution of genomes and genes. Notably, the majority of the most highly conserved non-coding sequences in mammalian genomes are clustered near a small subset of genes with important roles in development. Analysis of SNPs reveals long-range haplotypes across the entire dog genome, and defines the nature of genetic diversity within and across breeds. The current SNP map now makes it possible for genome-wide association studies to identify genes responsible for diseases and traits, with important consequences for human and companion animal health.

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Genome sequence, comparative analysis
and haplotype structure of the domestic
dog
Kerstin Lindblad-Toh1, Claire M Wade1,2, Tarjei S. Mikkelsen1,3, Elinor K. Karlsson1,4, David B. Jaffe1,
Michael Kamal1, Michele Clamp1, Jean L. Chang1, Edward J. Kulbokas III1, Michael C. Zody1, Evan Mauceli1,
Xiaohui Xie1, Matthew Breen5, Robert K. Wayne6, Elaine A. Ostrander7, Chris P. Ponting8, Francis Galibert9,
Douglas R. Smith10, Pieter J. deJong11, Ewen Kirkness12, Pablo Alvarez1, Tara Biagi1, William Brockman1,
Jonathan Butler1, Chee-Wye Chin1, April Cook1, James Cuff1, Mark J. Daly1,2, David DeCaprio1, Sante Gnerre1,
Manfred Grabherr1, Manolis Kellis1,13, Michael Kleber1, Carolyne Bardeleben6, Leo Goodstadt8, Andreas Heger8,
Christophe Hitte9, Lisa Kim7, Klaus-Peter Koepfli6, Heidi G. Parker7, John P. Pollinger6, Stephen M. J. Searle14,
Nathan B. Sutter7, Rachael Thomas5, Caleb Webber8, Broad Institute Genome Sequencing Platform*
& Eric S. Lander1,15
Here we report a high-quality draft genome sequence of the domestic dog (Canis familiaris), together with a dense map
of single nucleotide polymorphisms (SNPs) across breeds. The dog is of particular interest because it provides important
evolutionary information and because existing breeds show great phenotypic diversity for morphological, physiological
and behavioural traits. We use sequence comparison with the primate and rodent lineages to shed light on the structure
and evolution of genomes and genes. Notably, the majority of the most highly conserved non-coding sequences in
mammalian genomes are clustered near a small subset of genes with important roles in development. Analysis of SNPs
reveals long-range haplotypes across the entire dog genome, and defines the nature of genetic diversity within and
across breeds. The current SNP map now makes it possible for genome-wide association studies to identify genes
responsible for diseases and traits, with important consequences for human and companion animal health.
Man’s best friend, Canis familiaris, occupies a special niche in
genomics. The unique breeding historyof the domesticdog provides
an unparalleled opportunity to explore the genetic basis of disease
susceptibility, morphological variation and behavioural traits. The
position of the dog within the mammalian evolutionary tree also
makes it an important guide for comparative analysis of the human
genome.
The history of the domestic dog traces back at least 15,000 years,
andpossiblyasfarbackas100,000years,toitsoriginaldomestication
from the grey wolf in East Asia1–4. Dogs evolved through a mutually
beneficial relationship with humans, sharing living space and food
sources. In recent centuries, humans have selectively bred dogs that
excel at herding, hunting and obedience, and in this process have
createdbreedsrichinbehavioursthatbothmimichumanbehaviours
and support our needs. Dogs have also been bredfordesiredphysical
characteristics such as size, skull shape, coat colour and texture5,
producing breeds with closely delineated morphologies. This evolu-
tionary experiment has produced diverse domestic species, harbour-
ingmoremorphologicaldiversity thanexistswithintheremainderof
the family Canidae6.
As a consequence of these stringent breeding programmes and
periodic population bottlenecks (for example, during the World
Wars), many of the ,400 modern dog breeds also show a high
prevalence of specific diseases, including cancers, blindness, heart
disease, cataracts, epilepsy, hip dysplasia and deafness7,8. Most of
these diseases arealso commonly seen in the human population, and
clinical manifestations in the two species are often similar9. The high
prevalence of specific diseases within certain breeds suggests that a
limited number of loci underlie each disease, making their genetic
dissection potentially more tractable in dogs than in humans10.
Genetic analysis of traits in dogs is enhanced by the close
relationship between humans and canines in modern society.
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1Broad Institute of Harvard and MIT, 320 Charles Street, Cambridge, Massachusetts 02141, USA.2Center for Human Genetic Research, Massachusetts General Hospital,
185 Cambridge Street, Boston, Massachusetts 02114, USA.3Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts
02139, USA.4Program in Bioinformatics, Boston University, 44 Cummington Street, Boston, Massachusetts 02215, USA.5Department of Molecular Biomedical Sciences, College
of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, North Carolina 27606, USA.6Department of Ecology and Evolutionary Biology,
University of California, Los Angeles, California 90095, USA.7National Human Genome Research Institute, National Institutes of Health, 50 South Drive, MSC 8000, Building
50, Bethesda, Maryland 20892-8000, USA.8MRC Functional Genetics, University of Oxford, Department of Human Anatomy and Genetics, South Parks Road, Oxford OX1 3QX,
UK.9UMR 6061 Genetique et Developpement, CNRS—Universite ´ de Rennes 1, Faculte ´ de Me ´decine, 2, Avenue Leon Bernard, 35043 Rennes Cedex, France.10Agencourt
Bioscience Corporation, 500 Cummings Center, Suite 2450, Beverly, Massachusetts 01915, USA.11Children’s Hospital Oakland Research Institute, 5700 Martin Luther King Jr
Way, Oakland, California 94609, USA.12The Institute for Genomic Research, Rockville, Maryland 20850, USA.13Computer Science and Artificial Intelligence Laboratory,
Cambridge, Massachusetts 02139, USA.14The Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.15Whitehead
Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.
*A list of participants and affiliations appears at the end of the paper.
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Through the efforts of the American Kennel Club (AKC) and similar
organizations worldwide, extensive genealogies are easily accessible
for most purebred dogs. With the exception of human, dog is the
most intensely studied animal in medical practice, with detailed
family history and pathology data often available8. Using genetic
resources developed over the past 15 years11–16, researchers have
already identified mutations in genes underlying ,25 mendelian
diseases17,18. There are also growing efforts to understand the genetic
basis of phenotypic variation such as skeletal morphology10,19.
The dog is similarly important for the comparative analysis of
mammalian genome biology and evolution. The four mammalian
genomes that have been intensely analysed to date (human20–22,
chimpanzee23, mouse24and rat25) represent only one clade
(Euarchontoglires) out of the four clades of placental mammals.
The dog represents the neighbouring clade, Laurasiatheria26. It thus
serves as an outgroup to the Euarchontoglires and increases the total
branch length of the current tree of fully sequenced mammalian
genomes, thereby providing additional statistical power to search for
conserved functional elements in the human genome24,27–33. It also
helps us to draw inferences about the common ancestor of the two
clades, called the boreoeutherian ancestor, and provides a bridge to
the two remaining clades (Afrotheria and Xenarthra) that should be
helpfulforanchoringlow-coveragegenomesequencecurrentlybeing
produced from species such as elephant and armadillo28.
Here we report a high-quality draft sequence of the dog genome
covering ,99% of the euchromatic genome. The completeness,
nucleotide accuracy, sequence continuity and long-range connec-
tivity are extremely high, exceeding the values calculated for
the recent draft sequence of the mouse genome24and reflecting
improved algorithms, higher-quality data, deeper coverage and
intrinsic genome properties. We have also created a tool for the
formal assessment of assembly accuracy, and estimate that .99% of
the draft sequence is correctly assembled.
We also report an initial compendium of SNPs for the dog
population, containing .2.5million SNPs derived primarily from
partial sequence comparison of 11 dog breeds to a reference
sequence. We characterized the polymorphism rate of the SNPs
across breeds and the long-range linkage disequilibrium (LD) of
the SNPs within and across breeds.
We have analysed these data to study genome structure, gene
evolution, haplotypestructureandphylogenetics ofthedog.Ourkey
findings include:
†The evolutionary forces molding the mammalian genome differ
among lineages, with the average transposon insertion rate being
lowest in dog, the deletion rate being highest in mouse and the
nucleotide substitution rate being lowest in human.
†Comparison between human and dog shows that ,5.3% of the
human genome contains functional elements that have been under
purifying selection in both lineages. Nearly all of these elements are
confined to regions that have been retained in mouse, indicating
that they represent a common set of functional elements across
mammals.
†Fiftypercentofthemosthighlyconservednon-codingsequencein
the genome shows striking clustering in ,200 gene-poor regions,
most of which contain genes with key roles in establishing or
maintaining cellular identity, such as transcription factors or axon
guidance receptors.
†Sets of functionally related genes show highly similar patterns of
evolution in the human and dog lineages. This suggests that we
should be careful about interpreting accelerated evolution in
humanrelativetomouseasrepresentinghuman-specificinnovations
(for example, in genes involved in brain development), because
comparable acceleration is often seen in the dog lineage.
†Analysis across the entire genome of the sequenced boxer and
across 6% of the genome in ten additional breeds shows that linkage
disequilibrium (LD) within breeds extends over distances of several
megabases, but LD across breeds only extends over tens of kilobases.
These LD patterns reflect two principal bottlenecks in dog history:
early domestication and recent breed creation.
†Haplotypes within breeds extend over long distances, with ,3–5
alleles at each locus. Portions of these haplotypes, as large as
100kilobases (kb), are shared across multiple breeds, although they
are present at widely varying frequencies. The haplotype structure
suggests that genetic risk factors may be shared across breeds.
†The current SNP map has sufficient density and an adequate
within-breed polymorphism rate (,1/900 base pairs (bp) between
breeds and ,1/1,500bp within breeds) to enable systematic associa-
tion studies to map genes affecting traits of interest. Genotyping of
,10,000 SNPs should suffice for most purposes.
†The genome sequence can be used to select a small collection of
rapidly evolving sequences, which allows nearly complete resolution
of the evolutionary tree of nearly all living species of Canidae.
Generating a draft genome sequence
We sequenced the genome of a female boxer using the whole-
genomeshotgun(WGS)approach22,24(seeMethodsandSupplemen-
tary Table S1). A total of 31.5 million sequence reads, providing
,7.5-fold sequence redundancy, were assembled with an improved
version of the ARACHNE program34, resulting in an initial assembly
(CanFam1.0) used for much of the analysis below, and an updated
assembly (CanFam2.0) containing minor improvements (Table 1
and Supplementary Table S2).
Genome assembly. The recent genome assembly spans a total
distance of 2.41Gb, consisting of 2.38Gb of nucleotide sequence
with theremaining1%in capturedgaps.Theassembly has extremely
highcontinuity.TheN50contigsizeis180kb(thatis,halfofallbases
reside in a contiguous sequence of 180kb or more) and the N50
supercontig size is 45.0Mb (Table 1). In particular, this means that
most genes should contain no sequence gaps and that most canine
chromosomes (mean size 61Mb) have nearly all of their sequence
ordered and oriented within one or two supercontigs (Supplemen-
taryTableS2).Notably,thesequencecontigsare,50-foldlargerthan
the earlier survey sequence of the standard poodle16.
Theassemblywasanchoredtothecaninechromosomesusingdata
from both radiation hybrid and cytogenetic maps11,13,14. Roughly
97% of the assembled sequence was ordered and oriented on the
chromosomes, showing an excellent agreement with the two maps.
There were only three discrepancies, which were resolved by obtain-
ing additional fluorescence in situ hybridization (FISH) data from
the sequenced boxer. The 3% of the assembly that could not be
anchored consists largely of highly repetitive sequence, including
eight supercontigs of 0.5–1.0Mb composed almost entirely of
satellite sequence.
The nucleotide accuracy and genome coverage of the assembly is
high (Supplementary Table S3). Of the bases in the assembly,
98% have quality scores exceeding 40, corresponding to an error
rate of less than 1024and comparable to the standard for the
finished human sequence35. When we directly compared the assem-
bly to 760kb of finished sequence (in regions where the boxer is
Table 1 | Assembly statistics for CanFam1.0 and 2.0
CanFam1.0CanFam2.0
N50 contig size
N50 supercontig size
Assembly size (total bases)
Number of anchored supercontigs
Percentage of genome in anchored supercontigs
Sequence in anchored bases
Percentage of assembly in gaps
Estimated genome size*
Percentage of assembly in ‘certified regions’,
without assembly inconsistency
123kb
41.2Mb
2.360Gb
86
96
2.290Gb
0.9
2.411Gb
99.3
180kb
45.0Mb
2.385Gb
87
97
2.309Gb
0.8
2.445Gb
99.6
*Includes anchored bases, spanned gaps (21Mb in CanFam1.0, 18Mb in CanFam2.0) and
centromeric sequence (3Mb for each chromosome).
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homozygous, to eliminate differences attributable to polymorphisms;
seebelow),wefoundthatthedraftgenomesequencecovers99.8%of
the finished sequence and that bases with quality scores exceeding
40haveanempiricalerrorrateof2 £ 1025(SupplementaryTableS3).
Explainingthehighsequencecontinuity.Thedoggenomeassembly
has superior sequence continuity (180kb) than the WGS assemblyof
the mouse genome (25kb) obtained several years ago24. At least three
factors contribute to the higherconnectivityofthe dog assembly (see
Supplementary Information). First, we used a new version of
ARACHNE with improved algorithms. Assembling the dog genome
with the previous software version decreased N50 contig size from
180kb to 61kb, and assembling the mouse genome with the new
version increased N50 contig size from 25kb to 35kb. Second, the
amount of recently duplicated sequence is roughly twofold lower in
dog than mouse (Supplementary Table S4); this improves contiguity
because sequence gaps in both organisms tend to occur in recently
duplicated sequence. Third, the dog sequence data has both higher
redundancy (7.5-fold versus 6.5-fold) and higher quality (in terms of
read length, pairing rate and tight distribution of insert sizes) com-
paredwith mouse. The contigsizefor thedog genomedropsby about
32% when the data redundancy is decreased from 7.5-fold to 6.5-
fold. A countervailing influence is that the dog genome contains
polymorphism, whereas the laboratory mouse is completely inbred.
Assembly certification. Although ‘quality scores’ have been devel-
oped to indicate the nucleotide accuracy of a draft genome
sequence36, no analogous measures have been developed to reflect
the long-range assembly accuracy. We therefore sought to develop
such a measure on the basis of two types of internal inconsistencies
(see Supplementary Information). The first is haplotype incon-
sistency,involvingclearevidenceofthreeormoredistincthaplotypes
within an assembled region from a single diploid individual. The
second is linkage inconsistency, involving aclusterof reads for which
the placement of the paired-end reads is illogical. This includes
cases in which: (1) one end cannot be mapped to the region, (2) the
linkage relationships are inconsistent with the sequence within
contigs, or (3) distance constraints imply overlap between non-
overlapping sequence contigs. The linkage inconsistency tests are
most powerful when read pairs are derived from clone libraries
with tight constraints on insert size. A region of assembly is
defined as ‘certified’ if it is free of inconsistencies, and is otherwise
‘questionable’.
Approximately 99.6% of the assembly resides in certified regions,
with the N50 size of certified regions being ,12Mb or about one-
fifth of a chromosome. The remaining questionable regions are
typically small (most are less than 40kb), although there are a
handful of regions of several hundred kilobases (Supplementary
Fig. S1 and Supplementary Tables S5, S6). The questionable regions
typically contain many inconsistencies, probably reflecting mis-
assembly or overcollapse owing to segmental duplication. Chromo-
somes 2, 11 and 16 have 1.0–2.0% of their sequence in questionable
regions. The certified and questionable regions are annotated in
the public release of the dog genome assembly. With the concept
of assembly certification, the scientific community can have
appropriate levels of confidence in the draft genome sequence.
Genome landscape and evolution
Our understanding of the evolutionary processes that shape
mammalian genomes has greatly benefited from the comparative
analysis of sequenced primate21,23and rodent24,25genomes. However,
the rodent genome is highly derived relative to that of the common
ancestor of the eutherian mammals. As the first extensive sequence
from an outgroup to the cladethatincludes primates and rodents, the
dog genome offers a fresh perspective on mammalian genome evol-
ution. Accordingly, we examined the rates and correlations of large-
scale rearrangement, transposon insertion, deletion and nucleotide
divergence across three major mammalian orders (primates, rodents
and carnivores).
Conserved synteny and large-scale rearrangements. We created
multi-species synteny maps fromanchors of unique, unambiguously
aligned sequences (see Supplementary Information), showing
regions of conserved synteny among dog, human, mouse and rat
genomes. Approximately 94% of the dog genome lies in regions of
conserved synteny with the three other species (Supplementary
Figs S2–S4 and Supplementary Table S7).
Given a pair of genomes, we refer to a ‘syntenic segment’ as a
region that runs continuously without alterations of order and
orientation, and a ‘syntenic block’ as a region that is contiguous in
two genomes but may have undergone internal rearrangements.
Syntenic breakpoints between blocks reflect primarily interchromo-
somal exchanges, and breakpoints between syntenic segments reflect
intrachromosomal rearrangements. In the analysis below, we focus
on syntenic segments of at least 500kb.
We identified a total of 391 syntenic breakpoints across dog,
human, mouse and rat genomes (Fig. 1 and Supplementary
Figs S2, S5). With data for multiple species, it is possible to assign
events to specific lineages (Fig. 1 and Supplementary Table S8). We
counted the total number of breakpoints along the human, dog,
mouse and rat lineages, with the values for each rodent lineage
reflecting all breakpoints since the common ancestor with human
(Fig. 1). The total number of breakpoints in the human lineage is
substantiallysmallerthaninthedog,mouseorratlineages(83versus
100, 161 or 176, respectively). However, there are more intra-
chromosomal breakpoints in the human lineage than in dog (52
versus 33).
Although the overall level of genomic rearrangement has been
much higher in rodent than in human, comparison with dog shows
that there are regions where the opposite is true. In particular, of the
many intrachromosomal rearrangements previously observed
between human chromosome 17 and the orthologous mouse
Figure 1 | Conserved synteny among the human, dog, mouse and rat
genomes. a, Diagram of syntenic blocks (.500kb) along dog
chromosomes (Chr) 15, 16 and 31, with colours indicatingthe chromosome
containing the syntenic region in other species. Synteny breakpoints were
assigned to one of five lineages: dog (D), human (H), mouse (M), rat (R) or
the common rodent ancestor (Ro). b, Lineage-specific intrachromosomal
and interchromosomal breaks displayed on phylogenetic trees.
Intrachromosomal breaks are seen more frequently in the human lineage
than in mouse and rat, whereas interchromosomal breaks are somewhat
more common in dog and considerably more common in rodents than in
humans.
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sequence24, most have occurred in the human lineage (see Supple-
mentary Information). Human chromosome 17 is rich in segmental
duplications and gene families21, which may contribute to its
genomic fragility37,38.
Genomic insertion and deletion. The euchromatic genome of the
dog is ,150Mb smaller than in mouse, and ,500Mb smaller than
in human. The smaller total size is reflected at the local level, with
100-kb blocks of conserved synteny in dog corresponding to regions
for which the median size is ,3% larger in mouse and ,15% larger
in human.
To understand the balance of forces that determine genome size,
we studied the alignments of the human, mouse and dog genomes
(Fig. 2). In particular, we identified the lineage-specific interspersed
repeats within each genome, which consist of particular families of
short interspersed elements (SINEs), long interspersed elements
(LINEs) and other transposable elements that are readily recognized
by sequence analysis (Supplementary Tables S9, S10). The remaining
sequence was annotated as ‘ancestral’, consisting of both ancestral
unique sequence and ancestral repeat sequence; these two categories
were combined because the power to recognize ancient transposon-
derived sequences degrades with repeat age, particularly in the
rapidly diverging mouse lineage24.
This comparative analysis indicates that different forces account
for the smaller genome sizes in dog and mouse relative to human.
Thesmallersizeofthedoggenomeisprimarilyduetothepresenceof
substantially less lineage-specific repeat sequence in dog (334Mb)
than in human (609Mb) or mouse (954Mb). This reflects a lower
activity of endogenous retroviral and DNA transposons (,26,000
extant copies in dog versus ,183,000 in human), as well as the fact
that the SINE element in dog is smaller than in human (although of
similar length to that in mouse). As a consequence, the total
proportion of repetitive elements (both lineage-specific and ances-
tral) recognizable in the genome is lower for dog (34%) than for
mouse (40%) or human (46%). In contrast, the smaller size of the
mouse genome isprimarilydue to ahigherdeletion rate. Specifically,
the amount of extant ‘ancestral sequence’ is much lower in mouse
(1,474Mb) than in human (2,216Mb) ordog (1,997Mb). Assuming
an ancestral genome size of 2.8Gb (ref. 24) and also that deletions
occur continuously, we suggest that the rate of genomic deletion in
therodentlineagehasbeenapproximately2.5-foldhigherthaninthe
dog and human lineages (see Supplementary Information). As a
consequence, the human genome shares ,650Mb more ancestral
sequence with dog than with mouse, despite our more recent
common ancestor with the latter.
Active SINE family. Despite its relatively low proportion of trans-
posable element-derivedsequence,thedoggenomecontainsahighly
activecarnivore-specificSINEfamily(definedasSINEC_Cf;RepBase
release 7.11)16. The element is so active that many insertion sites are
still segregating polymorphisms that have not yet reached fixation.
Of ,87,000 young SINEC_Cf elements (defined by low divergence
fromtheconsensussequence),nearly8%areheterozygouswithinthe
draft genome sequence of the boxer. Moreover, comparison of the
boxer and standard poodle genome sequences reveals more than
10,000 insertion sites that are bimorphic, with thousands more
certain to be segregating in the dog population16,39. In contrast, the
number of polymorphic SINE insertions in the human genome is
estimated to be fewer than 1,000 (ref. 40).
The biological effect of these segregating SINE insertions is
unknown. SINE insertions can be mutagenic through direct disrup-
tion of coding regions or through indirect effects on regulation and
processing of messenger RNAs39. Such SINE insertions have already
been shown to be responsible for two diseases in dog: narcolepsy
and centronuclear myopathy41,42. It is conceivable that the genetic
variation resulting from these segregating SINE elements has pro-
vided important raw material for the selective breeding programmes
that have produced the wide phenotypic variations among modern
dog breeds16,43.
Sequence composition. The human and mouse genomes differ
markedly in sequence composition, with the human genome having
slightly lower average GþC content (41% versus 42% in mouse) but
much greater variation across the genome. The dog genome closely
resembles the human genome in its distribution of GþC content
(Fig. 3a; Spearman’s rho ¼ 0.85 for dog–human and 0.76 for dog–
mousecomparisons),evenifweconsideronlynucleotidesthatcanbe
aligned across all three species (Supplementary Fig. S6). The wider
distribution of GþC content in human and dog is thus likely to
reflect the boreoeutherian ancestor44,45, with the more homogeneous
composition in rodents having arisen primarily through lineage-
specific changes in substitution patterns46,47rather than deletion of
sequences with high GþC content.
Rate of nucleotide divergence. We estimated the mean nucleotide
divergence rates in 1-Mb windows along the dog, human and mouse
lineages on the basis of alignments of all ancestral repeats, using the
consensus sequence for the repeats as a surrogate outgroup (Fig. 3b;
see also Supplementary Information).
The dog lineage has diverged more rapidly than the human
lineage(medianrelativedivergencerateof1.18,longer branchlength
in 95% of windows), but at only half the rate of the mouse lineage
(median relative rate of 0.48, shorter branch length in 100% of
windows). The absolute divergence rates are somewhat sensitive to
the evolutionary model used and the filtering of alignment artefacts
(data not shown), but the relative rates appear to be robust and are
consistent with estimates from smaller sequence samples with
multiple outgroups28,48,49. The lineage-specific divergence rates
(human , dog , mouse) are probably explained by differences
in metabolic rates50,51or generation times52,53, but the relative
contributions of these factors remain unclear49.
Correlation in nucleotide divergence. As seen in other mammalian
genomes23–25, the average nucleotide divergence rate across 1-Mb
windows varies significantly across the dog genome (coefficient of
variation 0.11, compared with 0.024 expected under a uniform
distribution). This regional variation shows significant correlation
inorthologouswindowsacrossthedog,humanandmousegenomes,
but the strength of the correlation seems to decrease with total
branchlength(pair-wisecorrelationfororthologous1-Mbwindows:
Spearman’s rho ¼ 0.49 for dog–human and 0.24 for dog–mouse
comparisons). Lineage-specific variation in the regional divergence
Figure 2 | Venn diagram showing the total lengths of aligned and unique
sequences in the euchromatic portions of the dog, human and mouse
genomes. Lengths shown in Mb, as inferred from genome-wide BLASTZ
alignments (see Methods and Supplementary information). Overlapping
partitions represent orthologous ancestral sequences. Each lineage-specific
partition is further split into the total length of sequence classified as either
lineage-specificinterspersedrepeats(red)orancestralsequence(black).The
latter is assumed to primarily represent ancestral sequences deleted in the
two other species.
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rates may be coupled with changes in factors such as sequence
composition or chromosomal position23,54. Consistent with this,
the ratios of lineage-specific divergence rates in orthologous win-
dowsarepositivelycorrelatedwiththeratiosofcurrentGþCcontent
in the same windows (Spearman’s rho ¼ 0.16 for dog–human, 0.24
for dog–mouse).
Male mutation bias. Comparison of autosomal and X chromosome
substitution rates can be used to estimate the relative mutation rates
in the male and female germ lines (a), because the X chromosome is
present in females twice as often as in males. Using the lineage-
specific rates from ancestral repeats, we estimate a as 4.8 for the
lineage leading to human, and 2.8 for the lineages leading to both
mouse and dog. These values fall between recent estimates from
murids24,25and from hominids23, and suggest that male mutation
bias may have increased in the lineage leading to humans.
Mutational hotspots and chromosomal fission. Genome compari-
sonsofhumanwithbothchicken55andchimpanzee23havepreviously
revealed that sequences close to a telomere tend to have increased
divergence rates and G þ C content relative to interstitial sequences.
It has been unclear whether these increases are inherent character-
istics of the subtelomeric sequence itself or derived characteristics
causally connected with its chromosomal position. We find a similar
increaseinbothdivergence(medianincrease15%,P , 1025;Mann-
Whitney U-test) and GþC content (median increase 9%, P , 1029)
for subtelomeric regions along the dog lineage, with a sharp increase
towards the telomeres (Supplementary Fig. S7).
This phenomenon is manifested at other synteny breaks, not only
those at telomeres. We also observed a significant increase in
divergence and GþC content in interstitial regions that are sites of
syntenic breakpoints54,56(Supplementary Fig. S7). These properties
therefore seem correlated with the susceptibility of regions to
chromosomal breakage.
Proportion of genome under purifying selection
One of the striking discoveries to emerge from the comparison of the
human and mouse genomes21,24was the inference that ,5.2% of the
humangenomeshowsgreater-than-expected evolutionaryconserva-
tion (compared with the background rate seen in ancestral repeat
elements,whicharepresumedtobenonfunctional).Thisproportion
greatly exceeds the 1–2% that can be explained by protein-coding
regions alone. The extent and function of the large fraction of non-
coding conserved sequence remain unclear57, but this sequence is
likely to include regulatory elements, structural elements and RNA
genes.
Low turnover of conserved elements. We repeated the analysis of
conserved elements using the human and dog genomes. Briefly, the
analysisinvolvescalculatingaconservationscoreSHD,normalizedby
the regional divergence rate, for every 50-bp window in the human
genome that can be aligned to dog. The distribution of conservation
scores for all genomic sequences is compared to the distribution in
ancestral repeat sequences (which are presumed to diverge at the
local neutral rate), showing a clear excess of sequences with high
conservationscores.Bysubtractingascaledneutraldistributionfrom
the total distribution, one can estimate the distribution of conserva-
tion scores for sequences under purifying selection. Moreover, for a
given sequence with conservation score SHD, one can also assign a
probability Pselection(SHD) that the sequence is under purifying
selection (see ref. 24 and Supplementary Information).
The human–dog genome comparison indicates that ,5.3% of the
human genome is under purifying selection (Fig. 4a), which is
equivalenttotheproportionestimatedfromhuman–rodentanalysis.
Theobviousquestioniswhetherthebasesconservedbetweenhuman
and dog coincide with the bases conserved between humans and
rodents25,58. Because the conservation scores do not unambiguously
assign sequences as either selected or neutral (but instead only assign
probability scores for selection), we cannot directly compare the
conserved bases. We therefore devised the following alternative
approach.
We repeated the human–dog analysis, dividing the 1462Mb of
orthologous sequence between human and dog into those regions
with (812Mb) or without (650Mb) orthologous sequence in mouse
(Fig. 2). The first set shows a clear excess of conservation relative
to background, corresponding to ,5.2% of the human genome
(Fig. 4b). In contrast, the second set shows little or no excess
conservation, corresponding to at most 0.1% of the human genome
(Fig. 4c). This implies that hardly any of the functional elements
conserved between human and dog have been deleted in the mouse
lineage (see also Supplementary Information).
The results strongly suggest that there is a common set of
functional elements acrossall threemammalian species, correspond-
ing to ,5% of the human genome (,150Mb). These functional
elements reside largely within the 812Mb of ancestral sequence
common to human, mouse and dog. If we eliminate ancestral repeat
elements within this sharedsequenceas largely non-functional, most
functional elements can be localized to 634Mb, and constitute
approximately 24% of this sequence.
It should be noted that the estimate of ,5% pertains to conserved
elements across distantly related mammals. It is possible that there
are additional weakly constrained or recently evolved elements
within narrow clades (for example, primates) that can only be
detected by genomic sequencing of more closely related species29.
Clustering of highly conserved non-coding elements. We next
Figure 3 | Sequence composition and divergence rates. a, Distribution of
G þ C content in 10-kb windows across the genome in dog (green), human
(red) and mouse (blue). b, Median lineage-specific substitution rates based
on analysis of ancestral repeats aligning across all three genomes. Analysis
was performed in non-overlapping 1-Mb windows across the dog genome
that contained at least 2kb of aligned ancestral repeat sequence (median
8.8kb).Thetreewasrootedwiththeconsensussequencesfromtheancestral
repeats.Numbersinparenthesesgivethe20–80thpercentilerangeacrossthe
windows studied.
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explored the distribution of conserved non-coding elements (CNEs)
across mammalian genomes. For this purpose, we calculated a
conservation score SHMD based on simultaneous conservation
across all three species (see Methods). We defined highly conserved
non-coding elements (HCNEs) to be 50-bp windows that do not
overlap coding regions and for which Pselection(SHMD), the
probability of being under purifying selection given the conserva-
tion score, is at least 95%. We identified ,140,000 such windows
(6.5Mb total sequence), comprising ,0.2% of the human genome
and representing the most conserved ,5% of all mammalian
CNEs.
The densityof HCNEs shows striking peaks when plotted in 1-Mb
windows across the genome (Fig. 4d and Supplementary Figs S8 and
S9), with 50% lying in 204 regions that span less than 14% of the
human genome (Supplementary Table S11). These regions are
generally gene-poor, together containing only ,6% of all protein-
coding sequence.
The genes contained within these gene-poor regions are of
particular interest. At least 182 of the 204 regions contain genes
with key roles in establishing or maintaining cellular ‘state’. At least
156 of the regionscontain one or, in a fewcases, several transcription
factors involved in differentiation and development59. Another 26
regions contain a gene important for neuronal specialization and
growth, including several axon guidance receptors. The proportion
of developmental regulators is far greater than expected by chance
(P , 10231; see Supplementary Information).
We then tested whether the HCNEs within these regions tend to
cluster around the genes encoding regulators of development.
Analysis of the density of HCNEs in the intronic and intergenic
sequences flanking every gene in the 204 regions revealed that the
197 genes encoding developmental regulators show an average of
,10-foldenrichmentforHCNEsrelativetothefullsetof1,285genes
in the regions (Fig. 4e and Supplementary Fig. S10). The enrichment
sometimes extends into the immediately flanking genes.
We note that the 204 regions include nearly all of the recently
identified clusters of conserved elements between distantly related
vertebrates such as chicken and pufferfish55,59–62. For example, they
overlap 56 of the 57 large intervals containing conserved non-coding
sequence identified between human and chicken55. The mammalian
analysis, however, detects vastly more CNEs (.100-fold more
sequence than with pufferfish59and 2–3-fold more than with
chicken) and identifies many more clusters. The limited sensitivity
of these more distant vertebrate comparisons may reflect the diffi-
culty of aligning short orthologous elements across such large
evolutionary distances or the emergence of mammal-specific regu-
latory elements. In any case, mammalian comparative analysis may
beamorepowerfultoolforelucidatingtheregulatorycontrolsacross
these important regions.
Although the function of conserved non-coding elements is
unknown, on the basis of recent studies59,63–66it seems likely that
many regulate gene expression. If so, the above results suggest that
,50% of all mammalian HCNEs maybe devoted to regulating ,1%
of all genes. In fact, the distribution may be even more skewed, as
there are additional genomic regions with only slightly lower HCNE
density than the 204 studied above (Supplementary Fig. S8). All of
these regions clearly merit intensive investigation to assess indicators
of regulatory function. We speculate that these regions may harbour
characteristic chromatin structure and modifications that are poten-
tially involved in the establishment or maintenance of cellular state.
Genes
Accurate identification of the protein-coding genes in mammalian
genomes is essential for understanding the human genome, includ-
ing its cellular components, regulatory controls and evolutionary
Figure 4 | Conservation of orthologous sequence between human and
dog. a, Histogram of conservation scores, S, for all 50-bp windows across
the human genome with at least 20 bases of orthologous sequence aligning
to the dog genome, for all aligning sequences (red) and for ancestral repeat
sequence only (blue). b, Conservation scores for the subset of windows that
also have at least 20 bases of orthologous sequence aligning to the mouse
genome. c, Conservation scores of the complementary subset of windows
lacking such orthologous sequence in mouse. d, Density of 50-bp windows
not overlapping known coding regions, for which Pselection(S) . 95%,based
on comparisons between human and dog (HD), human and mouse (HM),
orbetweenhuman,mouseanddog(HMD),andthedensityofknowngenes,
all in 1-Mb sliding windows across human chromosome 3. e, Enrichment of
HCNEs in the immediate neighbourhood of genes encoding developmental
regulators in the 204 highly conserved regions. The histogram shows the
median number of HCNE bases in the intronic and surrounding intergenic
sequence, for the 197 known or putative development regulators (indicated
by top of red bar) and for all of the 1,285 genes (blue bar). The histogram is
centredatthe5
of the normalized distance to the flanking consecutive upstream genes
(marked 21, 22 and 23) or consecutive downstream genes (1, 2 and 3) as
indicated.Thesequencessurroundingthedevelopmentalgenesaretypically
longer, have more HCNE sequence and have a higher density of HNCE
sequence than other genes in the regions (see Supplementary Information).
0-endofthegene(marked0)andeachbincorrespondstohalf
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constraints. The numberof protein-coding genes in human has been
a topic of considerable debate, with estimates steadily falling from
,100,000 to 20,000–25,000 over the past decade21,22,67–70. We ana-
lysed the dog genome in order to refine the human gene catalogue
and to assess the evolutionary forces shaping mammals. (In the
Genes section, ‘gene’ refers only to a protein-coding gene.)
Genepredictionsindogandhuman.Wegeneratedgenepredictions
for the dog genome using an evidence-based method (see Sup-
plementary Information). The resulting collection contains 19,300
dog gene predictions, with nearly all being clear homologues of
known human genes.
The dog gene count is substantially lower than the ,22,000-gene
models in the current human gene catalogue (EnsEMBL build 26).
Formany predictedhuman genes, we find no convincing evidenceof
a corresponding dog gene. Much of the excess in the human gene
count is attributable to spurious gene predictions in the human
genome (M. Clamp, personal communication).
Gene duplications. Gene duplication is thought to contribute
substantially to functional innovation69,71. We identified 216 gene
duplications that are specific to the dog lineage and 574 that are
specific to the human lineage, using the synonymous substitution
rate KSas a distance metric and taking care to discard likely
pseudogenes. (The CanFam 2.0 assembly contains approximately
24 additional gene duplications, mostly olfactory receptors.) Human
genes are thus 2.7-fold more likely to have undergone duplication
than are dog genes over the same time period. This may reflect
increased repeat-mediated segmental duplication in the human
lineage72.
Although gene duplication has been less frequent in dog than
human, the affected gene classes are very similar. Prominent among
the lineage-specific duplicated genes are genes that function in
adaptive immunity, innate immunity, chemosensation and repro-
duction, as has been seen for other mammalian genomes24,25,69,71.
Reproductive competition within the species and competition
against parasites have thus been major driving forces in gene family
expansion.
The two gene families with the largest numbers of dog-specific
genesarethehistoneH2Bfamilyandthea-interferons,whichcluster
inmonophyleticcladeswhencomparedtotheirhumanhomologues.
This is particularly notable for the a-interferons, for which the gene
families within the six species (human, mouse, rat, dog, cat and
horse) are apparently monophyletic. This may be due either to
coincidental independent gene duplication in each of the six lineages
or to ongoing gene conversion events that have homogenized
ancestral gene duplicates73.
Evolution of orthologous genes across three species. The dog
genome sequence allows us for the first time to characterize the
large-scale patterns of evolution in protein-coding genes acrossthree
major mammalian orders. We focused on a subset of 13,816 human,
mouse and dog genes with 1:1:1 orthology. For each, we inferred the
number of lineage-specific synonymous (KS) and non-synonymous
(KA) substitutions along each lineage and calculated the KA/KSratio
(Table 2 and Supplementary Information), a traditional measure
of the strength of selection (both purifying and directional) on
proteins74.
The median KA/KSratio differs sharply across the three lineages
(P , 10244, Mann-Whitney U-test), with the dog lineage falling
between mouse and human. Population genetic theory predicts75
that the strength of purifying selection should increase with effective
population size (Ne). The observed relationship (mouse , dog ,
human)isthusconsistentwiththeevolutionaryprediction,giventhe
expectation that smaller mammals tend to have larger effective
population sizes76.
Wenextsearchedforparticularclassesofgenesshowingdeviations
from the expected rate of evolution for a species. Such variation in
rate (heterotachy) may point to lineage-specific positive selection or
relaxation of evolutionary constraints77. We developed a statistical
method similar to the recently described Gene Set Enrichment
Analysis (GSEA)78–80to detect evidence of heterotachy for sets of
functionally related genes (see Supplementary Information). Briefly,
the approach involves ranking all genes by KA/KSratio, testing
whether the set is randomly distributed along the list and assessing
the significance of the observed deviations by comparison with
randomly permuted gene sets. In contrast to previous studies,
which focused on small numbers of genes with prior hypotheses of
selection, this approach detects signals of lineage-specific evolution
inarelativelyunbiasedmannerandcanprovidecontexttotheresults
of more limited studies.
Atotalof4,950overlappinggenesetswerestudied,definedbysuch
criteria as biological function, cellular location or co-expression (see
Supplementary Information). Overall, the deviations between the
three lineages are small, and median KA/KSratios for particular gene
sets are highly correlated for each pair of species (Supplementary
Fig. S11). However, there is greater relative variation in human–
mouse and dog–mouse comparisons than in human–dog compari-
sons (Supplementary Fig. S12).
This suggests that observed heterotachy between human and
mouse must be interpreted with caution. For example, there is a
great interest in the identification of genetic changes underlying the
uniqueevolutionofthehumanbrain.Arecentstudy81highlighted24
genes involved in brain development and physiology that show signs
of accelerated evolution in the lineage leading from ancestral
primates to humans when compared to their rodent orthologues.
We observe the same trend for the 18 human genes that overlap
with the genes studied here, but find at least as many genes with
higher relative acceleration in the dog lineage (see Supplementary
Information). Heterotachy relative to mouse therefore does not
appear to be a distinctive feature of the human lineage. It may reflect
decelerated evolution in the rodent lineage, or possibly independent
adaptive evolution in the human and dog lineages82.
A small number of gene sets show evidence of significantly
accelerated evolution in the human lineage, relative to both mouse
and dog (32 sets at z $ 5.0 versus zero sets expected by chance,
P , 1024;Fig.5a).Thesesetsfallintotwocategories:genesexpressed
exclusively in testis, and (nuclear) genes encoding subunits of
the mitochondrial electron transport chain (ETC) complexes. The
former are believed to undergo rapid evolution as a consequence of
sperm competition across a wide range of species83–85, and lineage-
specific acceleration suggests that sexual selection may have been a
particularly strong force in primate evolution. The selective forces
acting on the latter category are less obvious. Because of the
importance of mitochondrial ATP generation for sperm motility86,
and the potentially antagonistic co-evolution of these genes with
maternally inherited mitochondrial DNA-encoded subunits87, we
Table 2 | Evolutionary rates for 1:1:1 orthologues among dog, mouse and human
Median (20–80th percentile range)Spearman’s rho
Dog*MouseHuman Dog–humanDog–mouseHuman–mouse
KS
KA
KA/KS
0.210 (0.138–0.322)
0.021 (0.006–0.051)
0.095 (0.030–0.221)
0.416 (0.310–0.558)
0.038 (0.013–0.087)
0.088 (0.031–0.197)
0.139 (0.0928–0.214)
0.017 (0.005–0.040)
0.112 (0.034–0.272)
0.47
0.87
0.80
0.50
0.87
0.85
0.52
0.86
0.82
*Estimates are based on unrooted tree. The dog branch thus includes the branch from the boreoeutherian ancestor to the primate–rodent split.
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propose that sexual selection may also be the primary force behind
the rapid evolution of the primate ETC genes. Given the ubiquitous
role of mitochondrial function, however, such sexual selection may
have led to profound secondary effects on physiology88.
We found no gene sets with comparably strong evidence for dog-
specificacceleratedevolution.Thereis,however,asmallexcessofsets
with moderately high acceleration scores (19 sets at z $ 3.0 versus 5
sets expected by chance, P , 0.02; Fig. 5b). These sets, which are
primarily related to metabolism, may contain promising candidates
for follow-up studies of molecular adaptation in carnivores.
Polymorphism and haplotype structure in the domestic dog
The modern dog has a distinct population structure with hundreds
of genetically isolated breeds, widely varying disease incidence and
distinctive morphological and behavioural traits89,90. Unlocking the
full potential of the dog genome for genetic analysis requires a dense
SNP map and an understanding of the structure of genetic variation
both within and among breeds.
Generating aSNPmap.WegeneratedaSNPmapofthedoggenome
containing .2.5 million distinct SNPs mapped to the draft genome
sequence, corresponding to an average density of approximately
one SNP per kb (Table 3). The SNPs were discovered in three
complementary ways (see Supplementary Information). (1) We
identified SNPs within the sequenced boxer genome (set 1;
,770,000 SNPs) by searching for sites at which alternative alleles
are supported by at least two independent reads each. We tested a
subset (n ¼ 40 SNPs) by genotyping and confirmed all as hetero-
zygous sites. (2) We compared the 1.5£ sequence from the standard
poodle16with the draft genome sequence from the boxer (set 2;
,1,460,000 SNPs). (3) We generated shotgun sequence data from
nine diverse dog breeds (,100,000 reads each, 0.02£ coverage), four
grey wolves and one coyote (,22,000 reads each, 0.004£ coverage)
and compared it to the boxer (set 3; ,440,000 SNPs). We tested a
subset (n ¼ 1,283 SNPs) by genotyping and confirmed 96% as true
polymorphisms.
The SNP rate between the boxer and any of the different breeds is
one SNP per ,900bp, with little variation among breeds (Table 3).
The only outlier (,1/790bp) is the Alaskan malamute, which is the
only breed studied that belongs to the Asian breed cluster91. The grey
wolf (,1/580bp) and coyote (,1/420bp) show greater variation
when compared with the boxer, supporting previous evidence of a
bottleneck during dog domestication, whereas that the SNP rate is
lower in the grey wolf than in the coyote reflects the closer relation-
ship of the grey wolf to the domestic dog1–3,92(see section ‘Resolving
canid phylogeny’).
The observed SNP rate within the sequenced boxer assembly is
,1/3,000bp. This underestimates the true heterozygosity owing to
the conservative criterion used for identifying SNPs within the boxer
assembly (requiring two reads containing each allele); correcting
for this leads to an estimate of ,1/1,600bp (see Supplementary
Information). This low rate reflects reduced polymorphism within a
breed, compared with the greater variation of ,1/900bp between
breeds.
To assess the utility of the SNPs for dog genetics, we genotyped a
subset from set 3a (n ¼ 1,283) in 20 dogs from each of ten breeds
(Supplementary Table S16). Within a typical breed, ,73% of the
SNPs were polymorphic. The polymorphic SNPs have minor allele
frequencies that are approximately evenly distributed between 5%
and 50% (allele frequencies less than 5% are not reliable with only 40
chromosomessampled).Inaddition,theSNPsfromsets2and3have
a roughly uniform distribution acrossthe genome (Fig. 6a, see below
concerning set 1). The SNP map thus has high density, even
distribution and high cross-breed polymorphism, indicating that it
should be valuable for genetic studies.
Expectations for linkage disequilibrium and haplotype structure.
Modern dog breeds are the product of at least two population
bottlenecks, the first associated with domestication from wolves
(,7,000–50,000 generations ago) and the second resulting from
intensive selection to create the breed (,50–100 generations ago).
This population history should leave distinctive signatures on the
patterns of genetic variation bothwithin and across breeds. We might
expectaspectsofboththelong-rangeLDseenininbredmousestrains,
with strain-specific haplotypes extending over multiple megabases,
and the short-range LD seen in humans, with ancestral haplo-
type blocks typically extending over tens of kilobases. Specifically,
Figure 5 | Gene sets showing accelerated evolution along the human and
doglineages. a,Distributionofaccelerationscoresalongthehumanlineage
relative to both mouse and dog, observed for 4,950 gene sets (red). The
expected distribution based on 10,000 randomized trials is shown in blue.
The dotted line shows the acceleration score for which the probability of
observingevenasingle setby randomchance(outofthe4,950setstested)is
less than 1%. In fact, 32 sets show acceleration scores on the human lineage
exceeding this threshold. b, The observed (red) and expected (blue)
distribution of acceleration scores for the dog lineage, relative to both
human and mouse.
Table 3 | SNPs discovered in dogs, wolves and coyotes compared to the
boxer assembly
Set numberBreed or speciesNumber of
SNPs
SNP rate
(one per x bases)
1Boxer versus boxer768,948 3,004 (observed)
1,637 (corrected)
8942
3a
Boxer versus poodle
Boxer versus breeds*
German shepherd
Rottweiler
Bedlington terrier
Beagle
Labrador retriever
English shepherd
Italian greyhound
Alaskan malamute
Portuguese water dog
Total distinct SNPs
Boxer versus Canids†
China grey wolf
Alaska grey wolf
India grey wolf
Spanish grey wolf
California coyote
Total distinct SNPs
Set 3 total distinct SNPs
Total distinct SNPs
1,455,007
45,271
44,097
44,168
42,572
40,730
40,935
39,390
45,103
45,457
373,382
900
917
913
903
926
907
954
787
896
900
3b
12,182
13,888
14,510
10,349
20,270
71,381
441,441
2,559,519
580
572
573
587
417
3
Total
*Based on ,100,000 sequence reads per breed.
†Based on ,20,000 sequence reads per wolf.
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long-range LDwouldbeexpected withindogbreedsandshort-range
LD across breeds.
Preliminary evidence of long-range LD within breeds has been
reported90. Five genome regions were examined (,1% of the
genome) in five breeds using ,200 SNPs with high minor allele
frequency. LD seemed to extend 10–100-fold further in dog than in
human, with relatively few haplotypes per breed.
With the availability of a genome sequence and a SNP map, we
sought to undertake a systematic analysis of LD and haplotype
structure in the dog genome.
Haplotype structure within the boxer assembly. We first analysed
thestructureofgeneticvariationwithinthesequencedboxergenome
by examining the distribution of the ,770,000 SNPs detected
between homologous chromosomes. Strikingly, the genome is a
mosaic of long, alternating regions of near-total homozygosity and
high heterozygosity (Fig. 6b, c), with observed SNP rates of ,14 per
Mb and ,850 per Mb, respectively. (The latter is close to that seen
within breeds and is indistinguishable when one corrects for the
conservative criterion used to identify SNPs within the boxer
assembly; see Supplementary Information.) The homozygous
regions have an N50 size of 6.9Mb and cover 62% of the genome,
and the heterozygous regions have an N50 size of 1.1Mb and cover
38% of the genome. The results imply that the boxer genome is
largely comprised of vast haplotype blocks. The long stretches of
homozygosityindicateregionsinwhichthesequencedboxergenome
carriesthesamehaplotypeonbothchromosomes.Theproportionof
homozygosity(,62%)reflectsthelimitedhaplotypediversitywithin
breeds.
Long-rangehaplotypesindifferentbreeds.Wesoughttodetermine
whether the striking haplotype structure seen in the boxer genome is
representative of most dog breeds. To this end, we randomly selected
ten regions of 15Mb each (,6% of the genome) and examined
linkage disequilibrium in these regions in a collection of 224 dogs,
consisting of 20 dogs from each of ten breeds and one dog from each
of 24 additional breeds (see Supplementary Tables S17–S19).
The ten breeds were chosen to represent all four clusters described
in ref. 91. The selected breeds have diverse histories, with varying
populationsizeandbottleneckseverity.Forexample,theBasenjiisan
ancientbreedfrom Africathathasasmall breeding population inthe
United States descending from dogs imported in the 1930s–1940s
(refs 93, 94). The Irish wolfhound suffered a severe bottleneck two
centuriesago,withmostdogstodaybeingdescendentsofasingledog
in the early 1800s (refs 5, 94). In contrast, the Labrador retriever and
golden retriever have long been, and remain, extremely popular dogs
Figure 6 | The distribution of SNPs is fairly uniform across breeds, but
non-uniform within the sequenced boxer assembly. a, SNPs across
chromosome3,generatedbycomparingtheboxerassemblywithWGSreads
fromninebreeds.b,TheSNPsonchromosome3oftheboxerassemblyshow
an uneven distribution (plotted in 500-kb windows). Note that boxer SNPs
were identified using a more conservative method, lowering the observed
SNP rate by roughly twofold. c, An alternating pattern of large homozygous
(lightblue,,62%ofgenome;N50size6.9Mb)andlargeheterozygous(dark
blue ,38% of genome; N50 size 1.1Mb) blocks indicates large identical or
divergent haplotypes across the boxer genome. White indicates centromeric
sequence.
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(with ,150,000 and ,50,000 new puppies registered annually,
respectively). They have not undergone such recent severe bottle-
necks,butsomelineshavelostdiversitybecauseoftherepeateduseof
popular sires89. The Glen of Imaal terrier represents the opposite end
of the popularity spectrum, with fewer than 100 new puppies
registered with the American kennel Club each year.
The 224 dogs were genotyped for SNPs across each of the ten
regions, providing 2,240 cases in which to assess long-range LD. The
SNPs (n ¼ 1,219; Supplementary Table S19) were distributed along
the regions to measure the fall-off of genetic correlation, with higher
density at the start of the region and lower densities at further
distances (Fig. 7a). In 645 cases, we also examined the first 10kb in
Figure 7 | Homozygous regions and linkage disequilibrium are nearly
100-fold longer within dog breeds than across the dog or human
populations. a, Sampling design for ten random regions of 15Mb each,
used to assess the haplotype structure of ,6% of the genome (see
Supplementary Information). For each region, we examined the first 10kb
through resequencing and dense genotyping. To detect long haplotypes, we
genotypedSNPsdistributedthroughout thenext1MbandsampledSNPsat
intervals of 1Mb for the next 14Mb. In total we genotyped 1,219 SNPs
across the ten regions in a collection of 224 dogs (20 dogs from each of 10
breeds and one dog from each of 24 breeds). b, Conditional on a dog being
homozygous for the initial 10-kb region (n ¼ 245), we assessed the
probability that the dog was homozygous for all SNPs within a given
distance. The average proportion remaining homozygous is compared for
the various breeds (green), for the boxer when sampled in the same ways as
the breeds (blue) and for the boxer using all SNPs found in the genome
sequence (red). About 50% of the individuals seem to be homozygous
throughout 1Mb both in the boxer and other breeds, indicating that other
breeds have comparable long-range homozygosity. c, Linkage
disequilibrium (LD) as a function of distance is shown as the r2statistic
within individual breeds (red), across various breeds (blue) and a human
population(black) taken from the CEPH collection genotyped as part of the
ENCODE component of the International HapMap Project118. For the
overall dog and human populations, LD falls rapidly, reaching the baseline
levelseenforunlinkedlociby,200kb.Incontrast,LDforindividualbreeds
falls initially but then stays at a moderately high level across several
megabases. d, The LD curves are broadly similar for most breeds, but the
proportion of long-range LD is correlated with known breed history. e, The
observed within-breed LD curve (averaged across breeds) is well fitted by a
simple model with a domestication bottleneck 10,500 generations ago and a
breed-creation bottleneck occurring 50 generations ago (see Supplementary
Information). f,LD curvesfor individual dogbreedscan befitted by models
with different breed-creation bottlenecks. The poorest fit is obtained for the
akita, the breeding history of which is known to involve two separate breed-
creation bottlenecks.
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greater detail by denser genotyping (with ,2 SNPs per kb) in 405
cases and complete resequencing in 240 cases. The resequencing data
yielded a heterozygosity rate of ,1 SNP per 1,500bp, essentially
equivalent to the rate seen in the sequenced boxer genome.
On the basis of examining the first 10kb, we found that ,38% of
instances seem to be completely homozygous and that all dogs seem
to be homozygous for at least one of the ten regions. We then
measured the distance over which homozygosity persisted. Of
instances homozygous in the initial 10-kb segment, 46% were
homozygous across 1Mb and 17% were still homozygous across
10Mb (Fig. 7b). The fall-off in homozygosity is essentially identical
to that seen in the boxer genome, provided that the boxer data are
sampled in an equivalent manner (see Supplementary Information).
This indicates that the long-range haplotype structure seen in the
boxer is typical of most dog breeds, although the precise haplotypes
vary with breed and the locations of homozygous regions vary
between individuals.
We also assessed long-range correlations by calculating r2,
a traditional measure of LD, across the 15-Mb regions. The r2
curve representing the overall dog population (one dog from each
of 24 breeds) drops rapidly to background levels. This is in sharp
contrast to the r2curves within each breed. Within breeds, LD is
biphasic, showing a sharp initial drop within ,90kb followed by an
extended shoulder that gradually declines to the background
(unlinked) level by 5–15Mb in most breeds (Fig. 7c). The basic
pattern is similar in all ten regions (Supplementary Fig. S13) and in
all breeds (Fig. 7d). (Labrador retrievers show the shortest LD,
probably due to their mixed aetiology and large population size.)
The biphasic r2curves within each breed thus consist of two
components (Fig. 7e), at scales differing by ,100-fold. The first
component matches the fall-off in the general dog population and is
likely to represent the short-range de-correlation of local haplotype
blocks in the ancestral dog population. The second component
represents long-range breed-specific haplotypes (Fig. 8a). Notably,
the first component falls off nearly twice as quickly as the LD in the
human population (,200kb), and the second component falls off
slightly slower than seen in laboratory mouse strains95.
Modelling the effects of population history. We tested this
interpretation by performing mathematical simulations on a dog
population that underwent an ancient bottleneck and recent breed-
creation bottlenecks, using the coalescent approach96(see Sup-
plementary Information). Our experimental results were well fitted
by models assuming an ancient bottleneck (effective domesticated
population size 13,000, inbreeding coefficient F ¼ 0.12) occurring
,9,000 generations ago (corresponding to ,27,000years) and
subsequent breed-creation bottlenecks of varying intensities occur-
ring 30–90 generations ago97(Supplementary Fig. S14). The model
closely reproduces the observed r2curves and the observed poly-
morphism rates within breeds, among breeds and between dog and
grey wolf. The model also yields estimates of breed-specific bottle-
necks that are broadly consistent with known breed histories. For
example, Labrador retrievers, and to a lesser extent golden retrievers
and English springer spaniels, show less severe bottlenecks.
Deterministically modelled results (Fig. 7e, f) indicate that a
simple, two-bottleneck model provides a close fit to the data for
the breeds. They do not rule out a more complex population history,
such as multiple domestication events, low levels of continuing gene
flow between domestic dog and grey wolf97,98or multiple bottlenecks
within breeds. Notably, the akita yields the poorest fit to the model,
with an r2curve that appears to be triphasic. This may reflect the
initial creation of the breed as a hunting dog in Japan ,450
generations ago, and a consecutive bottleneck associated with its
introduction into the United States during the 1940s (ref. 99).
Haplotypediversity.Wenextstudiedhaplotypediversitywithinand
among breeds, using the dense genotypes from the 10-kb regions.
Across the 645 cases examined, there is an average of ,10 distinct
haplotypes per region. Within a breed, we typically see four of
these haplotypes, with the average frequency of the most common
haplotype being 55% and the average frequency of the two most
common being 80% (Fig. 8c and Supplementary Fig. S18). The
haplotypes and their frequencies differ sharply across breeds. None-
theless, 80% of the haplotypes seen with a frequency of at least 5%
in one breed are found in other breeds as well (Supplementary
Table S26). This extends previous observations of haplotype sharing
across breeds90. In particular, the inclusion of all SNPs with a minor
allele frequency $5% across all breeds provides a more accurate
picture of haplotype sharing, because the analysis includes haplo-
typesthat arerarewithinasinglebreed butmore common acrossthe
population.
We then inferred the ancestral haplotype block structure in the
ancestral dog population (before the creation of modern breeds) by
combining the data across breeds and applying methods similar to
those used for haplotype analysis in the human genome100(see
Supplementary Information). In the 10-kb regions studied, one or
two haplotype blocks were typically observed. Additional data across
100-kb regions suggest that the ancestral blocks have an average size
of ,10kb. The blocks typically have ,4–5 distinct haplotypes
across the entire dog population (Fig. 8b). The overall situation
closely resembles the structure for the human genome, although
with slightly smaller block size (Supplementary Figs S15–S19 and
Supplementary Table S24–26).
Ancestral and breed-specific haplotypes. A clear picture of the
population genetic history of dogs emerges from the results detailed
above:
†The ancestral dog population had short-range LD. The haplotype
blocks were somewhat shorter than in modern humans (,10kb
versus ,20kb in human), consistent with the dog population being
somewhat older than the human population (,9,000 generations
versus,4,000 generations). Haplotypeblocksatlargedistanceswere
essentially uncorrelated (Fig. 8a).
†Breed creation introduced tight breed-specific bottlenecks, at least
for the breeds examined. From the great diversity of long-range
haplotype combinations carried in the ancestral population, the
founding chromosomes emerging from the bottleneck represented
only a small subset. These became long-range breed-specific haplo-
types (Fig. 8a).
†Although the breed-specific bottlenecks were tight, they did not
cause massive random fixation of individual haplotypes. Only 13%
of the small ancestral haplotypes are monomorphic within a typical
breed, consistent with the estimated inbreeding coefficient of ,12%.
Across larger regions ($100kb), we observed no cases of complete
fixation within a breed (Supplementary Fig. S20).
†There is notable sharing of 100-kb haplotypes across breeds, with
,60% seen in multiple breeds although with different frequencies.
On average, the probability of sampling the same haplotype on two
chromosomes chosen from different breeds is roughly twofold lower
than for chromosomes chosen within a single breed (Supplementary
Fig. S21).
Implications for genetic mapping. These results have important
implications for the design of dog genetic studies. Although early
efforts focused on cross-breeding of dogs for linkage analysis101–103, it
is now clear that within-breed association studies offer specific
advantages in the study of both monogenic and polygenic diseases.
First, they use existing dogs coming to medical attention and do not
require the sampling of families with large numbers of affected
individuals. Such studies should be highly informative, because
dog breeds have retained substantial genetic diversity. Moreover,
they will require a much lower density of SNPs than comparable
human association studies, because the long-range LD within breeds
extends ,50-fold further than in humans90,104,105.
Whereas human association studies require .300,000 evenly
spaced SNPs100,106,107, the fact that LD extends over at least 50-fold
greater distances in dog suggests that dog association studies would
require perhaps ,10,000 evenly spaced SNPs. To estimate the
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Figure 8 | Two bottlenecks, one old and one recent, have shaped the
haplotype structure and linkage disequilibrium of canine breeds.
a, Modern haplotype structure arose from key events in dog breeding
history. The domestic dog diverged from wolves 15,000–100,000years
ago97,119, probably through multiple domestication events98. Recent dog
breeds have been created within the past few hundred years. Both
bottleneckshaveinfluencedthehaplotypepatternandLDofcurrentbreeds.
(1) Before the creation of modern breeds, the dog populationhad the short-
range LD expected on the basis of its large size and time since the
domestication bottleneck. (2) In the creation of modern breeds, a small
subset of chromosomes was selected from the pool of domestic dogs. The
long-range patterns that happened to be carried on these chromosomes
became common within the breed, thereby creating long-range LD. (3) In
the short time since breed creation, these long-range patterns have not yet
been substantially broken down by recombination. Long-range haplotypes,
however, still retain the underlying short-range ancestral haplotype blocks
from the domestic dog population, and these are revealed when one
examines chromosomes across many breeds. b, c, Distribution of ancestral
haplotype blocks in a 10-kb window on chromosome 6 at ,31.4Mb across
24 breeds (b) and within four breeds (c). Ancestral haplotype blocks are
5–15kb in size (which is shorter than the ,25-kb blocks seen in humans)
and are shared across breeds. Typical blocks show a spectrum of ,5
haplotypes, with one common major haplotype. Blocks were defined using
the modified four-gamete rule (see Supplementary Information) and each
haplotype (minor allele frequency (maf) . 3%) within a block was given a
uniquecolour. d, e, Distribution of breed-derivedhaplotypes across a 10-kb
window on chromosome 6 at ,31.4Mb across 24 breeds (d) and within
fourbreeds(e).Eachcolourdenotesadistincthaplotype(maf . 3%)across
11 SNPs in the 10-kb window for each of the analysed dogs. Pairs of
haplotypes have an average of 3.7 differences. Most haplotypes can be
definitively identified on the basis of homozygosity within individual dogs.
Grey denotes haplotypes that cannot be unambiguously phased owing to
rare alleles or missing data. Within each of the four breeds shown, there are
2–5 haplotypes, with one or two major haplotypes accounting for the
majorityofthechromosomes.Acrossthe24breeds,thereareatotalofseven
haplotypes. All but three are seen in multiple breeds, although at varying
frequencies.
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number of SNPs required, we generated SNP sets from ten 1-Mb
regions by coalescent simulations using the bottleneck parameters
that generate SNP rates and LD curves equivalent to the actual data
(Supplementary Fig. S14 and Supplementary Table S20). We then
selected individual SNPs as ‘disease alleles’ and tested our ability
to map them by association analysis with various marker densities
(Fig. 9a).
Fordisease alleles causing a simple mendelian dominant trait with
high penetrance and no phenocopies, there is overwhelming power
to map the locus (Fig. 9a). Using ,15,000 evenly spaced SNPs and a
log likelihood odds ratio (LOD score) score threshold of 5, the
probability of detecting the locus is over 99% given a collection of
100 affected and 100 unaffected dogs. (The LOD score threshold
corresponds to a false positive rate of 3% loci per genome.)
For a multigenic trait, the power to detect disease alleles depends
on several factors, including the relative risk conferred by the allele,
the allele frequency and the interaction with other alleles. We
investigated a simple model of an allele that increases risk by a
multiplicative factor (l) of 2 or 5 (see Supplementary Information).
Using the above SNP density and LOD score threshold, the power to
detectalocuswithasampleof100affectedand100unaffecteddogsis
97% for l ¼ 5 and 50% for l ¼ 2 (Fig. 9b, c). Although initial
mapping will be best done by association within breeds, subsequent
fine-structure mapping to pinpoint the disease gene will probably
benefitfromcross-breedcomparison.Giventhegeneticrelationships
across breeds described above, it is likely that the same risk allele will
be carried in multiple breeds. By comparing risk-associated haplo-
typesinmultiplebreeds,itshouldbepossibletosubstantiallynarrow
the region containing the gene.
Resolving canid phylogeny
The dog family, Canidae, contains 34 closely related species that
diverged within the last ,10 million years1. Resolving the evolution-
ary relationships of such closely related taxa has been difficult
because a great quantity of genomic sequence is typically required
to yield enough informative nucleotide sites for the unambiguous
reconstruction of phylogenetic trees. We sought to streamline the
process of evolutionary reconstruction by exploiting our knowledge
of the dog genome to select genomic regions that would maximize
the amount of phylogenetic signal per sequenced base. Specifically,
we sought regions of rapidly evolving, unique sequence.
We first compared the coding regions of 13,816 dog genes with
human–dog–mouse1:1:1orthologuestofindthosewithhighneutral
evolutionary divergence (comparing KSand KA/KS). We selected 12
exons (8,080bp) for sequencing, based on the criteria that their
sequences (1) are consistent with the known phylogeny of human,
dog, mouse and rat, (2) have a high percentage of bases ($15%) that
are informative for phylogenetic reconstruction in the human, dog,
mouseandratphylogenies,and(3)couldbesuccessfullyamplifiedin
allcanids.Thechosenexonscontain3.3-foldmoresubstitutionsthan
random exonic sequence. Using our SNP database, we also evaluated
intronstoidentify thosewithhighvariationbetweendogandcoyote.
We selected four introns (3,029bp) that contained ,5-fold more
SNPs than the background frequency. We sequenced these exons and
introns (11,109bp) in 30 out of 34 living wild canids, and we
combined the data with additional sequences (3,839bp) from recent
studies3,92.
The resulting evolutionary tree has a high degree of statistical
support (Fig. 10), and uniquely resolves the topology of the dog’s
closest relatives. Grey wolf and dog are most closely related (0.04%
and 0.21% sequence divergence in nuclear exon and intron
sequences, respectively), followed by a close affiliation with coyote,
golden jackal and Ethiopian wolf, three species that can hybridize
withdogsinthewild(Fig.10).Closesttothisgrouparethedholeand
Africanwilddog,twospecieswithauniquelystructuredmeat-slicing
tooth, suggesting that this adaptation was later lost. The molecular
tree supports an African origin for the wolf-like canids, as the two
African jackals are the most basal members of this clade. The two
other large groupings of canids are (1) the South American canids,
which are clearly rooted by the two most morphologically divergent
canids,the manedwolfandbushdog;and(2)theredfox-like canids,
which are rooted by the fennec fox and Blanford’s fox, but now also
include the raccoon dog and bat-eared fox with higher support.
Together, these three clades contain93%of all living canids. Thegrey
fox lineage seems to be the most primitive and suggests a North
American origin of the living canids about 10 million years ago1.
These results demonstrate the close kinship of canids. Their
limited sequence divergence suggests that many molecular tools
developed for the dog (for example, expression microarrays) will
be useful for exploring adaptation and evolutionary divergence in
other canids as well.
Conclusions
Genome comparison is a powerful tool for discovery. It can reveal
unknown—and even unsuspected—biological functions, by sifting
the records of evolutionary experiments that have occurred over 100
years or over 100 million years. The dog genome sequence illustrates
the range of information that can be gleaned from such studies.
Mammaliangenomeanalysis ishelpingtodevelopaglobalpicture
of gene regulation in the human genome. Initial comparison with
rodents revealed that ,5% of the human genome is under purifying
selection, and that the majority of this sequence is not protein-
Figure 9 | Power to detect a disease locus by association mapping. One
SNPwasdesignatedasadiseasealleleunderoneofthreegeneticmodels:(a)
simple mendelian dominant, (b) fivefold multiplicative increase in risk and
(c) twofold multiplicative increase in risk. SNP genotypes across
surrounding chromosomal regions of 1Mb were simulated, using the
coalescent model corresponding to observed within-breed variation (see
text). Diploid genotypes across the chromosomal region were then
generated for 100 affected and 100 unaffected dogs, based on the disease
model, and association analysis was performed to detect the presence of the
disease allele. The distribution of the maximum LOD score across the 1-Mb
region is shown for analyses based on multi-SNP haplotypes (solid lines)
with SNP densities equivalent to a genome-wide map with a total of 7,500
(red), 15,000 (green) or 30,000 (blue) SNPs. Dotted curves show the null
distribution for a genome-wide search in which no disease locus is present
(see Supplementary Information). A LOD score of 5 corresponds to ,3%
chanceofafalsepositiveacrossthegenome.For thisthreshold,thepower to
detect a disease allele that increases risk by twofold using haplotype analysis
and a map with 15,000 SNPs is ,50%.
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coding. The dog genome is now further clarifying this picture, as our
data suggest that this ,5% represents functional elements common
toallmammals.Thedistributionoftheseelementsrelativetogenesis
highlyheterogeneous,withroughlyhalfofthemosthighlyconserved
non-coding elements apparently devoted to regulating ,1% of
human genes; these genes have important roles in development,
and understanding the regulatory clusters that surround them may
reveal how cellular states are established and maintained. In recent
papers32,108, the dog genome sequence has been used to greatly
expand the catalogue of mammalian regulatory motifs in promoters
and 3
used to substantially revise the human gene catalogue. Despite these
advances, it is clear that mammalian comparative genomics is still in
its early stages. Progress will be markedly accelerated by the avail-
ability of many additional mammalian genome sequences, initially
with light coverage28but eventually with near-complete coverage.
0-untranslated regions. The dog genome sequence is also being
In addition to its role in studies of mammalian evolution, the dog
has a special role in genomic studies because of the unparalleled
phenotypic diversity among closely related breeds. The dog is a
testament to the power of breeding programmes to select naturally
occurring genetic variants with the ability to shape morphology,
physiology and behaviour. Genome comparison within and across
breeds can reveal the genes that underlie such traits, informing basic
research on development and neurobiology. It can also identify
disease genes that were carried along in breeding programmes.
Potential benefits include insights into disease mechanism, and the
possibility of clinical trials in disease-affected dogs to accelerate new
therapeutics that would improve health in both dogs and humans.
The SNP map of the dog genome confirms that dog breeds show
the long-range haplotype structure expected from recent intensive
breeding. Moreover, our analysis shows that the current collection of
.2.5millionSNPsshouldbesufficienttoallowassociationstudiesof
Figure 10 | Phylogeny of canid species. The phylogenetic tree is based on
,15kb of exon and intron sequence (see text). Branch colours identify the
red-fox-like clade (red), the South American clade (green), the wolf-like
clade (blue) and the grey and island fox clade (orange). The tree shown was
constructed using maximum parsimony as the optimality criterion and is
the single most parsimonious tree. Bootstrap values and bayesian posterior
probability values are listed above and below the internodes, respectively;
dashes indicate bootstrap values below 50% or bayesian posterior
probability values below 95%. Horizontal bars indicate indels, with the
number of indels shown in parentheses if greater than one. Underlined
species names are represented with corresponding illustrations. (Copyright
permissions for illustrations are listed in the Supplementary Information.)
Divergence time, in millions of years (Myr), is indicated for three nodes as
discussed in ref. 1. For scientific names and species descriptions of canids,
see ref.119.Atreebasedonbayesianinferencediffersfromthetreeshownin
two respects: it groups the raccoon dog and bat-eared fox as sister taxa, and
groupsthegreyfoxandislandfoxasbasaltothecladecontainingthesesister
taxa. However, neither of these topological differences is strongly supported
(see text and Supplementary Information).
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nearly any trait in any breed. Realizing the full power of dog genetics
nowawaitsthedevelopmentofappropriategenotypingtools,suchas
multiplex ‘SNP chips’109—this is already underway. For millennia,
dogshaveaccompaniedhumans ontheir travels.Itisonly fittingthat
the dog should also be a valued companion on our journeys of
scientific discovery.
METHODS
Detailed descriptions of all methods are provided in the Supplementary
Information. Links to all of the data can be obtained via the Broad Institute
website (http://www.broad.mit.edu/tools/data.html).
WGS sequencing and assembly. Approximately 31.5million sequence reads
were derived from both ends of inserts (paired-end reads) from 4-, 10-, 40- and
200-kb clones, all prepared from primary blood lymphocyte DNA from a single
female boxer. This particular animal was chosen for sequencing because it had
the lowest heterozygosity rate among ,120 dogs tested at a limited set of loci;
subsequent analysis showed that the genome-wide heterozygosity rate in this
boxer is not substantially different from other breeds91. The assembly was carried
out using an interimversion of ARACHNE2þ (http://www.broad.mit.edu/wga/).
Genome alignment and comparison. Synteny maps were generated using
standard methods24from pair-wise alignments of repeat masked assemblies
using PatternHunter110on CanFam2.0. All other comparative analyses were
performed on BLASTZ/MULTIZ111,112genome-wide alignments obtained from
the UCSC genome browser (http://genome.ucsc.edu), based on CanFam1.0.
Known interspersed repeats were identified and dated using RepeatMasker and
DateRepeats113. The numbers of orthologous nucleotides were counted directly
from the alignments using human (hg17) as the reference sequence for all
overlapsexceptthedog–mouseoverlap, for whichpair-wise (CanFam1.0,mm5)
alignments were used.
Divergence rate estimates.Orthologous ancestral repeats were excisedfromthe
genome alignment and realigned with the corresponding RepBase consensus
using ClustalW. Nucleotide divergence rates were estimated from concatenated
repeat alignments using baseml with the REV substitution model114. Ortholo-
gous coding regions were excised from the genome alignments using the
annotated human coding sequences (CDS) from Ensembl and the UCSC
browser Known Genes track (October 2004) as reference. KAand KSwere
estimated for each orthologue triplet using codeml with the F3 £ 4 codon
frequency model and no additional constraints.
Detection and clustering of sequence conservation. Pair-wise conservation
scores and the fraction of orthologous sequences under purifying selection were
estimated as in ref. 24. The three-way conservation score SHMDwas defined as
SHMD¼ ðp2uÞ=
across all three genomes (human, mouse, dog) for each non-overlapping 50-bp
windowwithmorethan20alignedbases,pisthefractionofnucleotidesidentical
across all three genomes, and u is the mean identity of ancestral repeats within
500kb of the window. HCNEs were defined as windows with SHMD. 5.4 that
did not overlap a coding exon, as defined by the UCSC Known Genes track, and
HCNEclustersweredefinedasallrunsofoverlapping1-Mbintervals(50-kbstep
size) across the human genome with HCNE densities in the 90th percentile.
Genesetaccelerationscores.GeneannotationwasperformedonCanFam1.0.A
set of 13,816 orthologous human, mouse and dog genes were identified and
compiledinto4,950genesetscontaininggenesrelatedbyfunctionalannotations
or microarray gene expression data. For each gene set S, the acceleration score
A(S) along a lineage is defined by (1) ranking all genes based on KA/KSwithin a
lineage, (2) calculating the rank-sum statistic for the set along each lineage
(denoted adog(S), amouse(S), ahuman(S)), (3) calculating the rank-sum for
the lineage minus the maximum rank-sum the other lineages, for example,
ahuman(S)–max(adog(S), amouse(S)) and (4) converting this rank-sum difference
to a z-score by comparing it to the mean and standard deviation observed in
10,000 random sets of the same size. The expected number of sets at a given
z-score threshold was estimated by repeating steps (1)–(4) 10,000 times for
groups of 4,950 randomly permuted gene sets.
SNP discovery. The SNP discovery was performed on CanFam2.0. Set 1 SNPs
were discovered by comparison of the two haplotypes derived from the boxer
assembly using only high-quality discrepancies supported by two reads. SNPs in
sets 2 and 3 were discovered by aligning reads or contigs to the boxer assembly
and using the SSAHA SNP algorithm115.
Haplotype structure. The SNPs within the sequenced boxer genome
(CanFam2.0) were assigned to homozygous or heterozygous regions using a
Viterbi algorithm116. To determine whether the haplotype structure seen in the
boxer is representative of most dog breeds, we randomly selected ten regions of
15Mb each (,6% of the CanFam2.0 genome) and examined the extent of
homozygosity and linkage disequilibrium in these regions in a collection of 224
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðuð12uÞ=n
p
; where n is the number of nucleotides aligned
dogs, consisting of 20 dogs from each of 10 breeds (akita, basenji, bullmastiff,
English springerspaniel,Glen ofImaalterrier,goldenretriever,Irishwolfhound,
Labrador retriever, pug and rottweiler) and one dog from each of 24 additional
breeds (see Supplementary Information). For each instance in which a dog was
homozygous in a particular 10-kb region, we measured the distance from
the beginning of the 10-kb region to the first heterozygous SNP in the adjoining
100-kb, 1-Mb and 15-Mb data. This distance was used as the extent of
homozygosity. The boxer sequence was sampled in an identical manner to the
actual breed data. Linkage disequilibrium (represented by r2) across the ten
15-Mb regions was assessed using Haploview117.
Received 9 August; accepted 11 October 2005.
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