The origin of chow chows in the light of the East Asian breeds

Abstract

Background
East Asian dog breeds are one of the most ancient groups of dogs that radiated after the domestication of the dog and represent the most basal lineages of dog evolution. Among these, the Chow Chow is an ancient breed that embodies very distinct morphological and physiological features, such as sturdy build, dense coat, and blue/purple tongue. ResultsUsing a Restricted site Associated DNA (RAD) sequencing approach, we sequenced the genomes of nine Chow Chows from China. Combined with a dataset of 37 canid whole genome sequencing (WGS) from several published works, we found that the Chow Chow is one of the most basal lineages, which originated together with other East Asian breeds, such as the Shar-Pei and Akita. Demographic analysis found that Chow Chows originated from the Chinese indigenous dog about 8300 years ago. The bottleneck leading to Chow Chows was not strong and genetic migration between Chow Chows and other populations is low. Two classes of genes show strong evidence of positive selection along the Chow Chow lineage, namely genes related to metabolism and digestion as well as muscle/heart development and differentiation. Conclusions
Dog breeds from East Asia, including the Chow Chow, originated from Chinese indigenous dogs very early in time. The genetic bottleneck leading to Chow Chows and migrations with other populations are found to be quite mild. Our current study represents an early endeavor to characterize the origin of East Asian dog breeds and establishes an important reference point for understanding the origin of ancient breeds in Asia.

Full text

R E S E A R C H A R T I C L E Open Access
The origin of chow chows in the light of
the East Asian breeds
Hechuan Yang
1,2,3
, Guodong Wang
2
, Meng Wang
4
, Yaping Ma
4
, Tingting Yin
2,5,6
, Ruoxi Fan
4
, Hong Wu
4
, Li Zhong
4
,
David M. Irwin
7
, Weiwei Zhai
3*
and Yaping Zhang
2*
Abstract
Background: East Asian dog breeds are one of the most ancient groups of dogs that radiated after the domestication
of the dog and represent the most basal lineages of dog evolution. Among these, the Chow Chow is an ancient breed
that embodies very distinct morphological and physiological features, such as sturdy build, dense coat, and blue/
purple tongue.
Results: Using a Restricted site Associated DNA (RAD) sequencing approach, we sequenced the genomes of
nine Chow Chows from China. Combined with a dataset of 37 canid whole genome sequencing (WGS) from several
published works, we found that the Chow Chow is one of the most basal lineages, which originated together with other
East Asian breeds, such as the Shar-Pei and Akita. Demographic analysis found that Chow Chows originated from the
Chinese indigenous dog about 8300 years ago. The bottleneck leading to Chow Chows was not strong and genetic
migration between Chow Chows and other populations is low. Two classes of genes show strong evidence of positive
selection along the Chow Chow lineage, namely genes related to metabolism and digestion as well as muscle/heart
development and differentiation.
Conclusions: Dog breeds from East Asia, including the Chow Chow, originated from Chinese indigenous dogs very early
in time. The genetic bottleneck leading to Chow Chows and migrations with other populations are found to be quite
mild. Our current study represents an early endeavor to characterize the origin of East Asian dog breeds and establishes
an important reference point for understanding the origin of ancient breeds in Asia.
Keywords: Dog domestication, RAD sequencing, Demographic history, Artificial selection
Background
Animal and plant domestication, one of the greatest
innovations in recent human history, is a fundamental
basis for modern civilization [1]. Of all the large mammals
that are candidates for domestication (about 148 species
with body weight greater than 90 lb), only a few species
were successfully domesticated (14 species) [2]. Among
these, the domestic dog (Canis lupus familiaris)isthe
only large carnivore that was able to thrive in a human-
created environment [3]. Dog domestication represents
one of the most enchanting evolutionary processes com-
posed by human beings.
Even though, extensive efforts have been put into
understanding the history of dog domestication, the
conclusions are still greatly debated. For example,
mtDNA, Y chromosome and whole genome sequencing
(WGS) have pointed to southern East Asia as the region
where dog originated from [4–8]. However, genetic
comparisons between gray wolves and domestic dogs
using SNP array suggested that the Middle East and
Central Asia were important sources for dog domestica-
tion [9, 10]. Moreover, an ancient mtDNA work also
suggested Europe as another site for the origin of dog
domestication [11]. Thus, four geographic locations on
the Eurasian continent have been suggested as the
birthplace of dog domestication and the origin of dogs seem
to be a great mystery in the light of these different studies.
* Correspondence: zhaiww1@gis.a-star.edu.sg;zhangyp@mail.kiz.ac.cn
3
Human Genetics, Genome Institute of Singapore, A*STAR, 60 Biopolis Street,
Genome #02-01, Singapore 138672, Singapore
2
State Key Laboratory of Genetic Resources and Evolution, and Yunnan
Laboratory of Molecular Biology of Domestic Animals, Kunming Institute of
Zoology, Chinese Academy of Sciences, Kunming 650223, China
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Yang et al. BMC Genomics (2017) 18:174
DOI 10.1186/s12864-017-3525-9
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Although multiple centers have been proposed for dog
domestication, the indigenous dogs from China and
several ancient breeds from East Asia embody the
highest amounts of genetic variability and are identified
as the basal lineages after arising from gray wolves
[4–8, 12]. There are several endemic classes of breeds
in East Asia, including working dogs (Tibetan Mastiff,
Akita, Samoyed and Siberian Husky), toy dogs (Pekingese,
Pug, Shih Tzu and Japanese Chin) and various other
breeds with very diverse temperament and appearance
(Chow Chow, Shar-Pei, Lhase Aspo, Shiba Inu and Jindo)
[13]. Compared with many European breeds, which were
selected recently in the past few hundred years through
intense artificial selection [13], East Asian ancient breeds
tend to carry substantially more genetic variability and are
morphologically as distinctive as the European breeds
[14]. Given the wide geographic distribution of Chinese
indigenous dogs across the country side of China (see
Wang et al. for a description of Chinese indigenous dogs
[8]), how East Asian dogs originated and evolved from
each other is an interesting question awaiting answers.
One of the most interesting ancient breeds in East
Asia is the Chow Chow. In Chinese history, the Chow
Chow often appears as a symbol similar to the trad-
itional stone guardians (stone lions) found in front of
Buddhist temples and palaces. It has a sturdy build with
a very dense coat, particularly thick in the neck area. In
addition, Chow Chows also have several distinguishing
features including an extra pair of teeth (44 instead of
42), an unusual blue-black/purple tongue and straight
hind legs, resulting in a rather stilted gait. In this study,
we conducted Restricted site Associated DNA (RAD)
sequencing [15] on nine Chow Chows sampled from
China. Combining these sequences with WGS data from
many other dogs and relatives, we inferred the origin of
the Chow Chow in light of the East Asian dogs and
identified adaptively evolving genes along the Chow
Chow lineage.
Methods
Sample collection and RAD sequencing experiment
We collected blood samples from nine Chow Chows,
three of which were collected in Beijing and the other
six from Kunming. In order to balance sequencing cost
and the number of individuals we can study, we chose
the RAD sequencing approach to survey the Chow
Chow genomes. After simulating the cut sites in the dog
reference genome Canfam3.0 using all type II restriction
enzyme from REBASE [16], we selected SpeI (a six mer,
A^CTAGT), which has 340,847 predicted cutting sites
across the dog reference genome. After extracting the
genomic DNA using QIAamp DNA Blood Mini Kit from
QIAGEN, SpeI was used to incubate the genomic DNA
for 16 h. The resulting short fragments were ligated to
the sequencing adaptor primers (P1). Subsequently,
DNA fragments were sonicated to shorten the frag-
ments. Following size selection using electrophoresis, we
used NEBNext Ultra DNA Library Prep Kit from
Illumina to repair the fragments and ligate the Y
adapters to the sonicated fragments. Paired PCR primers
with one complementary to the P1 adaptor and the
other containing both the barcode as well as the comple-
mentary sequence to one arm of the Y adaptor were
used to amplify the target genomic segments, where one
end has the P1 adaptor and the other end has a Y
adaptor. After PCR amplification, the resulting library
was quantified using an Agilent 2100 bioanalyzer. Equal
amount of DNA were subsequently pooled for sequen-
cing using the Hiseq 2000 platform at the Kunming
Institute of Zoology.
Public data curation
Sets of WGS data were collected from four previous
studies. The first study [7] included four gray wolves,
three Chinese indigenous dogs and three dog breeds
(Tibetan Mastiff, Belgian Malinois, German Shepherd).
We collected six breed dogs from the second study [17].
They are one Afghan Hound, one Labrador Retriever,
one Chow Chow, one Tornjack (Croatian Shepherd
Dog), one Istrian Shorthaired Hound and one Caucasian
Ovcharka. Genome sequences from 10 Tibetan Mastiffs
and 10 Chinese indigenous dogs from Yingjiang (Yunnan,
China) were obtained from the third study [18]. We also
collected data for one Jindo dog from Korea published in
2012 [19]. In total, we collected genome sequences from
37 canids, which included four gray wolves, 13 Chinese
indigenous dogs, 11 Tibetan Mastiffs, one Chow Chow
and eight other dog breeds (Additional file 1: Table S1). In
addition to the sequencing data, we also used a SNP array
dataset, which contains about 48,000 SNPs from 1191
canids [20].
Read mapping and variant calling
After downloading the WGS data from the NCBI/DDBJ
SRA repository (Additional file 1: Table S1), we mapped
the short reads as well as our RAD sequencing data to
the reference genome (Canfam3.0) using BWA (version
0.6.2-r126) [21]. Picard (version 1.87) [22] was used to
mark duplications and GATK (version 2.7-2-g6bda569)
[23] was used to perform base recalibration and local
realignment. BAM files from both the WGS and RAD
sequencing were conjugated to call variants jointly using
mpileup in SAMtools package (version 0.1.19-44428 cd)
[24]. Subsequently, Perl script vcfutils.pl in SAMtools
package was used to extract the high fidelity variants for
the downstream analysis.
Yang et al. BMC Genomics (2017) 18:174 Page 2 of 13
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Experimental verification
We randomly selected 14 E1 regions (from read1 that is
adjacent to the restriction enzyme cutting site) and 11
SNPs from outside E1 regions to validate our SNP set.
We used Sanger sequencing for all of those regions in
eight Chow Chows. In order to calculate false positive
and false negative for the SNP calling, we first identify
genomic regions of each Sanger reads. SNPs found in
Sanger, but not in the SNP set is designated as false
negative. SNPs private to the SNP set, but not found in
the Sanger sequencing is designated as the false positive.
Genetic diversity, kinship, principle component analysis
and population structure analysis
Heterozygous sites within each individual were used to
calculate the genetic diversity. For the WGS data,
genetic diversity was calculated as the percentage of
heterozygous sites within each window. For the RAD
data, due to the non-uniform coverage across the
genome, we exclude sites where coverage was less than
five times and the genetic diversity was calculated focus-
ing solely on the remaining sites. We used a window size
of 1 Mb and step size of 200 kb.
We used the software KING [25] to investigate the
relationship between nine Chow Chows sequenced in
this study. As a comparison, we also computed the
kinship between Chow Chows (12 individuals) from the
SNP array data [20].
We combined all the sequencing data (RAD and WGS)
with a SNP array dataset [20], and then performed
principle component analysis (PCA) and population struc-
ture analysis over the combined datasets. PCA was carried
out using smartPCA in EIGENSOFT (version 4.2) [26].
Population structure analysis was carried out using
ADMIXTURE (Version 1.23) [27] and the results of the
population structure analysis were plotted using CLUMPAK
(Version 1.1) [28].
Linkage disequilibrium
In order to compare linkage disequilibrium (LD) across
different dog and gray wolf populations, we combined
the WGS data with the array data [20] and extracted
45,766 SNPs, which are genotyped in both datasets. Sub-
sequently, we selected populations that had at least nine
individuals. After randomly selecting nine individuals
from each of these populations, linkage disequilibrium
in terms of correlation coefficient (r-square) was calcu-
lated between all sites whose distances between each
other is less or equal to 500 kb using PLINK (Version
1.07) [29]. After processing each population, we binned
distances into discrete 5 kb windows (500 kb/5 kb = 100)
and calculated the mean r-square for all windows of
different sizes. In order to measure the overall level of
linkage disequilibrium, we defined a H statistic which is
the sum of all the LD values for different 5 kb windows
(from 5 kb to 500 kb at step of 5 kb). In other words, H
statistic is an analog of the area under the curve for
mean LD across 500 kb windows and captures overall
levels of LD within each population.
TreeMix and the three-population test
In order to focus our analyses on the East Asian breeds,
we combined all samples that were sequenced by WGS
in our collection with a set of East Asian breeds (those
that locate in the group 1 cluster in the PCA analysis of
all samples) and the Samoyed, which has been thought
to be a potential ancestor population for the Chow
Chow due to morphological similarities between the two
breeds [30], from the SNP array data. Since Chow
Chows from different sources are quite concordant, we
choose to use the Chow Chows from the SNP array as
the representative set to do this inference (combining
the SNP array with the RAD data will leave us with too
few SNPs for the TreeMix analysis). TreeMix (Version
1.1) [31] was used to perform the analysis. The three-
population test was conducted using ADMIXTOOLS
(Version 1.1) [32] across all population combinations.
Demographic inference
We used G-PhoCS (Version 1.2.2) [33] to infer the dem-
ography history of Chow Chows together with gray
wolves and Chinese indigenous dogs. First of all, we used
a series of filters to select independently evolving neutral
loci across the genome. For most SpeI cutting sites, both
the upstream and downstream regions adjacent to them
will be sequenced. We selected all the SpeI cut sites with
high coverage (i.e. 100 bp flanking regions on both sides
were sequenced at least five times in each individual
among the nine RAD sequenced Chow Chows). The
100 bp sequences flanking the cutting sites were joined
together (with the sequence motif ‘ACTAGT’) and 3 bp
subsequently trimmed from both ends of the sequences
(bases at these ends tend to have lower quality scores),
producing 200 bp loci at each restriction enzyme cutting
site. All the extracted loci overlapping with CpG islands,
repeat regions, gap regions in Canfm3.0 were also
removed (annotation was downloaded from UCSC gen-
ome browser [34]). In order to focus our analysis on the
neutrally evolving regions of the genome, we retained all
the sequences at least 10 kb distance from exons and
more than 100 bp away from conserved noncoding
elements (CNEs). We used the gene annotation informa-
tion from both UCSC [34] and NCBI database [35]. We
extracted the CNE information of dog genome similar to
the method described in a previous study [36], using an
updated dataset of the multi-species alignment located
at UCSC [37]. Since G-PhoCS requires independently
evolving loci across the genome, we took one locus every
Yang et al. BMC Genomics (2017) 18:174 Page 3 of 13
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100 kb. These filters identified 13,468 regions, each with
200 bp in length, which could be used for the demo-
graphic inference.
We extracted sequences from all these 13,468 regions
from all four gray wolves, eight Chinese indigenous dogs
(subgroup 1) and all 10 Chow Chows. We randomly
selected 2000 loci across the genome and picked four
individuals from each population to perform the demo-
graphic inference. The Markov Chains were run for
5,200,000 iterations, with the first 200,000 iterations
treated as burn-in. Chains were sampled every 10 itera-
tions. We randomly subsampled five replicate datasets
and results from these randomly selected datasets were
then combined for the final result.
Mutation rate calibration
Mutation rate is a very important parameter for the demo-
graphic inference. Using multiple species as the outgroup
to the canids, we calibrated the mutation rate along the
dog lineage using neutral sequences across the genome
similar to one of the recent study [8] (Additional file 2:
Note S1). To translate the results into real units, we used
this calibrated mutation rate from comparative genomic
analysis (2.2*10
−9
per site per year) and a generation time
of3years[7,8,38].
Selective sweeps and Gene Ontology analysis
We used both population branch statistic (PBS) [39] and
SweepFinder [40] to identify the selective sweep regions
in the genomes of Chow Chow. For PBS, pairwise
window-based Fst values (window size 100 kb and step
size 20 kb) were calculated using VCFtools (Version:
0.1.11) [41] among the Chinese indigenous dogs, Tibetan
Mastiff and Chow Chows. The PBS for the Chow Chows
was calculated as (T
CI
+T
CT
-T
IT
)/2 [39]. T was
computed as -log(1-F
ST
)andthesubscriptsCstands
for the Chow Chow, I stands for the Chinese indigen-
ousdogsandTstandsfortheTibetanMastiff.Higher
PBS values represent long evolutionary distances in
terms of allele frequency differences along the Chow
Chow lineage.
For the SweepFinder, we used a dhole’sgenomeasthe
outgroup [7] to identify the ancestral states for all of the
SNP positions. Then, using the genome background site
frequency spectra as a control (all of the autosomal
chromosomes), we employed SweepFinder [40] as an
independent approach to identify traces of selective sweeps.
We selected the intersection of the top 3% of the PBS
regions and the top 3% of the SweepFinder regions as
the candidate regions for the final set. Gene annotation
was based on the Ensembl annotation [42]. We then
converted those dog gene IDs to their associated human
gene IDs using ensemble homologous mapping extracted
from Ensembl BioMart portal [42]. Gene Ontology (GO)
analysis was conducted using DAVID [43].
In order to investigate the genetic basis of super-
numerary teeth in Chow Chows, we conducted literature
survey and found that four important pathways (BMP,
FGF, SHH and WNT) are involved in the teeth develop-
ment [44]. Genes associated with these pathways were
extracted from WikiPathways [45] (for the BMP, SHH
and WNT pathway) and the literature [46] (for the FGF
pathway). In addition, we also curated from the
literature a list of genes causing tooth abnormalities in
transgenic mice [44]. These genes were combined and
used as the list of candidates responsible for the teeth
development. We subsequently overlapped the list of
selected genes with this gene list, looking for possible
candidate genes responsible for the different number of
teeth in Chow Chows. For the genetic basis of the blue
tongue of Chow Chows, we extracted all pigmentation
genes from Color Genes database [47].
Results
Sample collection
Using a modified RAD construction protocol [15],
sequencing libraries from nine individuals were pooled
and sequenced using the Illumina platform. In RAD
sequencing, one end of the paired-end sequencing (de-
noted as E1) is strictly positioned at the same restriction
cutting site and has uniform coverage, while the other end
(denoted as E2) is variable in position depending on the
insert size. On average, each individual is sequenced to
about 36-fold at the E1 site (Additional file 1: Table S1)
and the E1 reads cover about 2.7% of the whole genome.
The sequence data generated by our RAD sequencing
was combined with a dataset consisting of whole
genome sequences for 37 canids curated from four
published studies [7, 17–19] (Additional file 1: Table S1,
Fig. 1). In total, we called 16,716,649 SNPs across the
whole genome (not limited to the RAD regions). The
transition/transversion ratio of this set is 2.186, indicat-
ing good quality results from the variant calling proced-
ure implemented in SAMtools [48]. We denote this SNP
set as the whole genome SNP set (i.e. WG SNP set).
Since the sequence coverage from the RAD sequencing
will be restricted to certain genomic regions, we further
filter the SNP set by targeting on the genomic regions
with good coverage from the RAD individuals (i.e. geno-
type quality > =20 in at least six out of nine individuals)
and extracted 1,130,910 high quality SNPs (denoted as
RAD SNP set). Using Sanger validation, we found that,
the false positive and false negatives in variant calling in
the RAD data are 5.2 and 6.4%, respectively (Additional
file 1: Table S2). The subsequent population genetic
analysis was conducted using different combination of
these two SNP sets (Additional file 1: Table S3). Using a
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kinship estimation procedure [25], we found that Chow
Chows in our collection are not closely related and show
similar kinship value to the individuals from the SNP
array (Additional file 3: Figure S1).
Genetic diversity across the genome
Using heterozygous SNPs called from each individual,
we calculated the genetic diversity for each individual
across the genome. In Fig. 1, we plotted the genome
wide distribution of variation for all 46 individuals. As
we can see, there is a general trend of decreasing diver-
sity from gray wolves to Chinese indigenous dogs,
Tibetan Mastiff and Jindo. Chow Chow together with
many other dog breeds, most of which are from the
Middle East and Europe have lower genetic diversity.
Among dog breeds, genetic diversity varies quite
extensively. For example, most of the dog breeds from
outside East Asia have genetic diversity similar to the
Chow Chow. The only two exceptions are shepherd dogs
from Germany and Croatia which possess much reduced
genetic diversity. Among the East Asian dogs, the Chinese
indigenous dogs, Tibetan Mastiff and Jindo have compar-
able levels of diversity, which are higher than the diversity
of the Chow Chow.
Principle component analysis
To explore the genetic relationships among these dogs,
we combined the RAD SNP set of the 46 individuals (37
WGS samples and nine RAD sequenced Chow Chows)
with a previously published SNP array dataset [20] and
performed a principle component analysis (PCA) on this
combined dataset. In Fig. 2a, we plotted the relationships
of these samples. The first PC, which accounts for 8.8%
of the total variation, separates the dogs from the gray
wolves and other canids.
Across all worldwide dog populations, we can clearly
see a wide distribution in the genetic differences, includ-
ing a dense cluster of individuals that are much closer to
the gray wolves than to other dogs (denoted as group 1,
Fig. 2a). This dense cluster includes a large group of East
Asian breeds and a few other breeds from other
geographic locations. For example, New Guinea Singing
Dog and Dingo are currently from Australia, but are
found to have spread from south East Asia [49]. The
Alaskan Malamute is from Alaska, which is close to East
Asia. The only exception is the Basenji, which is an
African dog breed that previously was found to have
admixture from the gray wolf [31]. Quite reassuringly,
multiple sources of Chow Chows are concordant with
each other in the PCA plot. This suggests that the
sample quality among the diverse sets of data collected
for the Chow Chow is very consistent.
To further dissect the relationships among the group 1
individuals, PCA analysis was conducted among these
individuals (Fig. 2b). We found that the clustering
pattern correlates quite well with the geographic origin
of these individuals. For example, the Basenji stays
distinct from the remaining individuals along the first
axis (PC1). Subsequently, breeds from the arctic regions
(Siberian Husky, Alaskan Malamute) are separated from
the others along the PC2 axis. The rest of the East Asian
breeds stay close to each other.
Within the core East Asian cluster (Fig. 2b), there are
still different degrees of closeness. Running from the top
of the PCA plot are a) Dingo and New Guinea Singing
dog, b) Chow Chow, c) Shar-Pei, Akita, Chinese indigen-
ous dogs and Jindo, and d) Tibetan Mastiff. Among the
Chinese indigenous dogs, the distribution is rather
heterogeneous and they are quite scattered across the
genetic landscape (also see later sections). Genetically,
the Chow Chow is slightly differentiated from the other
East Asian breeds (e.g. Shar-Pei, Akita, Chinese indigen-
ous dogs, Jindo), which could be related to their unique
history of origin.
When we examined the extent of linkage disequilibrium
(LD) across the groups, especially using a numerical
Fig. 1 Genetic diversity (Heterozygosity) across 46 canids. Boxplot for heterozygosity across 46 canids were plotted in this graph. The middle line
represents the median, and the box represents the interquartile range; bars extend to 1.5 times the interquartile range. The color corresponds to
different population groupings
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measurement (denoted as the H statistic) to capture the
overall extent of linkage disequilibrium for each population,
we found that group 1 dogs have a greatly reduced level of
linkage disequilibrium compared to other dog breeds
(Fig.2c).Interestingly,theLDleveloftheChowChowis
fairly low compared with other group 1 breeds. The low
LD in the Chow Chow suggests its ancient origin or a rela-
tively mild bottleneck at the time of origin.
Population structure analysis
Population structure analysis provides a powerful alter-
native approach for exploring the relationships among
multiple individuals. When combining the sequenced
East Asian dogs with a large number of canids from the
SNP array collection [20], we see that the East Asian
individuals are the ones most similar to the gray wolves
(Fig. 3a), matching our observation from the PCA plot
(Fig. 2a). To further explorethegeneticrelationships
among the East Asian lineages, we conducted a structure
analysis restricted only to the East Asian breeds (Fig. 3b).
When partitioning the set into two groups, Chow Chow
and Siberian Husky are the two extremes of the landscape
(Fig. 3b). The other populations are intermediates between
these two groups, which matches the earlier PCA analysis
(Fig. 2b). Further partitioning the set into more groups,
leads to the separation of the Akita (K = 3), Samoyed
(K = 4), Shar-Pei (K = 5), Tibetan Mastiff (K = 6), and the
Chinese indigenous dogs (K = 7), which contain a subset of
mixed constituent individuals [8]. The Korean breed Jindo
shows a similar profile to the Chinese indigenous dogs,
matching the earlier results from the PCA analysis (Fig. 2b).
We found two distinct subgroups in the Chinese
indigenous dogs. One group (denoted as subgroup 1)
has relatively pure genetic constitutions and the other
Fig. 2 Principle component analysis of canids and linkage disequilibrium across populations. aPCA results for the first two PCs were plotted here
for 1237 canids. The percentage of variances explained by the two PCs are also shown. Different symbols corresponding to different populations are
shown in the legend (* marks sequenced samples in this study). bPCA analysis of group 1 dogs. The percentage of variances explained by the PCs
and the symbols are shown similar to panel a. In the legend, * marks sequenced samples in this work. cLinkage disequilibrium of the gray wolves,
group 1 dogs as well as other dogs are plotted as boxplots. Six wolf populations, eight group 1 dog populations as well as 66 other dog populations
are shown in this figure. Chow Chow is shown as an asterisk. The Y axis (the H statistic) is the numerical measurement of linkage disequilibrium across
a 500 kb window (see Methods)
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subgroup (denoted as subgroup 2) from northern China
tends to have more mixed genetic components. This also
agrees with the earlier observation that the Chinese
indigenous dogs show a wide range of distributions
across the PCA plot (Fig. 2a and b).
TreeMix analysis
The population structure and PCA analyses allowed us
to explore the genetic closeness of these groups, but it
does not provide detailed evolutionary relationships
among these populations. To explore the phylogenetic
relationship among these individuals, we conducted a
TreeMix analysis [31] (Fig. 4 and Additional file 4:
Figure S2) of all the populations suggested by the struc-
ture analysis. Given that we are particularly interested in
the East Asian breeds, we combined the WGS dataset of
37 individuals with a few East Asian breeds from the
SNP array study.
In Fig. 4a, we see that there are two deeply divergent
lineages among the dogs (denoted as clade 1 and 2,
Fig. 4). One clade represents a subgroup of breeds from
East Asia while the other clade includes the Tibetan
Mastiff, Arctic groups and many of the other Breeds
from the Eurasian continent (Fig. 4a). It is interesting to
observe that, the Tibetan Mastiff clustered at the
basal position of the two clades and grouped with the
non-Asia clade (clade 2). Surprisingly, the two subgroups
(subgroup 1 and 2) of indigenous dogs informed by
the structure analysis were separated in the TreeMix
analysis.
Fig. 3 Structure analysis of the canids. aStructure analysis of all the canid data for K = 2-3 (Populations with only one sample are not included for
this analysis except the Jindo). The groups of different dog types were extracted from Vondolt et al. [9]. The focal group 1 populations are marked on
top of the panel. bStructure analysis of all the East Asian breeds with different number of groups (K= 2-7). Colors mark different groupings from the
structure analysis
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Using the three-population test implemented in
ADMIXTOOLS [32], we tested for the possibility of
admixture events across all of these populations. We
found that only subgroup 2 of the Chinese indigenous
dogs bear a strong signal of admixture, and that the two
source populations contributing to the admixture are
always one population from clade 1 and one from clade
2 (Additional file 1: Table S4).
In light of the three-population test results, we allowed
one migration track in the TreeMix analysis (Fig. 4b).
We see that the two subgroups of Chinese indigenous
dogs are now clustered together and that a migration
track from clade 2 contributed a source component for
the tentatively admixed subgroup in the Chinese indi-
genous dogs (subgroup 2). There seems to be at least
two subgroups in the Chinese indigenous dogs, where
one subgroup is pure in genetic constitution, while the
other bears some migratory/admixture signal from the
clade 2 lineages [8]. In addition, Chow Chows are the
breed that is closest to the Chinese indigenous dog com-
paring to other Asian breeds.
Demographic inference
As an ancient breed originated in China, the time and
process that gave rise to the Chow Chow should be in-
formative for our understanding of breed formation in
East Asia. Using a Markov Chain Monte Carlo approach
based on the divergence between the multiple sequences
[33] and a well calibrated mutation rate (Additional file
2: Note S1, Additional file 1: Table S5), we dated the
origin of the Chow Chow from the Chinese indigenous
dogs (Fig. 5 and Additional file 1: Table S6). We found
that dogs separated from the gray wolves in East Asia
about 31,700 years ago, matching several earlier discover-
ies [7, 8]. After the separation of these two populations,
the ancestral Chinese indigenous dogs maintain a rela-
tively small population. The time of origin for the Chow
Chow from the Chinese indigenous dogs was estimated to
be 8300 years ago. Interestingly, the population size of the
Chinese indigenous dogs increased quite rapidly after the
split from the Chow Chow, while the Chow Chow’s overall
Fig. 4 TreeMix analysis of the East Asian breeds together with our WGS
collection. aTreeMix results for the analysis without allowing for any
migration track. The x-axis corresponds to the amount of genetic drift.
Clade 1 is all from East Asia while clade 2 has a mixture of East Asian
breeds and non-Asian breeds. bTreeMix results for the analysis allowing
one migration track. The inferred migration track is shown as a red arrow
in the phylogenetic tree. The weight of migration component is scaled
according to the rainbow in the left
Fig. 5 Demographic model for the origin of the Chow Chow. The
results from the G-PhoCS analysis are depicted in this figure. Divergence
time (in years), population size and migration rate (2 Nm) were shown
together with the demographic history
Yang et al. BMC Genomics (2017) 18:174 Page 8 of 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

population size show a slight decrease in size comparing
to the population size of the ancestral Chinese indigenous
dogs. The levels of migration estimated between the
wolves and dogs, as well as among dog groups are quite
low (2 Nm ~1 or lower). This suggests that the East Asian
lineages stayed relatively distinct from each other during
the history of dog evolution.
Candidates for artificial selection
Given the Chow Chow’s unique morphological and
physiological features such as sturdy build, dense coat,
supernumerary teeth and blue/purple tongue, we wanted
to understand the genetic basis of these traits, especially
whether there are traces of positive selection at the loci
responsible for these interesting phenotypes. We used
both an Fst based method PBS [39] and a composite likeli-
hood method SweepFinder [40] to scan the Chow Chow
genomes for traces of recent adaptation. In order to be
conservative in our discoveries, we required that the
candidate regions be within the top 3% for both measure-
ments. After annotating these regions (0.81% of the
genome), we identified 226 genes with strong signal for
adaptive evolution along the Chow Chow lineage (Fig. 6).
Classifying these genes using Gene Ontology (GO)
[43], we found that there are two major functional
categories that were selected along the Chow Chow
lineage after separating from the Chinese indigenous dog.
The first class of genes is related to digestion and metabol-
ism (Table 1 and Additional file 1: Table S7). Genes
involved in multiple amino acid digesting processes (e.g.
Proline and Glutamine) as well as lipid metabolism are
strongly enriched in this first class. It is quite interesting
to observe this, since strong selection in genes involved in
metabolism and digestion was also observed along the
Chinese indigenous dog lineage [7, 8]. This might suggest
that digestion and metabolism are fundamental to the
evolution of dogs, and are constantly being tuned for their
new diets in a rapid paced human environment.
The second class of genes is related to muscle and heart
development (Table 1 and Additional file 1: Table S7). For
example, multiple GO terms, ranging from muscle cell
differentiation to multiple categories of muscle develop-
ment, are associated with this class of genes. Among the
genes related to muscle development, SMARCD3 is
particularly interesting and is expressed specifically in the
heart and somites in the early mouse embryo. Experimental
silencing of this gene in mice using RNA interference
resulted in abnormal cardiac and skeletal muscle differenti-
ation[50].Inhumans,theprotein product of this gene
jointly acts with the muscle determination factor MyoD to
reprogram hESCs into skeletal muscle cells [51].
In addition, multiple other genes related to muscle
and heart development such as TSC1 [52], MKL2 [53],
ADRB2 [54] and COL5A1 [55] also show evidence of
adaptive evolution (Additional file 1: Table S8).
Other than muscle development, adaptive evolution of
genes involved in biological processes such as respiratory
gaseous exchange and adult behavior are also quite
interesting (Table 1 and Additional file 1: Table S7).
FUT8 encodes an enzyme belonging to the family of
fucosyltransferases, and homozygous deletion of this
gene in mouse shows emphysema-like changes in the
lung [56]. ABAT is a gene responsible for the catabolism
of an important neurotransmitter gamma-amino butyric
acid (GABA). TSHR is an important gene with multiple
functions including the regulation of metabolism and
the photoperiod control of reproduction in vertebrates.
This gene has been found to be selected across many
other domesticated species including cat [57], chicken
[58] as well as sheep [59].
In order to look into the possible genetic underpin-
nings of teeth developments (Chow Chows have 44 teeth
instead of 42), we collected all the genes involved in
teeth development. Overlapping these genes and the list
of 226 positively selected genes yielded two candidate
genes OSR2 and ROR1. ROR1 (homologous to another
receptor tyrosine kinase ROR2) was a member of the
WNT signaling pathway. Both Ror1 and Ror2 genes
expressed in molar tooth primordia in mouse [60], and
Ror2(−/−) mice exhibited defective differentiation of
Fig. 6 Genome wide evidence for selection using both PBS and SweepFinder. A Manhattan plot across different chromosomes for the evidence
of positive selection. The top panel is for the PBS statistic and the bottom panel is for the SweepFinder. Shadowed area is the region on
chromosome 6 with the strongest signal for selection. A selected gene list is marked on the genome wide plot
Yang et al. BMC Genomics (2017) 18:174 Page 9 of 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

tooth [61]. Mice lacking Osr2 gene developed super-
numerary teeth lingual to their molars [62].
Discussion
Using the Chow Chow as an example breed, we have
made a systematic study of an ancient East Asian dog
breed. There are several interesting observations that are
worth discussing here. First of all, the time of origin of
the East Asian breeds is perhaps quite old. For example,
in the phylogenetic relationship presented in Fig. 4, the
Chow Chow is the breed closest to the Chinese indigen-
ous dogs. If all other dog breeds also begin from the
Chinese indigenous dogs, then they all must have
originated even earlier than 8300 years ago (Fig. 4). The
mild population bottleneck leading to the Chow Chow
suggests a gradual process leading to this breed. Histor-
ically, based on the morphological feature of the dense
coat, Chow Chow has often been thought of a breed of
high latitude origin [58]. However, the analysis here
showed that, Chow Chow was selected from Chinese
indigenous dogs, which are of Southern origin [8]. Given
the fact that agriculture started in East Asia around
11,000 to 9000 years ago near the Yangtze River [63], the
sedentary environment of humans could have facilitated
the selection of the Chow Chows from Chinese indigen-
ous dogs. Studying the historical context of the East
Asian breeds together with modern human development
is an enchanting picture waiting to be unveiled.
Secondly, even though the arise of Chow Chows has
been hypothesized to be gradual, the amount of overall
gene flow found between the Chinese indigenous dogs
and Chow Chows is surprisingly low. It is possible that
when an incipient breed is under development, the
amount of genetic exchange between the source population
and the population of interest could be quite high, as the
amount of differentiation is still low [64]. However, the
inferenceresultsshowtheoppositepattern(lowmigration).
This suggests that the creation of Chow Chows can be very
fast and subsequent interbreeding was restricted (possibly
disfavored by human beings or there were behavior
differences between breeds). Given the overall low migra-
tion rate found among East Asian breeds, this mode of
breed formation might be quite general across many
ancient breeds.
Thirdly, our gene-based analysis using gene ontology
matches only a subset of the expectations from the
phenotypes that are unique to Chow Chow. For
example, the blue tongue and thick coats are not
Table 1 Gene Ontology (GO) analysis of positively selected genes in Chow Chow
GO terms PValue Enrichment fold Genes mentioned in the main text
Proline biosynthetic process 0.002 42.946 -
Proline metabolic process 0.004 30.062 -
Adult behavior 0.01 5.826 ASIP, TSHR, ABAT
Glutamine family amino acid biosynthetic
process
0.015 15.822 -
Oxidation reduction 0.025 2.039 -
Lysosomal transport 0.025 12.025 -
Intracellular transport 0.03 1.983 -
Feeding behavior 0.031 5.809 ASIP
Muscle cell differentiation 0.032 4.141 SMARCD3, TSC1, MKL2
Heart morphogenesis 0.036 5.491 SMARCD3, COL5A1, MKL2
Positive regulation of striated muscle development 0.039 50.104 ADRB2, MKL2
Positive regulation of muscle development 0.039 50.104 ADRB2, MKL2
Vacuolar transport 0.04 9.394 -
Lipid biosynthetic process 0.042 2.482 -
Respiratory gaseous exchange 0.049 8.351 FUT8
PcG protein complex 0.005 28.404 -
Pyrroline-5-carboxylate reductase activity 0.001 55.247 -
Oxidoreductase activity, acting on the CH-NH group
of donors, NAD or NADP as acceptor
0.016 15.346 -
Oxidoreductase activity, acting on the CH-NH group
of donors
0.039 9.525 -
Steroid dehydrogenase activity 0.044 8.911 -
Hsa00150:Androgen and estrogen metabolism 0.005 10.779 -
Yang et al. BMC Genomics (2017) 18:174 Page 10 of 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

strongly indicated in the GO analysis results. A possible
explanation for this is that the genes responsible for
these phenotypes can be quite simple (only limited to a
few genes) and cannot be easily be picked up by the GO
based analysis. Very interestingly, we found a large
region on chromosome 6 that showed a very strong
signal for selection (around 7 Mb, Fig. 6). Inspecting all
genes in this region with a database of pigmentation
genes [47] found one gene PDPK1, which encodes 3-
phosphoinositide dependent protein kinase 1. Mutations
in PDPK1 cause abnormal pigmentation in mouse em-
bryos [65]. The origin of this large sweep region should be
worth pursuing in future studies. Another gene related
with pigmentation on our list of selected genes is ASIP,
which is located on chromosome 24. It affects the pig-
mentation phenotype in many different animals [66–70].
Extensive selection in genes involved in muscle and
heart development as well in adult behavior is consistent
with the fact that the Chow Chow was kept as a sporting
dog during its early development [58]. Interestingly,
other animals such as horses also show evidence of
strong positive selection in genes involved with muscle
and cardiac development [71]. Future studies in other
sport dogs should unveil a more dynamic picture of
positive selection, some of which might be quite similar
to that seen in the Chow Chow.
Lastly, using multiple public datasets, we revealed the
existence of two subgroups of dogs within the Chinese
indigenous dogs. One group is rather pure in terms of
the genetic makeup while the second group shows
admixture between the East Asian lineage and the non-
Asian lineage (clade 2), matching the finding from a
recent study [8]. Given the fact that most of the samples
were taken from the southern part of China, under-
standing the genetic makeup of the Chinese indigenous
dogs across China and East Asia will be quite important
for our understanding of the origin of dogs in relation to
these other lineages.
Conclusions
Using RAD sequences from nine Chow Chows together
with whole genome sequences from 37 canids, we
characterized the origin of this ancient breed. Demo-
graphic inferences found that, Chow Chows originated
from Chinese indigenous dogs 8300 years ago. The
evolutionary process leading to the Chow Chow is
accompanied by low levels of gene flow and mild
population bottleneck. Two classes of genes showed
strong evidence of positive selection along the Chow
Chow lineage, namely genes related to metabolism and
digestion and those related to muscle/heart development
and differentiation. The study of Chow Chows offered an
important insight into the history and process giving rise
to East Asian breeds.
Additional files
Additional file 1: Table S1. Sample information. Table S2. Sanger
sequencing validation in SNP calling. Table S3. SNP sets used in different
analyses. Table S4. The three-population test result for the Chinese indi-
genous dogs (subgroup 2). Table S5. Mutation rate estimation. Table S6.
G-PhoCS estimation of the demographic history. Table S7. Gene Ontol-
ogy (GO) analysis of the positively selected genes in Chow Chows. Table
S8. The list of selected genes with highlighted functions. (XLSX 27 kb)
Additional file 2: Note S1. Mutation Rate Estimation. (DOCX 44 kb)
Additional file 3: Figure S1. Boxplot of the kinship coefficient between
pairs of Chow Chows in the RAD set and from the SNP array data.
Different levels of relatedness will yield different kinship coefficients.
For example, it is suggested that the estimated kinship coefficient
range [0.354, 1], [0.177, 0.354], [0.0884, 0.177] and [0.0442, 0.0884]
correspond to duplicate/monozygotic twin, 1st-degree, 2nd-degree,
and 3rd-degree relationships respectively. All kinship coefficients from
the RAD sequenced Chow Chows are smaller than 0.0442, and are
comparable to the Chow Chows from the SNP array data. (PDF 4 kb)
Additional file 4: Figure S2. Residual of the TreeMix analysis presented in
Fig. 4. Panel A and B correspond to the panels in the Fig. 4. (PDF 116 kb)
Acknowledgements
We want to thank Prof. Robert Wayne for providing us the SNP array dataset
used in the earlier publication. We are grateful to Drs. Caihong Zheng, Xu
Shen and Guojing Liu for experimental advice. We also thank the owners of
the Chow Chows for providing samples.
Funding
This study was supported by the National Natural Science Foundation of
China [91531303, 31000957], the Breakthrough Project of Strategic Priority
Program of the Chinese Academy of Sciences [XDB13000000] and the
National Basic Research Program of China [2013CB835200 and
2013CB835202]. WZ and HCY are supported by the Genome Institute of
Singapore. GDW is supported by the Youth Innovation Promotion
Association, Chinese Academy of Sciences.
Availability of data and materials
The datasets supporting the conclusions of this article are available in the
National Center for Biotechnology Information (NCBI) Sequence Read
Archive (SRA) database under the accession SRP068254 (https://
trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP068254).
Authors’contributions
YPZ, WZ, GDW and HCY conceived and designed the experiments. HCY,
GDW, MW, YPM, HW, TTY, RXF and LZ performed the experiments. HCY GDW
and WZ analyzed the data. HCY DMI and WZ wrote the paper. All authors
have read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
All the samples used in this study were obtained and handled following the
guidelines of the by-laws on experimentation on animals, and was approved
by the Ethics and Experimental Animal Committee of Kunming Institute of
Zoology, Chinese Academy of Science, China (KIZ_YP201002).
Author details
1
Department of Molecular and Cell Biology, School of Life Sciences,
University of Science and Technology of China, Hefei 230026, China.
2
State
Key Laboratory of Genetic Resources and Evolution, and Yunnan Laboratory
of Molecular Biology of Domestic Animals, Kunming Institute of Zoology,
Chinese Academy of Sciences, Kunming 650223, China.
3
Human Genetics,
Genome Institute of Singapore, A*STAR, 60 Biopolis Street, Genome #02-01,
Singapore 138672, Singapore.
4
Laboratory for Conservation and Utilization of
Yang et al. BMC Genomics (2017) 18:174 Page 11 of 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Bio-resource & Key Laboratory for Microbial Resources of the Ministry of
Education, Yunnan University, Kunming 650091, China.
5
University of Chinese
Academy of Sciences, Beijing 100049, China.
6
Kunming College of Life
Science, University of Chinese Academy of Sciences, Kunming 650223, China.
7
Laboratory Medicine & Pathobiology, University of Toronto, 1 King’s College
Circle, Rm 6211, Toronto, ON M5S 1A8, Canada.
Received: 2 April 2016 Accepted: 28 January 2017
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