Differences in Selection Drive Olfactory Receptor Genes in Different Directions in Dogs and Wolf

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Differences in Selection Drive Olfactory Receptor Genes inDifferent Directions in Dogs and Wolf

Rui Chen,1,2 David M. Irwin,1,3 and Ya-Ping Zhang1,4,*1State 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, China2School of Life Science, University of Science and Technology of China, Hefei, China3Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada4Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming, China

*Corresponding author:E-mail: [email protected].

Associate Editor:John H. McDonald

Abstract

The olfactory receptor (OR) gene family is the largest gene family found in mammalian genomes. It is known to evolve through abirth-and-death process. Here, we characterized the sequences of 16 segregating OR pseudogenes in the samples of the wolf andthe Chinese village dog (CVD) and compared them with the sequences from dogs of different breeds. Our results show that thesegregating OR pseudogenes in breed dogs are under strong purifying selection, while evolving neutrally in the CVD, and show amore complicated pattern in the wolf. In the wolf, we found a trend to remove deleterious polymorphisms and accumulatenondeleterious polymorphisms. On the basis of protein structure of the ORs, we found that the distribution of different types of

polymorphisms (synonymous, nonsynonymous, tolerated, and untolerated) varied greatly between the wolf and the breed dogs.In summary, our results suggest that different forms of selection have acted on the segregating OR pseudogenes in the CVD sincedomestication, breed dogs after breed formation, and ancestral wolf population, which has driven the evolution of these genes indifferent directions.

Key words:dog, wolf, polymorphism, purifying selection, tolerated, untolerated.

Introduction

Olfaction plays a large role in mammals, helping these animalsdiscriminate noxious foods, mates, prey, marking territories,etc. (Firestein 2001; Mombaerts 2004). Olfaction is initiated bythe binding of ligands to an olfactory receptor (OR) in the

nasal cavity, with the OR gene family being the largest genefamily in mammals (Glusman et al. 2001). Although mam-mals typically have a large number of OR genes, the numberof intact genes varies considerably between species (Haydenet al. 2010). The previous studies have identified OR pseudo-genes, and the generation of these genes have been correlatedwith evolutionary changes in specific groups. For example, inprimates, the rapid and specific loss of OR genes (Gilad et al.2003) is associated with the acquisition of trichromatic vision(Gilad et al. 2004). The fraction of the OR gene complementsthat are pseudogenes is determined by selective constraints(Rouquier et al. 2000), and these constraints have been foundto vary between human populations (Gilad and Lancet 2003).Unlike Drosophila, where specific OR genes correspond tospecific olfactory neurons (Ray et al. 2007), in mammals,the expression of OR genes is stochastically determined in aspecific olfactory neuron, and an alternate OR gene will beactivated if the first gene selected is a pseudogene (Shykind2005). It is also known that specific amino acid residues in theOR sequences are required for guiding axons to the Glomeruliin the olfactory bulb (Feinstein and Mombaerts 2004).

The dog diverged from the wolf more than 10,000 years

ago (Vila et al. 1997;Savolainen et al. 2002;Pang et al. 2009).Mitochondrial DNA studies indicate that the dog was domes-ticated from several hundred wolves in southern China (Pang

et al. 2009). During dog evolution, two bottlenecks have been

described; the first accompanied the domestication process,while the second occurred during the recent formation of themultiple modern breeds (Lindblad-Toh et al. 2005). The vil-

lage dogs, including the Chinese village dog (CVD), arethought to represent an ancestral state of dog before breed

formation and thus would not have experienced the secondbottleneck associated with breed formation (Boyko et al.

2009; Boyko et al. 2010). Thus, analysis of village dogsshould yield insight into the ancestral populations of dogsand, in comparison with modern breed dogs, the process of

breed formation. After domestication, the dog has experi-

enced both artificial and natural selections, where the selec-tion on some genes has been relaxed (Bjornerfeldt et al.

2006), whereas for others, it has been strengthened (Sutteret al. 2007). During breed formation, selection on some geneshas been greatly strengthened by artificial selection (Vila et al.

1997;Vila et al. 2005;Mosher et al. 2007), and extensive link-age disequilibrium has been found in dog breeds (Sutter et al.

2004). As there have been two major bottlenecks in dogevolutionary history, most of the phenotypes of breed dogs

can be explained by a small number of quantitative trait loci

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Mol. Biol. Evol.29(11):34753484 doi:10.1093/molbev/mss153 Advance Access publication June 7, 2012 3475


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(Lindblad-Toh et al. 2005;Gray et al. 2009;Boyko et al. 2010).The dog is an olfactory sensitive animal and has an OR generepertoire of more than 1,000 genes; however, about 20% ofthese OR genes are pseudogenes (Quignon et al. 2005). Thefraction of OR genes that are pseudogenes in the dog is muchlower than the 60% found in humans (Glusman et al. 2001)and is similar to the 20% found in the rats (Quignon et al.2005). The small fraction of pseudogenes implies that the OR

genes of dog have been under fairly intense selection pressurethrough most of their evolutionary history (Gilad et al. 2003,2004). Whether degeneration of olfaction has occurred sincedomestication is a fascinating question.

Although pseudogenes are generally considered to beunder neutral selection (Balakirev and Ayala 2003; Podlahaand Zhang 2010), it has been found that some pseudogenesplay a regulatory role for their paralogs (Balakirev and Ayala2003). The benefit or harmfulness of a gene can change de-pending on environment (Zhang 2008), and gene functioncan be lost or regained at different times (Bekpen et al. 2009).Segregating pseudogenes, genes that are intact in some indi-

viduals but pseudogenes in others within a population, wereused byGilad and Lancet (2003)to study the selection actingin different human populations. Segregating pseudogeneshave a very recent origin (Walsh 1995), thus are good candi-dates to study the loss and gain of gene function. Investigationinto OR genes has found that different domains of the se-quence are under different selection pressures (Emes et al.2004). Studies in the Pea Aphid indicated that most of thepositively selected sites in OR genes arein the transmembrane(TM) domain(Smadja et al. 2009). OR ligand-bindingsites arelocated in the extracellular regions (ECRs) between TM do-mains 3 and 6 (Vaidehi et al. 2002), and these residues arehypervariable and have variable Ka:Ks ratios in rodents (Emeset al. 2004). Differences in the selection pressure acting ondifferent domains of a protein make it possible to determinewhether a gene is under a neutral selection. Previous studieshave shown that some amino acid substitution in OR genesequences are deleterious, and the encoded protein does notfunction despite appearing to be an intact gene (Menasheet al. 2006), thus it is necessary to discriminate nondeleteriouspolymorphisms from deleterious polymorphisms. In thisstudy, we used SIFT (Ng and Henikoff 2001) to predictwhether mutations in an OR gene are untolerated, thusimpair function, or tolerated and compatible with function.We then analyzed the dynamics of the polymorphisms of the

segregating OR genes among wolves, CVDs, and dogs of dif-ferent breeds.

Materials and Methods

DNA Samples and PCR Amplification of SegregatingOR Pseudogenes

Blood from six wolves (three from Heilongjiang province,China, two from Tver province, Russia, and one fromKrasnoyarskiy Krai, Russia) and six CVDs were collected,and genomic DNA was extracted using a phenolchloroformmethod. The primers (available on request) and conditionsfor the polymerase chain reaction (PCR) amplification are

from a previous study (Robin et al. 2009). PCR productswere purified using the Watson PCR Purification Kit(Watson BioTechnologies, Shanghai). PCR products were an-alyzed on an ABI PRISM 3730 DNA Analyzer (AppliedBiosystems), and DNA sequences were edited usingDNASTAR 5.0 (DNASTAR).

Data Acquisition

We focused on 16 segregating OR pseudogenes that werepreviously identified from a sample of 109 different ORgenes in dogs of diverse breeds (the segregating OR pseudo-genes are listed in table 8 of the study byRobin et al. [2009])(supplementary table S1, Supplementary Material online). ORgene nomenclatures are from the study byQuignon et al.(2005). OR gene sequences from the wolves and CVD wereobtained in our study, with haplotypes acquired through theuse of PHASE (Scheet and Stephens 2006). The reference se-quences were from the database of a previous study (http://dogs.genouest.org/ORrepertoire.html)(Quignon et al. 2005);haplotypes and SNP frequencies for 16 segregating OR pseu-

dogenes in dogs of different breeds were obtained from aprevious study (Robin et al. 2009), and the sequences forbreed dogs were recovered with a program written in Clanguage.

Data Analysis

On the basis of the phylogenetic tree of OR genes (Quignonet al. 2005), we identified and obtained the sequences of the29 most closely related genes of each of the segregating ORgenes amplified in this study (supplementary table S2,Supplementary Materialonline). The 30 OR sequences, seg-regating pseudogene and 29 close relatives, were then aligned

with MEGA5 (Tamura et al. 2011). Alignments were thenanalyzed with SIFT (http://sift.jcvi.org) (Ng and Henikoff2001) to predict whether the observed amino acid changesin the OR protein sequences are likely to be tolerated.Insertions and deletions were not considered. Due to thepoor alignment of the N-terminal and C-terminal regions,we only considered sequences between amino acids 32 and300, in accord with the previous study (Menashe et al. 2006).The JC69 model was used to count the numbers of synony-mous and nonsynonymous sites and tolerated and untoler-ated sites. (Jukes and Cantor 1969). If all the nonsynonymouspolymorphisms between alleles at a specific position were

tolerated, then we defined this polymorphism as a toleratedpolymorphism. If at least one of the nonsynonymous poly-morphisms at a position was found to be untolerated (dam-aging), then this is an untolerated polymorphism. Thenucleotide diversity and numbers of synonymous and non-synonymous sites were calculated with DnaSP 5.1 (Libradoand Rozas 2009), and the raw data produced from SIFT wereused to count the numbers of tolerated and untolerated sites.The Fishers exact test was applied to determine whether thepolymorphism ratios were significantly different from the siteratios. We divided the dataset into a wolf population, a CVDpopulation, and the dog breed (dogs of different breeds) pop-ulation. If a polymorphism existed in only one population,

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then it was defined as a population-specific polymorphism,and thus, it was likely acquired after the foundation of thatpopulation. If a polymorphism exists in two populations, thenit was defined as a polymorphism that was lost in the thirdpopulation. Polymorphisms that were shared by more thanone population were defined as ancestral polymorphisms.

The topology of the ORs proteins was predicted usingHMMTOP (Tusnady and Simon 2001). The numbers of

each type of polymorphism in the TM domains 17 (TM17), intracellular regions (ICRs), and ECRs were counted. Thedistributions of each type of polymorphism were defined asthe number of that type in TM17, ICRs, and ECRs. As mostpolymorphisms in the NH2 and COOH tails are removedfrom the tolerated and untolerated polymorphisms analysis,we did not consider polymorphisms in the two tails duringthe polymorphism distribution analysis. We analyzed the dis-tributions of polymorphisms using two approaches. First,polymorphisms were divided into four groups: ancestral poly-morphisms, wolf-specific polymorphisms, CVD-specific poly-morphisms, and breed dog-specific polymorphisms. In each

group, the distributions of synonymous polymorphisms werecompared with the distributions of the other types (nonsy-nonymous, tolerated, and untolerated polymorphisms).Second, the numbers of synonymous, nonsynonymous, tol-erated, and untolerated polymorphisms and sites werecounted in each of the three domains (TM17, ECRs, andICRs) of the protein, and then the distributions of the num-bers of polymorphisms of each type in the four groups (an-cestral, wolf-specific, CVD-specific, and breed dog-specificpolymorphisms) were compared with the distribution ofthe sites of that type. A chi-square test with 2 degrees offreedom was used to determine whether the distribution ofsynonymous, nonsynonymous, tolerated, and untoleratedpolymorphisms across groups is significantly different fromdistribution of sites or synonymous polymorphisms.

Tajimas D (Tajima 1989), Fu and Lis D*, Fu and Lis F*, Fuand Lis D, and Fu and Lis F tests (Fu and Li 1993;Fu 1997)were used to test whether the genes in the different popula-tions were evolving neutrally using DnaSP 5.1 (Librado andRozas 2009).

Results

Differences in the Levels of Gene Diversity betweenthe Wolf and Dogs

To examine gene diversity in dogs and wolves, we investigated16 segregating OR pseudogenes that had been identified in aprevious study (Robin et al. 2009). Gene sequences were ob-tained from all CVDs and wolves for each of the segregatingOR pseudogenes except OR gene CfOR04c05 in two of theCVDs. Combining our new sequences with those from dog ofdifferent breeds, which were from a previous study (Robinet al. 2009), a total of 118 nonsynonymous and 85 synony-mous polymorphisms and 14 indels (insertions and deletions)were found. Of the polymorphisms and indels identified, 65polymorphisms and 1 indels were newly identified in the wolfand the CVD sequences (supplementary tables S3 andS4,Supplementary Material online). Of the 9 nonsense

mutations and 13 indels identified in the previous study(Robin et al. 2009), 5 of the indels and 2 of the nonsensemutations were not found in either the wolves or theCVDs. The new indel that we found was a deletion in aCVD, and two of the new polymorphisms were nonsensemutations found in wolves (supplementary table S3,Supplementary Material online). Base C at position 702 ofCfOR5912, from the OR gene repertoire (Quignon et al.

2005), is absent in all our new sequences. This base is presentin the 1.5x Poodle genome (Kirkness et al. 2003) but absent inthe 7.5x boxer genome (Lindblad-Toh et al. 2005) raising thepossibility that this is a sequencing error and that this geneshould be 900 bp long. Due to missing data after base 872, wehave included only 872 bases of this gene in our analysis.

We also calculated the proportion of the alleles at the16 segregating OR genes that were pseudogenes in thewolves, CVD, and 6 different breeds of dogs (table 1).The proportion of the alleles that were pseudogenesranged from 0% to 100% for the different subpopulation,with average pseudogenes proportions ranging from 28.7%

for the genes in CVD to 40.9% in the Labrador retriever.However, the differences in pseudogene proportions wasnot statistically significant different among the eight groups(Friedman test P = 0.629).

The geneCfOR04c05failed to produce an intact sequencefrom two individuals of CVD. In these two individuals, a PCRproduct of the expected size was generated for CfOR04c05,but the inner sequencing primer failed to generate a clearsequence due to the deletion of one base in one of the allelesin each individual. To calculate nucleotide diversity, we com-bined all gene sequences, exceptCfOR04c05. Wolves showedhigher nucleotide diversity (0.00248) than CVD, who had in-termediate nucleotide diversity (0.00228), whereas breed dogs

Table 1. Pseudogene Frequency in Wolf, CVD, and Dog Breeds for

Segregating OR Pseudogenes.

OR Name Wolf CVD BM ESS GREY GSD LR PEK

CfOR0004 0.58 0.42 0.63 0.56 0.88 0.88 0.56 0.56

CfOR0043 1.00 0.25 0.88 0.81 1.00 0.81 0.94 0.44

CfOR0135 0.00 0.00 0.00 0.06 0.00 0.00 0.00 0.00

CfOR0180 0.08 0.17 0.06 0.06 0.00 0.00 0.00 0.00

CfOR0401 0.00 0.00 0.00 0.06 0.00 0.00 0.00 0.00

CfOR0438 0.08 0.50 1.00 0.88 0.81 0.63 0.56 0.94

CfOR04C05 0.17 0.33 0.00 0.00 0.00 0.00 0.00 0.13

CfOR0519 1.00 1.00 1.00 1.00 0.94 1.00 1.00 1.00

CfOR0565 0.08 0.00 0.06 0.06 0.00 0.00 0.00 0.00

CfOR0821 0.00 0.25 0.00 0.00 0.00 0.00 0.00 0.13

CfOR08G02 0.17 0.08 0.38 0.13 0.19 0.81 0.44 0.06

CfOR12F06 1.00 0.50 0.50 0.38 0.25 0.88 0.75 0.38

CfOR14A11 0.08 0.00 0.44 0.25 0.50 0.06 0.91 0.25

CfOR16C11 0.00 0.00 0.19 0.44 0.25 0.13 0.19 0.00

CfOR3109 0.75 0.92 1.00 0.63 0.56 1.00 0.88 1.00

CfOR5912 0.08 0.17 0.19 0.00 0.19 0.25 0.31 0.06

NOTE.Data for dog breed are from the haplotypes from a previous study (Robin

et al. 2009). BM, Belgian Malinois; ESS, English Springer Spaniel; GREY, Greyhound;GSD, German Shepherd Dog; LR, Labrador Retriever; PEK, Pekingese.

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showed the lowest diversity (0.00201) (table 2). These resultsare in accord with dogs representing a bottlenecked subpop-ulation of wolves, withbreed dogs being further bottlenecked.These results are consistent with the CVD being more repre-

sentative of an ancestral dog population before breed forma-tion (Brown et al. 2011). When the ratio of nonsynonymouspolymorphisms to synonymous polymorphisms was exam-ined, it was found that wolves have the highest ratio, whereasbreed dogs have the lowest ratio (supplementary table S4,Supplementary Materialonline).

Selection on the Different Types of Polymorphisms

To analyze whether the segregating OR genes are evolvingneutrally in the different groups (wolves, CVDs, and breeddogs), we compared the ratios of nonsynonymous poly-morphisms to synonymous polymorphisms with the ratio

of nonsynonymous sites to synonymous sites. The ratio ofnonsynonymous polymorphisms to synonymous polymor-phisms is significantly lower than the ratio of nonsynon-ymous sites to synonymous sites for ancestralpolymorphisms (P< 0.01, Fishers exact test one tailed) in-dicating that the OR genes in the ancestral populations ofthe three groups (wolves, CVD, and breed dogs) were notevolving neutrally and likely were functional (fig. 1).Population-specific polymorphisms of breed dogs alsohave a ratio of nonsynonymous polymorphisms to synon-ymous polymorphisms significantly lower than the ratio ofnonsynonymous sites to synonymous sites (P< 0.01,

Fishers exact test one tailed), thus purifying selection con-tinued to occur in the breed dogs since their divergencefrom the village dogs.

Because some nonsynonymous mutations are not delete-rious (tolerated), whereas some are deleterious (untolerated),the ratio of nonsynonymous polymorphisms to synonymouspolymorphisms may not be powerful enough to detect pos-itive selection, because at best only a small proportion oftolerated mutations are potentially under positive selection.Therefore, we divided the nonsynonymous polymorphismsinto tolerated and untolerated polymorphisms. As describedin the Materials and Methods, we analyzed amino acid resi-dues 32 to 300 of the OR sequences, where a total of 102

nonsynonymous polymorphisms were identified. Of the 102

polymorphisms, 40 generate untolerated amino acid substi-tutions, whereas 62 generate tolerated amino acid substitu-

tions (supplementary table S5, Supplementary Material

online). A total of 4192 nonvariable amino acid sites (sitesthat do not have nonsynonymous polymorphisms) wereidentified from amino acid 32 to 300 (from amino acid 32to 290 inCfOR5912) of the intact copies of the 16 segregating

pseudogenes. Of these 4192 nonvariable amino acid sites, 14were identified as being untolerated amino acids by SIFT (Ngand Henikoff 2001). If the intact copies of these segregating

pseudogenes are indeed functional, then SIFT has incorrectlyassessed the functional impact of these 14 residues. However,

this result also indicates that the likelihood that functionalresidues being assessed as untolerated by SIFT is less than

0.33% (14/4192). Further strengthening our conclusion that

SIFT identified untolerated mutations that impair functionwas the observation that the mutational direction of all the

untolerated substitutions was from a tolerated amino acidancestor to an untolerated amino acid derived sequence.

Next, we compared the pairs of ratios. First, we comparedthe ratio of tolerated polymorphisms to synonymous poly-morphisms with the ratio of tolerated sites to synonymous

sites, and second, we compared the ratio of untolerated poly-morphisms to synonymous polymorphisms with the ratio ofuntolerated sites to synonymous sites. For the ancestral poly-

morphisms, we found that ratios of tolerated polymorphisms

to synonymous polymorphisms (P< 0.01, Fishers exact test

one tailed) and untolerated polymorphisms to synonymouspolymorphisms (P< 0.01, Fishers exact test one tailed) are all

significantly lower than the ratios of their sites (fig. 1). Forpolymorphisms that were specific to breed dogs, we alsofound that ratios of tolerated polymorphisms to synonymous

polymorphisms (P< 0.01, Fishers exact test one tailed) anduntolerated polymorphisms to synonymous polymorphisms

(P< 0.01, Fishers exact test one tailed) were significantlylower than the ratios of their sites (fig. 2). These observationssuggest that selection is acting strongly against both tolerated

and untolerated polymorphisms within the different breedsof dog. In the specific polymorphisms of CVD and wolf, sta-tistical tests were not significant for either the ratio of

Table 2. Test Statistics from the Neutrality Analysis of All Segregating OR Pseudogenes Except CfOR04c05.

Tajimas D Nucleotide Diversity Fu and Lis D* Fu and Lis F* Fu and Lis D Fu and Lis F

All samples 0.40907 0.00232 0.95052 0.83822

Wolf 0.43860 0.00248 0.46486 0.52246 0.46892 0.57499

CVD 0.15074 0.00228 0.20661 0.21890 0.15941 0.19310

Breeds 0.72885 0.00201 0.58063 0.77230 0.72911 0.56698

BM 0.35535 0.00170 0.12812 0.22285 0.29702 0.23305

ESS 0.29364 0.00198 0.06332 0.04436 0.11361 0.03280

GREY 0.76230 0.00150 0.20054 0.41620 0.76024 0.97887

GSD 0.51345 0.00160 0.20012 0.33429 0.24500 0.36662

LR 0.45971 0.00181 0.53464 0.59341 0.49581 0.57840

PEK 0.46256 0.00170 0.06292 0.10008 0.00942 0.16172

NOTE.Summary statistics for Tajimas D, Fu and Lis D*, Fu and Lis F*, Fu and Lis D, and Fu and Lis F were calculated for the entire sample and the different populations (wolf,

CVD, and different breeds of dog). BM, Belgian Malinois; ESS, English Springer Spaniel; GREY, Greyhound; GSD, German Shepherd Dog; LR, Labrador Retriever; PEK, Pekingese.

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tolerated polymorphisms to synonymous polymorphisms orthe ratio of untolerated polymorphisms to synonymous poly-morphisms, when compared with the ratios of their sites.However, for specific polymorphisms within wolves, the

number of observed tolerated polymorphisms (19) wasgreater than expected (11.1), whereas the number of untol-erated polymorphisms (8) was fewer than expected (14.6)under the expectation that the number of these polymor-phisms should be proportional to the numbers of these sites.A similar observation is seen within CVD. These observationssuggest that within CVD and wolves, there was a greaterlikelihood that tolerated changes were maintained, but thatselection was still acting strongly against untolerated changes.The differences in the ratios of the different types of polymor-phisms and their sites ratios in the three subpopulations(wolves, CVD, and breed dogs) suggest that different selectionpressures are acting on the different subpopulations since

their divergences.The protein encoded by CfOR0180 was predicted by

HMMTOP to have only six rather than the expected sevenTM domains; therefore, it was removed from our analysis ofthe distributions of polymorphisms. The distributions of non-synonymous, tolerated, and untolerated polymorphisms inthe ancestral polymorphisms were different from the distri-bution of synonymous polymorphisms, as fewer nonsynon-ymous, tolerated, and untolerated polymorphisms found inTM17 and more being located in the ECRs and ICRs of theprotein. When the polymorphisms that were specific to thesubpopulations were examined, differences were observed. In

the wolf, the distribution of the nonsynonymous polymor-phisms is different from that of the synonymous polymor-phisms (fig. 3); however, in breed dogs, we found that thedistributions of all four types of polymorphisms were almostthe same. These observations suggest that selection againstnonsynonymous, tolerated, and untolerated polymorphismsin TM17 has changed in breed dogs. Then the distributionsof the ancestral, wolf-specific, CVD-specific, and breed dog-specific polymorphisms were compared with the sites distri-butions of the four polymorphic types (synonymous, nonsy-nonymous, tolerated, and untolerated polymorphisms)(fig. 4). Synonymous polymorphisms were found only inTM17 of CVD. For synonymous polymorphisms in the

other three subpopulations (ancestral, wolf-specific, and

breed-specific polymorphisms), no statistically significant dif-ference was found. In the distribution of the other three typespolymorphisms (nonsynonymous, tolerated, and untolerated

polymorphisms), statistically significant (P< 0.05) differenceswere only found in the ancestral and wolf-specific polymor-

phisms, suggesting that selection has changed sincedomestication.

Neutral Test on Segregating Pseudogenes in DifferentPopulations

To attempt to identify the type of selection that may haveoccurred in the three subpopulations, we applied Tajimas D,

Fu and Lis D, and Fu and Lis F tests. However, these tests canbe influenced by population demographic dynamics in addi-

tion to selection. Negative values from these tests indicate anexcess of rare alleles, which may be caused by positive selec-

tion, negative selection, or population expansion. Positivevalues indicate an excess of intermediate alleles, which may

be due to balancing selection or a population bottleneck.Negative values for these tests were generated by the wolfand CVD data, whereas the breed dogs yield positive values

(table 2). To distinguish selection from population dynamics,

we separated nonsynonymous polymorphisms from synony-mous polymorphisms, and for the nonsynonymous polymor-phisms, we separated them into tolerated and untolerated

polymorphisms (supplementary table S6, Supplementary

Material online). Synonymous polymorphisms should be neu-tral, so that they can be used to detect population history.Negative values from these tests using synonymous poly-morphisms for the wolf suggest that they are experiencing

a population expansion, whereas positive value for the breed

dogs suggests that they experienced a bottleneck. The teststatistics for the CVD is near zero, which suggests that the

CVD population size has remained nearly constant. Becausepopulationdynamics do not influence the test statistics in theCVD, in contrast to the wolf and breed dogs, we can then

attribute the positive values from Tajimas D, Fu and Lis D,and Fu and Lis F tests for balancing selection acting in the

CVD population.

FIG. 1. Polymorphisms in the segregating OR pseudogenes in the wolf, CVD, and breed dogs. Venn diagram shows the sharing of polymorphisms in the

segregating OR pseudogenes. (A) Pairs of numbers represent nonsynonymous/synonymous polymorphisms. (B) Pairs of numbers represent tol-

erated/untolerated polymorphisms.

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Discussion

Nucleotide Diversities of Segregating OR Pseudogenes

OR gene families adapt to the physical requirements of dif-

ferent species (Hayden et al. 2010), since olfaction is very

important for mammals (Szetei et al. 2003). The ecologicalniche of dog has changed greatly since domestication fromthe wolf (Morey 1994); thus, it is likely that changes in the

composition of the OR gene family have occurred between

wolf and dog. The dog only recently diverged from the wolf,about 16,000 years ago (Pang et al. 2009), and there has beensome backcrossing since domestication (Savolainen et al.2002;Vila et al. 2005), which might explain why no significant

difference in the OR pseudogene fraction between the wolf

and dog was found (Zhang et al. 2011). In this study, wefocused on 16 OR genes that had been previously shown tobe segregating pseudogenes in breed dogs (Robin et al. 2009).

We characterized these 16 genes in wolves, CVDs, and breeddogs and examined distribution patterns of polymorphisms

to identify whether the selective forces acting on these se-quences differ between the subpopulations.

The nucleotide diversities of the segregating OR pseudo-

genes, except CfOR04c05, were found to range from 0.00201 inthe breed dogs to 0.00248 in wolves (table 2), values that are alittle higher than the nucleotide diversity (y

p= 0.0018) calcu-

lated in the previous study of all 105 OR genes in breed dogs

(Robin et al. 2009). Because the expected yof genes based onthe entire dog genome is 0.0005 (based on y = 4Ne)(Lindblad-Toh et al. 2005), there may be three reasons for

the elevated levels seen in the segregating OR pseudogenes.First, the necessity to interact with an enormous diversity of

potential ligands may be the cause of the high nucleotidediversity of OR genes (Hayden et al. 2010). Second, balancing

selection is known to be an important element in maintaininghigh nucleotide diversity, and this type of selection might be

different between functional genes and segregating pseudo-genes (Alonso et al. 2008). Third, the reduced effective pop-

ulation size of dog, especially breed dogs, may have led tolower nucleotide diversity in those dogs (Lindblad-Toh et al.2005).

The gray wolf has experienced population contraction andexpansion in the past 10,000 years (Lucchini et al. 2004), while

FIG. 2. Gain and loss of polymorphisms in the segregating OR pseudogenes during the evolutionary history of the wolf, CVD, and dog breeds. (AandC).

Numbers in the rectangular box are nonsynonymous to synonymous (nonsynonymous/synonymous) polymorphism ratios. Numbers above the line

are the numbers of polymorphisms gained, whereas the numbers below the line are the numbers of polymorphisms lost. (BandD). Numbers in the

rectangular box are ratios of tolerated to untolerated polymorphisms (tolerated/untolerated). Numbers above the line are the numbers of polymor-

phisms gained, whereas the numbers below the line are the numbers of polymorphisms lost. BM, Belgian Malinois; ESS, English Springer Spaniel; GREY,

Greyhound; GSD, German Shepherd Dog; LR, Labrador Retriever; PEK, Pekingese. Double or single asterisks indicate that the ratios of polymorphisms

(nonsynonymous, tolerated, or untolerated polymorphisms) to synonymous polymorphisms are significantly different (**P< 0.05) or show a trend(*0.05< P< 0.1) from the sites ratios (nonsynonymous, tolerated, or untolerated sites to synonymous sites).

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at the same time, the domesticated dog has gone throughtwo bottlenecks due to domestication and breed formation(Lindblad-Toh et al. 2005). Negative values for synonymouspolymorphisms are consistent with the expansion of the wolfpopulation, whereas the near zero values indicate that theCVD population has remained nearly constant. Positivevalues for synonymous polymorphisms demonstrate thatthe breed dogs have experienced a bottleneck. Previous stud-ies have shown that the bottleneck associated with breedformation was much more intensive than the bottleneckthat occurred during domestication (Boyko et al. 2010);thus, the much lower values for nucleotide diversity observed

within breeds in our data is in accord with this conclusion.However, population demography alone cannot explain thelower ratios of nonsynonymous polymorphisms to synony-mous polymorphisms and suggests that selection continuesto act on the segregating pseudogenes.

Selection on the Segregating OR Pseudogenes

OR genes in humans are maintained by balancing selection(Alonso et al. 2008). Higher values from the neutrality tests onnonsynonymous polymorphisms indicate that the segregat-ing OR pseudogenes may be under balancing selection; how-ever, the values was not significant, hence, we cannot make a

strong conclusion. To refine our results, we divided the non-synonymous polymorphisms into tolerated and untoleratedpolymorphisms based on their predicted effects on proteinfunction. Untolerated substitutions are predicted to disruptfunction (Ng and Henikoff 2001), whereas tolerated substitu-tions are not incompatible with function. However, accuracyof this analysis depends on the power of SIFT, and specificcharacters of OR genes may influence the results of this anal-ysis. Because ORs must interact with a huge number of po-tential ligands in nature, tolerated polymorphisms in theextracellular domains may be beneficial for generating thediversity of OR function(Alonso et al. 2008). Discrimination

of untolerated polymorphisms from tolerated polymor-phisms in the nonsynonymous polymorphism analysisshould allow a more accurate detection of selection, as puri-fying selection on the deleterious polymorphisms shouldreduce the ratio of nonsynonymous to synonymous polymor-phisms. On the other hand, positive selection acting on ben-eficial polymorphisms would increase the nonsynonymous tosynonymous ratio. With this analysis, by using the ratios ofuntolerated polymorphisms to synonymous polymorphismsand tolerated polymorphisms to synonymous polymor-phisms, we can distinguish purifying from positive selection.In the wolf, selection is removing the untolerated polymor-phisms and preserving the tolerated polymorphisms, whereas

FIG. 3. Distribution of polymorphisms in three domains of OR proteins (TM17, ICRs, and ECRs). Distribution of the four polymorphism types

(synonymous polymorphisms [syn], nonsynonymous polymorphisms [nonsyn], tolerated polymorphisms [tole], untolerated polymorphisms [untole])

are shown for each domain. (A) Analysis of ancestral polymorphisms. (B) Analysis of specific polymorphisms of wolf. (C) Analysis of specific poly-

morphisms of CVD. (D) Analysis of specific polymorphisms of breed dogs. Significant differences in the patterns of distributions of sites to synonymous,

nonsynonymous, tolerated, or untolerated polymorphisms were determined by a chi-square test with 2 degrees of freedom. P value of the statistical

significance is shown above each significant type.

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in the breed dogs, purifying selection is acting on both theuntolerated and tolerated polymorphisms. Gene conversionand recombination may help OR pseudogenes to becomefunctional genes (Sharon et al. 1999), and our findings dem-

onstrate that selection may also remove pseudogenes to pre-serve the functional genes.

Selection acts differently on the different domains of che-mosensory receptors and on the orthologous genes in differ-ent species (Shi and Zhang 2006; Smadja et al. 2009). We

analyzed the distribution patterns of polymorphisms acrossthe different domains of the OR protein for the segregatingOR pseudogenes and found there were significant differences

between the distribution patterns of the synonymous poly-morphisms with polymorphisms of other types (nonsynon-ymous, tolerated, and untolerated) for the ancestral

polymorphisms. However, in the subpopulation-specific poly-morphisms, within breed dogs, the patterns of distribution forthe three types of polymorphisms (nonsynonymous, toler-

ated, and untolerated) are almost the same as the patternof distribution for synonymous polymorphisms (fig. 3). Incontrast, in the wolf, the patterns of distribution for thethree types of polymorphisms differ from that for synony-

mous polymorphisms, with the wolf having a pattern moresimilar to the pattern of distributions for the ancestral poly-morphisms (fig. 3). When the distribution patterns of the

polymorphisms are compared with the patterns for the dis-tribution of the sites, our statistical analysis did not find anysignificant differences for the CVD- or breed dog-specific(fig. 4), nonsynonymous, tolerated, and untolerated polymor-

phisms. In contrast, the pattern of distributions for thewolf-specific and ancestral nonsynonymous and untoleratedpolymorphisms are statistically significant (P< 0.05) or have a

significant trend (0.05< P< 0.1), that is, different from thedistribution patterns for the sites, suggesting that the selec-tion acting on these sites has changed since domestication.

In my previous analysis, a low ratio of nonsynonymouspolymorphisms to synonymous polymorphisms in ancestral

polymorphisms and specific polymorphisms of breed dogswas found. Our polymorphism distribution analysis suggeststhat the reasons for the similar patterns for ancestral and wolf

polymorphisms compared with breed dogs are not identical.The low ratio for the ancestral polymorphisms may be ex-plained by accelerated fixation of beneficial mutations due to

positive selection and elimination of deleterious mutations bypurifying selection. It is not simply the result of purifyingselection. Accumulation of tolerated polymorphisms wasalso enhanced in wolves, with the number of untolerated

polymorphisms reduced, in accord with this conclusion.The low ratio observed in the specific polymorphisms ofbreed dogs is likely the result of artificial selection. The history

FIG. 4. Distribution of sites and polymorphisms across three domains of OR proteins (TM17, ICRs, and ECRs). Distributions were analyzed in four

groups (ancestral polymorphisms [AP], specific polymorphisms of wolf [WP], specific polymorphisms of CVD [CP], specific polymorphisms of dog

breeds [BP]). (A) Synonymous (syn) sites and polymorphisms. (B) Nonsynonymous (nonsyn) sites and polymorphisms. (C) Tolerated (tole) sites and

polymorphisms. (D) Untolerated (untole) sites and polymorphisms. Significant differences in the patterns of distributions of sites to synonymous,

nonsynonymous, tolerated, or untolerated polymorphisms were determined by a chi-square test with 2 degrees of freedom. P value of the statistical

significance is shown above each significant type.

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of most dog breeds is not more than 400 years (Parker et al.2004). A great amount of linkage disequilibrium is found inthe breed dogs (Sutter et al. 2004). Artificial selection, a formof purifying selection in our study, is not as complex as naturalselections (Boyko et al. 2010) and may not be powerfulenough to discriminate all tolerated and untolerated muta-tions. Our results suggest that selection on the OR genes haschanged a great deal since the divergence of the dog breeds,

whereas the analysis of the segregating OR pseudogenes inwolves indicates that they are still undergoing selection thatfavors functional genes.

Conclusion

Segregating pseudogenes exist during a subtle period of time,where they could become a functional or pseudogene withina population. In our investigation, we found that in naturalpopulations, segregating OR pseudogene and pseudoallelesshowed a trend to be eliminated in competition with func-tional alleles, whereas within domesticated dog, this trend haschanged. In the CVD, these genes are evolving neutrally,

whereas in breed dogs, they are evolving under artificial pu-rifying selection. Genes can be beneficial in one period of timeand then become deleterious in another period (Olson 1999;Zhang 2008). Our analysis demonstrates the selection actingon segregating OR pseudogenes has changed during the do-mestication of dogs, and thus, these genes are evolving indifferent ways among wolves, CVD, and breed dogs.

Supplementary Material

Supplementary tables S1S6and sequence information areavailable at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).

Acknowledgments

The authors thank Guodong Wang for valuable advice andStephanie Robin and Francis Galibert for their kind help. Theyalso thank Dr John H. McDonald and anonymous reviewersfor their comments and suggestions. This work was sup-ported by grants from the National Basic Research Programof China (973 Program), Chinese Academy of Sciences, Bureauof Science and Technology of Yunnan Province, and NationalNatural Science Foundation of China.

ReferencesAlonso S, Lopez S, Izagirre N, de la Rua C.. 2008. Overdominance in the

human genome and olfactory receptor activity. Mol Biol Evol. 25:9971001.

Balakirev ES, Ayala FJ. 2003. Pseudogenes: are they "junk" or functional

DNA?Annu Rev Genet. 37:123151.

Bekpen C, Marques-Bonet T, Alkan C, Antonacci F, Leogrande MB,

Ventura M, Kidd JM, Siswara P, Howard JC, Eichler EE. 2009.

Death and resurrection of the human IRGM gene.PLoS Genet. 5:

e1000403.

Bjornerfeldt S, Webster MT, Vila C. 2006. Relaxation of selective con-

straint on dog mitochondrial DNA following domestication.

Genome Res. 16:990994.

Boyko AR, Boyko RH, Boyko CM, et al. (15 co-authors). 2009. Complex

population structure in African village dogs and its implications for

inferring dog domestication history.Proc Natl Acad Sci U S A. 106:

1390313908.

Boyko AR, Quignon P, Li L, et al. (24 co-authors). 2010. A simple genetic

architecture underlies morphological variation in dogs.PLoS Biol.8:

e1000451.

Brown SK, Pedersen NC, Jafarishorijeh S, Bannasch DL, Ahrens KD, Wu

JT, Okon M, Sacks BN. 2011. Phylogenetic distinctiveness of Middle

Eastern and Southeast Asian village dog Y chromosomes illuminates

dog origins.PLoS One 6:e28496.Emes RD, Beatson SA, Ponting CP, Goodstadt L. 2004. Evolution and

comparative genomics of odorant- and pheromone-associated

genes in rodents.Genome Res. 14:591602.

Feinstein P, Mombaerts P. 2004. A contextual model for axonal sorting

into glomeruli in the mouse olfactory system. Cell 117:817831.

Firestein S. 2001. How the olfactory system makes sense of scents.

Nature 413:211218.

Fu YX. 1997. Statistical tests of neutrality of mutations against popula-

tion growth, hitchhiking and background selection. Genetics 147:

915925.

Fu YX, Li WH. 1993. Statistical tests of neutrality of mutations. Genetics

133:693709.

Gilad Y, Lancet D.. 2003. Population differences in the human functional

olfactory repertoire.Mol Biol Evol. 20:307314.

Gilad Y, Man O, Paabo S, LancetD. 2003. Human specific loss of olfactory

receptor genes.Proc Natl Acad Sci U S A. 100:33243327.

Gilad Y, Przeworski M, Lancet D. 2004. Loss of olfactory receptor genes

coincides with the acquisition of full trichromatic vision in primates.

PLoS Biol.2:E5.

Glusman G, Yanai I, Rubin I, Lancet D. 2001. The complete human

olfactory subgenome.Genome Res.11:685702.

Gray MM, Granka JM, Bustamante CD, Sutter NB, Boyko AR, Zhu L,

Ostrander EA, Wayne RK. 2009. Linkage disequilibrium and demo-

graphic history of wild and domestic canids. Genetics 181:

14931505.Hayden S, Bekaert M, Crider TA, Mariani S, Murphy WJ, Teeling EC.

2010. Ecological adaptation determines functional mammalian

olfactory subgenomes.Genome Res. 20:19.

Jukes TH, Cantor CR. 1969. Evolution of protein molecules. In: Munro

HN, editor. Mammalian protein metabolism. New York: Academy

Press. p. 21123.

Kirkness EF, Bafna V, Halpern AL, et al. (11 co-authors). 2003. The dog

genome: survey sequencing and comparative analysis.Science 301:

18981903.

Librado P, Rozas J. 2009. DnaSP v5: a software for comprehensive analysis

of DNA polymorphism data.Bioinformatics 25:14511452.

Lindblad-Toh K, Wade CM, Mikkelsen TS, et al. (234 co-authors). 2005.

Genome sequence, comparative analysis and haplotype structure ofthe domestic dog. Nature 438:803819.

Lucchini V, Galov A, Randi E. 2004. Evidence of genetic distinction and

long-term population decline in wolves (Canis lupus) in the Italian

Apennines.Mol Ecol. 13:523536.

Menashe I, Aloni R, Lancet D. 2006. A probabilistic classifier for olfactory

receptor pseudogenes.BMC Bioinformatics 7:393.

Mombaerts P. 2004. Genes and ligands for odorant, vomeronasal and

taste receptors.Nat Rev Neurosci. 5:263278.

Morey DF. 1994. The early evolution of the domestic dog. Am Sci. 82:

336347.

Mosher DS, Quignon P, Bustamante CD, Sutter NB, Mellersh CS, Parker

HG, Ostrander EA. 2007. A mutation in the myostatin gene

3483

http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-


10/10

increases muscle mass and enhances racing performance in hetero-

zygote dogs.PLoS Genet. 3:e79.

Ng PC, Henikoff S. 2001. Predicting deleterious amino acid substitutions.

Genome Res. 11:863874.

Olson MV. 1999. When less is more: gene loss as an engine of evolu-

tionary change.Am J Hum Genet. 64:1823.

Pang JF, Kluetsch C, Zou XJ, et al. (14 co-authors). 2009. mtDNA data

indicate a single origin for dogs south of Yangtze River, less than

16,300 years ago, from numerous wolves. Mol Biol Evol. 26:28492864.

Parker HG, Kim LV, Sutter NB, Carlson S, Lorentzen TD, Malek TB,

Johnson GS, DeFrance HB, Ostrander EA, Kruglyak L. 2004.

Genetic structure of the purebred domestic dog. Science 304:

11601164.

Podlaha O, Zhang J. 2010. Pseudogenes and their evolution. In:

Encyclopedia of life sciences (ELS). Chichester (United Kingdom):

John Wiley & Sons, Ltd.

Quignon P, Giraud M, Rimbault M, et al. (11 co-authors). 2005. The dog

and rat olfactory receptor repertoires.Genome Biol. 6:R83.

Ray A, van Naters WG, Shiraiwa T, Carlson JR. 2007. Mechanisms of odor

receptor gene choice in Drosophila.Neuron 53:353369.

Robin S, Tacher S, Rimbault M, Vaysse A, Dreano S, Andre C, Hitte C,

Galibert F. 2009. Genetic diversity of canine olfactory receptors.BMC

Genomics10:21.

Rouquier S, Blancher A, Giorgi D. 2000. The olfactory receptor gene

repertoire in primates and mouse: evidence for reduction of the

functional fraction in primates. Proc Natl Acad Sci U S A. 97:

28702874.

Savolainen P, Zhang YP, Luo J, Lundeberg J, Leitner T. 2002. Genetic

evidence for an East Asian origin of domestic dogs. Science 298:

16101613.

Scheet P, Stephens M. 2006. A fast and flexible statistical model for

large-scale population genotype data: applications to inferring

missing genotypes and haplotypic phase. Am J Hum Genet. 78:

629644.

Sharon D, Glusman G, Pilpel Y, Khen M, Gruetzner F, Haaf T, Lancet D.

1999. Primate evolution of an olfactory receptor cluster: diversifica-

tion by gene conversion and recent emergence of pseudogenes.

Genomics 61:2436.

Shi P, Zhang J.. 2006. Contrasting modes of evolution between verte-

brate sweet/umami receptor genes and bitter receptor genes.Mol

Biol Evol. 23:292300.

Shykind BM. 2005. Regulation of odorant receptors: one allele at a time.

Hum Mol Genet. 14:R33R39.

Smadja C, Shi P, Butlin RK, Robertson HM. 2009. Large gene family

expansions and adaptive evolution for odorant and gustatory re-

ceptors in the pea aphid.Acyrthosiphon pisum. Mol Biol Evol. 26:

20732086.

Sutter NB, Bustamante CD, Chase K, et al. (21 co-authors). 2007. A single

IGF1 allele is a major determinant of small size in dogs. Science316:

112115.Sutter NB, Eberle MA, Parker HG, Pullar BJ, Kirkness EF, Kruglyak L,

Ostrander EA. 2004. Extensive and breed-specific linkage disequilib-

rium in Canis familiaris.Genome Res. 14:23882396.

Szetei V, Miklosi A, Topal J, Csanyi V. 2003. When dogs seem to lose their

nose: an investigation on the use of visual and olfactory cues in

communicative context between dog and owner.Appl Anim Behav

Sci. 83:141152.

Tajima F. 1989. Statistical method for testing the neutral mutation hy-

pothesis by DNA polymorphism.Genetics 123:585595.

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S.. 2011.

MEGA5: molecular evolutionary genetics analysis using maximum

likelihood, evolutionary distance, and maximum parsimony meth-

ods.Mol Biol Evol. 28:27312739.Tusnady GE, Simon I. 2001. The HMMTOP transmembrane topology

prediction server.Bioinformatics17:849850.

Vaidehi N, Floriano WB, Trabanino R, Hall SE, Freddolino P, Choi EJ,

Zamanakos G, Goddard WA III. 2002. Prediction of structure and

function of G protein-coupled receptors.Proc Natl Acad Sci U S A.

99:1262212627.

Vila C, Savolainen P, Maldonado JE, Amorim IR, Rice JE, Honeycutt RL,

Crandall KA, Lundeberg J, Wayne RK. 1997. Multiple and ancient

origins of the domestic dog.Science 276:16871689.

Vila C, Seddon J, Ellegren H. 2005. Genes of domestic mammals aug-

mented by backcrossing with wild ancestors. Trends Genet. 21:

214218.

Walsh JB. 1995. How often do duplicated genes evolve new functions?Genetics 139:421428.

Zhang H-h, Wei Q-g, Zhang H-x, Chen L. 2011. Comparison of the

fraction of olfactory receptor pseudogenes in wolf (Canis lupus)

with domestic dog (Canis familiaris).J Forest Res. 22:275280.

Zhang J. 2008. Positive selection, not negative selection, in the pseudo-

genization of rcsA inYersinia pestis.Proc Natl Acad Sci U S A. 105:

E69.

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Differences in Selection Drive Olfactory Receptor Genes in Different Directions in Dogs and Wolf