Artificial Selection Drives SNPs of Olfactory Receptor Genes into Different Working Traits in Labrador Retrievers

Labs as guide dogs or sniffer dogs in usage have been introduced into China for more than 20 years. These two types of working dogs own blunt or acute olfactory senses, which have been obtained by artificial selection in relatively closed populations. In order to attain stable olfactory attributes and meet use-oriented demands, Chinese breeders keep doing the same artificial selection. Though olfactory behavior is canine genetic behavior, genotypes of OR genes formed by breeding schemes are largely unknown. Here, we characterized 26 SNPs, 2 deletions, and 2 insertions of 7 OR genes between sniffer dogs and guide dogs in order to find out the candidate alleles associated with working specific traits. The results showed that there were candidate functional SNP alleles in one locus that had statistically severely significant differences between the two subpopulations. Furthermore, the levels of polymorphism were not high in all loci and linkage disequilibrium only happened within one OR gene. Hardy–Weinberg equilibrium (HWE) tests showed that there was a higher ratio not in HWE and lower FST within the two working dog populations. We conclude that artificial selection in working capacities has acted on SNP alleles of OR genes in a dog breed and driven the evolution in compliance with people's intentions though the changes are limited in decades of strategic breeding.


Background
Artificial selection has been largely responsible for breedspecific and strain-specific traits of modern dogs [1]. Compared with early domestication of a dog breed driven by natural factors such as dietary conditions and geographical environments, the recent subpopulation differentiations are more dramatically caused by artificial goal-directed breeding practices, especially during the last two centuries [2]. Straindependent differences of dogs are mainly presented in body size, coat color, and work capacity [3,4]. Labrador Retriever (Lab) is a traditional waterdog in Newfoundland. During the latter half of the nineteenth century, British breeders had refined and standardized Labs that had become a popular breed because they were famously friendly and exuberant around the world [5][6][7]. Many Labs play an important role such as sniffer dogs or guide dogs in many countries. ese two types of work abilities are based on either enhanced olfaction or blunt olfaction. In order to attain stable olfactory attributes and meet use-oriented demands, Labs are bred in relatively closed populations by strict selection [8,9]. e offsprings of pure breed Labs as guide dogs or sniffer dogs abroad were purchased from Japan by guide dogs training organizations and from Germany by police departments into China two decades ago. e same as foreign ones did, Chinese working dog organizations continued to breed Labs following the principle of closed-flock reproduction and occasionally adopted some excellent bloodlines of Labs from amateurs in the civilian clubs into Lab working dogs populations. Based on Labs' health, a temperament test of scent interest and a search test divide Labs into candidates of guide dogs with no scent interest or sniffer dogs displaying great scent interest and eager searching during early training [10][11][12].
en, behavioral suitability of a working dog is shaped by specialized training [8,13]. Subsequently, the olfactory detection phenotype of qualified sniffer dogs is that the dog indicates a target odor in the manner in which it was trained while qualified guide dogs could navigate by avoiding obstacles [14][15][16]. As far as disqualified Labs are concerned, their outcome is to be screened out of the subpopulations. erefore, the offsprings did not degenerate and gradually became more competent for mobility support for the visually impaired/blind or scent detection for law implement. Detailed subgroup analyses are expected to explain the genetic basis for variation in olfactory behavior and to establish protocols for the reproduction of the two Labs' working strains.
Olfactory capacity and sensitivity are related to canine olfactory receptor gene polymorphism in dog breeds and individuals [9,13]. Selections of different forms have acted on OR genes in dogs since domestication. e genetic diversity of 109 OR genes in six breeds including Labs was studied, which were representative of a large number of families, subfamilies, and clusters [17]. rough OR genes sequencing data analysis, the SNPs were identified and its Minor Allele Frequency (MAF) values testified that the 8 SNPs in 7 OR genes were private and restricted to only Labs. Because most of private SNPs arose following dog-breed formation under the selection from breeding practices and SNP alleles might present a pair of phenotypes' pros and cons in olfactory cognition, 30 SNPs including 7 private SNPs and 23 representative SNPs in the same OR genes might be studied to find out how the artificial selection drives them into phenotypic differentiation of olfactory behavior in Labs. Identifying the distributions of SNP alleles between the two subgroups might provide insights into genetic variation that might explain specific working traits and then efficiently obtain a desired phenotype [18].

Ethical Statement.
is study was carried out in Police-Dog Technology Department, Criminal Investigation Police University of China. e protocol for the animal experiment was reviewed and approved by the Ethics Committee of Police-Dog Technology Department (JQJS2019-0001), and all applicable institutional and governmental regulations concerning the ethical use of animals were followed.

Animals and Blood Sample Collection.
e trial included two subpopulations, 49 guide dogs (males, n � 36; females, n � 13) and 43 sniffer dogs (males, n � 31; females, n � 12) of Labs, whose ages ranged from 2 to 4 years old. e Labs were reproduced by China Guide Dog Training Center and Police-Dog Technology Department, Criminal Investigation Police University of China. According to the certificates of the dogs' pedigree, the two groups sampled were unrelated. All the dogs treated in a humane manner passed the qualification assessments of working dogs and had been competent for the tasks assigned. Blood samples were obtained from each dog, and then Universal Genomic DNA Extraction Kit (Takara, China) was used to extract DNAs.

SNP and Primer
Design. 30 SNP loci lying in exonic regions of 7 OR genes were analyzed and are shown in Table 1. Asterisks marked out the breed-specific SNPs [17]. No asterisks meant that the SNPs could be found in other dog breeds besides Labs. Because non-breed-specific SNPs in the 7 OR genes led to amino-acid substitutions, frameshift variants, protein altering variants, and splicing donor variants, they were studied too. Pairs of specific primers for fraction amplification of 7 OR genes were designed using the Primer-BLAST and Primer Premier 5 program. e primer sequences, fragment sizes, and annealing temperatures (Tm) above mentioned are shown in Table S1.

PCR and Sequencing.
Polymerase chain reaction (PCR) was conducted using a Veriti thermal cycler (ABI, USA) in a total volume of 50 μL that included 2 μL 100 ng of genomic DNA, 25 μL Premix Taq (Takara, China), and 1 μM (final conc.) of each specific primer and water up to 50 μL. PCR products were loaded onto a 2% agarose gel stained with GeneFinder (Biov, China) and DNAs were extracted from the gel. e purified products of PCR were cloned into pMD18-T vector (Takara, China) and positive clones specific for each of the seven amplicons were sequenced in both directions. Nucleotide BLAST of the sequences was done.
en the purified products of PCR were massively directly sequenced by the forward or reverse primers.
Sequencing PCR reaction was performed in a 10 μl reaction system that consisted of Premix 4 μL, the purified PCR products 0.5 μL, primer 1 μL (1 μM), and 4.5 μL ddH 2 O, starting with 30 cycles of denaturation at 96°C for 20 secs, annealing at 55°C for 20 secs, and extension at 60°C for 4 mins, followed by an extra stop reaction initiated by adding 0.5 μL of glycogen, 1 μL of 3M sodium acetate (pH5.2), and 1 μL of 100 mM Na 2 -EDTA (pH8.0) per reaction. 40 μL of sample loading solution was added into DNA precipitates with ethanol in individual tubes to resuspend the samples. e capillary electrophoresis was analyzed on Beckman GeXP Genetic Analysis System according to Genome-Lab Dye Terminator Cycle Sequencing with Quick Start Kit (Beckman, USA).

Results
Allele frequencies and genotype frequencies of 30 SNP loci are given in Table 2, which ranged from 0 to 1. Overall, the most of the SNP loci had a major allele whose frequencies were beyond 0.8 except for six loci in guide dogs, seven loci in sniffer dogs, and seven loci in the collective. Furthermore, a unique homozygous genotype was presented in 12 SNP loci, while the only heterozygous genotype was found in 2 SNP loci within all Labs. So, the 14 SNP loci were excluded in the chi-square test for differences of alleles on a single SNP locus in a pairwise study. Among the 12 homozygous loci, the deletions of the loci OR16C11:c.633del and OR7215: c.398del that could result in no expression of gene OR16C11 and a splice donor variant of gene OR7215 did not happen and the insertion of OR7215:c.272ins producing a protein altering variant did not happen either. Noticeably the insertion of OR7215:c.273insCTTCCA producing a frameshift variant occurred to all Labs. Regarding the remaining 16 loci, there were differences either in genotypes or in genotype frequencies between sniffer dogs and guide dogs. All three genotypes were found in the two loci OR0007:c.691 G > C and OR0007:c.830 C >T only in guide dogs while sniffer dogs presented GG or GC genotype in the locus OR0007: c.691 G > C and were all homozygotes with TT genotype in the locus OR0007:c.830 C >T. Consequently, the alleles in seven SNP loci showed significant differences (P < 0.05), and highly significant differences (P < 0.001) were shown in the four SNP loci of which the three SNP loci were breed-specific between the two working groups given in Table 3. On the basis of differences of the SNP alleles between sniffer dogs and guide dogs, a binary logistic regression analysis was carried out, and it found that the alleles and genotypes in five SNP loci could affect types of working dogs (P < 0.05) given in Table 4.
In this study, except for 12 homozygous monogenotype loci, PIC values of 18 SNPs ranged from 0 to 0.375. Among the 18 SNPs, 10 SNPs belonged to low polymorphism (PIC<0.25) and 5 SNPs belonged to moderate polymorphism (0.25 < PIC<0.5) for all Labs. As far as the other three SNPs, OR0007:c.331 T >C, OR0007:c.430 G > A, and

Genetics Research
OR7215:c.251 C >T, were concerned, they belonged to either low or moderate polymorphism, which differed between guide dogs and sniffer dogs (seen in Table 5). As could be seen in Table 5, Ho values were within the wide scope of 0 to 1 and He values were not more than 0.5. ere were unanimously low Ho values in 12 SNP loci (<0.5) and high Ho values in 4 SNP loci (≥0.5) in the two working groups except that the values varied in the SNP loci of OR0007: c.331 T >C and OR0007:c.829C>T between guide dogs and sniffer dogs. P values of HWE tests for 18 SNP loci were given in Table 5. e total number of genotyped Labs was 92 and so HWE P � 0.0005 emphasized the need for multiple comparisons by applying Bonferroni correction (the significant level � 0.05/92). Except 12 homozygous monogenotype loci, the results revealed that 5 SNP loci were out of HWE (P < 0.0005) and 5 SNP loci were in the state of Hardy-Weinberg balance (P > 0.0005) for all Labs. e other eight SNP loci showed either significant departure or no significant deviations from HWE between working populations, while six of the eight SNP loci were in HWE in the collective. After the two working groups and the collective were analyzed, respectively, there was a higher ratio 73.3% not in HWE in sniffer dogs than 66.7% in guide dogs and 63.3% in the collective. Furthermore, the value of genetic differentiation between the two working populations was 0.054 (0.05﹤FST﹤0.15) when the loci OR0006:c.524 G > T, OR0007:c.331 T >C, OR0007:c.430 G > A, OR0007: c.793 G > C, and OR16C11:c.294 G > A in HWE were considered.
e F-values of average inbreeding coefficient were −0.226 for guide dogs and −0.241 for sniffer dogs given in Table 6. Linkage disequilibrium values for OR0007 gene, OR16C11 gene, DOPRX09 gene, and OR7215 gene were calculated for every working dog population. e results displayed that the r 2 value that was more than 0.80 was shown only within OR0007 gene for the two working dog groups (see Figure 1).
Nonsynonymous mutations are divided into two kinds of being tolerated (>0.05) and deleterious (≤ 0.05) with SIFT.
e biological impact effects of amino acid substitution induced by nonsynonymous SNPs were considered "deleterious" (<−2.5) and "neutral" (>−2.5) by PROVEAN v.1.1.3. We estimated 24 nonsynonymous SNPs using SIFT,  Genetics Research 5 PROVEAN, and Align GVGD. 6 SNPs were predicted to be "untolerated" with a score below 0.05 by SIFT, 7 SNPs are predicted to be "deleterious" by PROVEAN and 10 SNPs were classified as a class C65 change, the first highest pathogenicity category by Align GVGD (see Table 7). OR0006:c.524 G > T, OR16C11:c.368 C >A, OR7215: c.251 C >T, and OR04B06 : 215T >G were considered to affect protein function or structure with all of the three analysis software.

Discussion
e aim of this study was to explain how use-oriented selection could lead to phenotypic changes of olfactory behavior in Labs. Olfactory traits of guide Labs and sniffer Labs are expected to play a role in canine behavioral genetics [27]. We focused on 26 SNPs, 2 deletions, and 2 insertions in 7 OR genes from the two Labs working dog populations. e genotyping test found that one population presented only one type of homozygote compared with the other population presenting two or three genotypes in 7 loci, suggesting that the alleles were purified and needed for working traits of guide dogs and sniffer dogs by rapid evolution [3]. ese regions of extended homozygosity might be best explained by selection rather than genetic drift [28]. On the contrary, heterozygotes or one type of homozygote in a certain locus could occur in any working dog group, which indicated that putative SNP alleles and genotypes were chosen and possibly contributed to the common working traits under artificial selection. e significant differences between sniffer dogs and guide dogs happened to 36.7% of these loci, which explained that behavioural phenotypes in a dog breed might be affected by genetic background and controlled by multiple SNP alleles with large effects, which were preserved by artificial selection. So, special alleles might play functional roles that shaped working dog traits within a dog breed [28]. Moreover, functional loss or alternation of OR genes resulted from deleterious mutations could have been avoided through maintaining a large population size that enabled the effects of genetic drift to be negligible [29,30]. Because the differences of these alleles were analyzed in small volume samples and the association between the alleles and olfactory behavior phenotypes was not analyzed, their effects remained potential.
Meanwhile, the loss of genomic diversity (PIC<0.375) resulted from strong selective pressures of olfactory behavior through gene pathways [31]. In addition, the data sets that Ho values were higher than He values except the locus OR7215:c.578 A >T in any Labs group showed that selective breeding in working dog populations had tried to gain popular hereditary features and avoid inbreeding in a limited degree. Furthermore, the mean F-values of inbreeding coefficients verified the negative regression in the two working dog groups. Low levels of linkage disequilibrium obtained for every working dog subpopulation were consistent with homogeneity within a dog breed [32,33].
is highlighted that the most of SNP alleles within individual OR genes were not inherited as a block and suggested an ongoing gene conversion process for desirable working traits although it took people about 150 years to develop a dog breed.  Genetics Research e analysis of 26 SNPs by the collective and two working Labs groups showed that the proportions of being in HWE were lower than 52.6% in a former study of Labs that was a lower proportion among dog breeds [34]. It was a possible explanation that the selection of desirable phenotypic traits depending on alleles had screened the disqualified Labs out of the populations. Consequently, the artificial selection concurrently indirectly caused assortative mating and worsened the state of being out of HWE since a large number of working dogs had not made up for the shortcoming of olfactory behavior selection thoroughly in the relatively closed populations. e degree of genetic differentiation (Fst) between the two working Labs groups was 0.054, a little higher than the borderline 0.05 above  which a dog breed showed moderate differentiation among its subpopulations [35]. An important reason was that breeders introduced ideal sires and dams from other Labs' subpopulations which had the improvement of some working characteristics instead of some other dog breeds with very close genetic relationship. In silico prediction tools, SIFT, PROVEAN, and Align GVGD evaluated the pathogenicity of missense variants of 24 SNPs because bioinformatics analysis alone was usually not sufficient [36]. In order to decrease the proportion of false positive results, the positive judgments in agreement with each other were accepted. So the four missense variants residing in EC1, EC2, and TM2 domains of OR protein were not tolerated on the basis of the comprehensive analysis. Substituting amino acids by missense mutations in OR genes could contribute to increase or decrease in the sensitivity towards target odorants [37]. Among them, OR16C11:p.A123D residing in EC1 domain showed a significant difference and OR7215:p.P84 L residing in TM2 domain showed a severely significant difference between the two working Labs groups. In addition, OR7215:p.P84 L showed a possible functional role in typing working Labs subpopulations. So the functional allele is possibly related to working capacities of Labs, which might help breeders to pick out Labs of different levels in olfactory acuity early and accurately and set up different working Labs' strains. But the similar study is not still carried out in other sniffer dogs, for example, Springer Spaniels and bloodhounds. Additionally, the functions of these alleles might be examined by the transduction of the olfactory message in the future [38].

Conclusion
A number of analyses were conducted to reveal canine breed phylogeny or relation with some complicated qualities due to SNP variation. However, few studies aimed at subpopulation differentiations within a same dog breed. e strong artificial selection was only performed urgently to gain working attributes. Labs working in different fields acted as the sample model. SNP loci in OR genes presented indexes on the positive and negative selection, namely, a preference for acute sniffing and being numb about odor. Our analysis demonstrated that possibly functional alleles were quickly evolving in compliance with people's intentions though the changes were limited in decades of artificial selection.

Data Availability
e data used to support the findings of this study are included within the article and the supplementary information file.

Conflicts of Interest
e authors declare that they have no conflicts of interest regarding the publication of this paper.