1. Background
Artificial selection has been largely responsible for breed-specific and strain-specific traits of modern dogs [Reference Ostrander, Wayne, Freedman and Davis1]. 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 [Reference Lampi, Donner, Anderson and Pohjoismäki2]. Strain-dependent differences of dogs are mainly presented in body size, coat color, and work capacity [Reference Cagan and Blass3, Reference Plassais, Kim and Davis4]. 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 [Reference Asher, Harvey, Green and England5–Reference Shouldice, Edwards, Serpell, Niel and Robinson7]. Many Labs play an important role such as sniffer dogs or guide dogs in many countries. These 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 [Reference Harvey, Craigon, Blythe, England and Asher8, Reference Lesniak, Walczak, Jezierski, Sacharczuk, Gawkowski and Jaszczak9].
The 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. The 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 [Reference Arata, Momozawa, Takeuchi and Mori10–Reference Lazarowski, Rogers, Krichbaum, Haney, Smith and Waggoner12]. Then, behavioral suitability of a working dog is shaped by specialized training [Reference Harvey, Craigon, Blythe, England and Asher8, Reference Yang, Geng, Zhang, Cui, Zhang and Zheng13]. 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 [Reference Bogaerts, Moons, Van Nieuwerburgh, Peelman, Saunders and Broeckx14–Reference Gaunet and Besse16]. As far as disqualified Labs are concerned, their outcome is to be screened out of the subpopulations. Therefore, 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 [Reference Lesniak, Walczak, Jezierski, Sacharczuk, Gawkowski and Jaszczak9, Reference Yang, Geng, Zhang, Cui, Zhang and Zheng13]. Selections of different forms have acted on OR genes in dogs since domestication. The 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 [Reference Robin, Tacher and Rimbault17]. Through 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 [Reference Friedenberg, Meurs and Mackay18].
2. Materials and Methods
2.1. Ethical Statement
This study was carried out in Police-Dog Technology Department, Criminal Investigation Police University of China. The 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.
2.2. Animals and Blood Sample Collection
The 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. The 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.
2.3. 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 [Reference Robin, Tacher and Rimbault17]. 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. The primer sequences, fragment sizes, and annealing temperatures (Tm) above mentioned are shown in Table S1.
TABLE 1: Characteristics of the analyzed SNPs.
∗Asterisks marked out the breed-specific SNPs. No asterisks meant that the SNPs could be found in other dog breeds besides Labs. MAF: minor allele frequency.
2.4. 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. The 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. Then 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 ddH2O, 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 Na2-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. The capillary electrophoresis was analyzed on Beckman GeXP Genetic Analysis System according to Genome-Lab Dye Terminator Cycle Sequencing with Quick Start Kit (Beckman, USA).
2.5. Statistical Analysis
Allele frequency, Genotype frequency, observed heterozygosity (Ho), and polymorphism information content (PIC) were calculated by PowerStats software v1.2 [Reference Wang, Jin and Xiong19]. Expected heterozygosity (He) was counted by CERVUS 3.0.3 [Reference Kalinowski, Taper and Marshall20] software. The F-value of an inbreeding coefficient was derived by [1- (Ho/He)] by EXCEL software too [Reference Pedersen, Pooch and Liu21]. Hardy–Weinberg equilibrium (HWE) tests were performed by ARLEQUIN software v3.5 [Reference Ossowski, Piatek and Parafiniuk22]. Linkage disequilibrium analyses (correlation coefficient, r 2) and the chi-square test about alleles on single locus in a pairwise study were analyzed by SHEsis online tool. A logistic regression analysis of alleles and genotypes was performed on IBM SPSS Statistics 25 software. Fst values were calculated by GenAlEx 6.5 software [Reference Peakall and Smouse23]. The tools including Protein Variation Effect Analyzer (PROVEAN) http://provean.jcvi.org/index.php [Reference Won, Kim and Do24], Align-Grantham variation and Grantham deviation (Align GVGD) http://agvgd.hci.utah.edu/ [Reference Tavtigian, Greenblatt, Lesueur and Byrnes25], and Sorting Intolerant from Tolerant (SIFT) https://sift.bii.astar.edu.sg/ [Reference Gharahkhani, O’Leary, Kyaw-Tanner, Sturm and Duffy26] were used to predict the effect of nonsynonymous SNPs in the encoded proteins.
3. 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.
TABLE 2: Allele frequency and genotype frequency of 30 SNP loci.
∗ Asterisks marked out the breed-specific SNPs. No asterisks meant that the SNPs could be found in other dog breeds besides Labs.
TABLE 3: Differences of SNP alleles between sniffer dogs and guide dogs.
Asterisks marked out the breed-specific SNPs. No asterisks meant that the SNPs could be found in other dog breeds besides Labs.
TABLE 4: Logistic regression analysis of alleles and genotypes between sniffer dogs and guide dogs.
Asterisks marked out the breed-specific SNPs. No asterisks meant that the SNPs could be found in other dog breeds besides Labs.
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 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. There 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.
TABLE 5: Genetic polymorphism and HWE analysis of the collective and working dog subpopulations.
Asterisks marked out the breed-specific SNPs. No asterisks meant that the SNPs could be found in other dog breeds besides Labs. Observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC), and Hardy–Weinberg equilibrium (HWE) values.
P values of HWE tests for 18 SNP loci were given in Table 5. The 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. The 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.
The 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. The 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).
TABLE 6: F-value of an inbreeding coefficient in sniffer dogs and guide dogs.
FIGURE 1: Linkage disequilibrium analyses were performed between pairwise SNP loci in OR0007 gene in the two working populations of Labs. Actual correlation coefficient (r 2) values were magnified 100 times in the small box.
Nonsynonymous mutations are divided into two kinds of being tolerated (>0.05) and deleterious (≤ 0.05) with SIFT. The 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, 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.
TABLE 7: Prediction of the deleterious effect of the nonsynonymous polymorphism in OR genes by SIFT, PROVEAN, and Align GVGD.
Asterisks marked out the breed-specific SNPs. No asterisks meant that the SNPs could be found in other dog breeds besides Labs. SIFT scores range from 0 to 1. The amino acid substitution is predicted to be damaging if the score is ≤0.05 and tolerated if the score is > 0.05. N/A: not available, GD ≥ C65 = most likely affected, and GD ≥ C0 = less likely affected. The amino acid substitution is predicted to be deleterious (<−2.5) and neutral (>−2.5) by PROVEAN.
4. Discussion
The 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 [Reference Houpt27]. We focused on 26 SNPs, 2 deletions, and 2 insertions in 7 OR genes from the two Labs working dog populations. The 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 [Reference Cagan and Blass3]. These regions of extended homozygosity might be best explained by selection rather than genetic drift [Reference Vaysse, Ratnakumar and Derrien28]. 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. The 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 [Reference Vaysse, Ratnakumar and Derrien28]. 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 [Reference Hu29, Reference Marsden, Ortega-Del Vecchyo and O'Brien30]. 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 [Reference Proschowsky, Olsen, Jepsen and Fredholm31]. 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 [Reference Lindblad-Toh, Wade, Karlsson, Jaffe, Kamal and Clamp32, Reference Sutter, Eberle and Parker33]. This 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.
The 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 [Reference Short, Kennedy and Barnes34]. 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. The 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 [Reference Jordana, Piedrafita, Sanchez and Puig35]. 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 [Reference Ernst, Hahnen and Engel36]. 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 [Reference Wu and Lo37]. 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 [Reference Benbernou, Robin, Tacher, Rimbault, Rakotomanga and Galibert38].
5. 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. The 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
The data used to support the findings of this study are included within the article and the supplementary information file.
Conflicts of Interest
The authors declare that they have no conflicts of interest regarding the publication of this paper.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (31702028).
Supplementary materials
Table S1. Primer sequences of 7 OR genes.
Open access
