SHORT COMMUNICATION doi: 10.1111/age.12389 SNP genotypes of olfactory receptor genes associated with olfactory ability in German Shepherd dogs † ‡ – M. Yang*, G.-J. Geng , W. Zhang , L. Cui§, H.-X. Zhang and J.-L. Zheng‡ † *Police-dog Technology Department, National Police University of China, Shenyang, Liaoning 110034, China. Technology Department, ‡ Shenyang Traffic Police Detachment, Shenyang, Liaoning 110001, China. Forensic Medicine Department, National Police University of China, Shenyang, Liaoning 110854, China. §Document Inspection Department, National Police University of China, Shenyang, Liaoning – 110854, China. Mark Inspection Department, National Police University of China, Shenyang, Liaoning 110854, China. Summary To find out the relationship between SNP genotypes of canine olfactory receptor genes and olfactory ability, 28 males and 20 females from German Shepherd dogs in police service were scored by odor detection tests and analyzed using the Beckman GenomeLab SNPstream. The representative 22 SNP loci from the exonic regions of 12 olfactory receptor genes were investigated, and three kinds of odor (human, ice drug and trinitrotoluene) were detected. The results showed that the SNP genotypes at the OR10H1-like:c.632C>T, OR10H1-like:c.770A>T, OR2K2-like:c.518G>A, OR4C11-like: c.511T>G and OR4C11-like:c.692G>A loci had a statistically significant effect on the scenting abilities (P < 0.001). The kind of odor influenced the performances of the dogs (P < 0.001). In addition, there were interactions between genotype and the kind of odor at the following loci: OR10H1-like:c.632C>T, OR10H1-like:c.770A>T, OR4C11-like:c.511T>G and OR4C11-like:c.692G>A(P< 0.001). The dogs with genotype CC at the OR10H1-like: c.632C>T, genotype AA at the OR10H1-like:c.770A>T, genotype TT at the OR4C11-like: c.511T>G and genotype GG at the OR4C11-like:c.692G>A loci did better at detecting the ice drug. We concluded that there was linkage between certain SNP genotypes and the olfactory ability of dogs and that SNP genotypes might be useful in determining dogs’ scenting potential. Keywords correlation, dog, scenting behavior, genetic markers Certain dog breeds, such as German Shepherd dogs, are Chen et al. 2012; Randi et al. 2014). Gene polymorphisms very popular in the field of police work because of their are associated with behavior of dogs in the fields of social sensitive nose. Because of the willingness of these dogs to behavior, scenting behavior, activity–impulsivity, etc. cooperate with humans, police forces make use of this trait (Hejjas et al. 2007; Kubinyi et al. 2012; Kis et al. 2014). in dogs as tools for detecting a variety of odors such as Over half of the canine olfactory receptor (cOR) genes explosives, drugs, humans and even molds (Lesniak et al. were found to show high polymorphism, some of which 2008; Pinc et al. 2011; Lippi and Cervellin 2012). Whether were breed specific and some of which might affect the olfactory sensation of dogs is acute or not is often individual olfactory perception (Keller and Vosshall 2008; evaluated through the use of many kinds of behavior tests, Robin et al. 2009; Quignon et al. 2012; Kim et al. 2012). which have raised questions about the objectivity the tests Therefore, the correlation between the genetic polymor- in the absence of a ‘gold standard’ (Doty and Kamath phism of cOR genes and olfactory ability was further 2014). The spread of different phenotypes in dogs has studied. If a certain allele is likely to endow olfactory resulted from restricting gene flow and artificially selecting receptors with ligand-binding capacity, that might con- offspring (Spady and Ostrander 2008; Akey et al. 2010; tribute to the olfaction sensitivity of dogs. Lesniak et al. (2008) verified that the specific alleles at two loci, cOR9S13: Address for correspondence c.592G>A and cOR52N9:c.176A>G, were statistically sig- nificantly linked to odor recognition capabilities of dogs. M. Yang, Police-dog Technology Department, National Police University of China, 4 Baishan Road, Yuhong District, Shenyang, However, it is possible that a certain polymorphism at a Liaoning 110034, China. given locus does not fully determine a dog’s odor sensing E-mail: [email protected] skills. Understanding the genetic mechanism of olfactory Accepted for publication 13 October 2015 behavior traits could help breeders to breed dogs that cater 240 © 2015 Stichting International Foundation for Animal Genetics, 47, 240–244 OR genotype associated with olfactory ability 241 to human requirements and create a genetic method of Yij ¼ l þ Gi þ Oj þ (GO)ij þ eij; picking out excellent individuals. l An experimental group of 48 German Shepherd dogs where Yij is the score of the smelling ability, is the total (males, n = 28; females, n = 20), 2–3 years old, were mean values, Gi is the major effect on genotype i level, Ej is selected from the Police Dog Technology School of Ministry the major effect on odor j level, (GO)ij is the interaction of Ɛ of Public Security, China. All the dogs had reached the genotype with the kind of odor on smelling ability and ij is standard for police dogs and were treated in a humane the effect of random error. manner (Lesniak et al. 2008). Experienced handlers Overall, each SNP locus had one main effective allele recorded the sniffing ability of each dog, which had been (Table S5) with a frequency between 0.594 and 0.917. In > trained to detect specific kinds of odor, including the odors particular, the OR2K2-like:c.72 C/T, cOR52E17: > > > of human, ice drug and trinitrotoluene, and were in the last c.791G A, OR2M5:c.98T C, OR2M5:c.170C T, OR2M5: > > phase of training. The standards for scoring are shown in c.862G A and OR08H10:c.241G A loci presented as Table S1. homozygous, reflecting a unique genotype because some A blood sample was obtained from each dog, and then alleles are breed specific or rare in the dog population, DNA was isolated. Twenty-two representative SNP sites, of whereas the other loci presented two or three genotypes which five were breed specific in German Shepherd dogs, lay (Robin et al. 2009; Kim et al. 2012). According to the allele in exonic regions of 12 cOR genes (Robin et al. 2009) frequencies, genetic disequilibrium was found at the > > (Table S2). Pairs of specific primers for 12 cOR gene fraction OR52J3:c.469G A, OR2K2-like:c.518G A and OR4C11- > v2 > < amplification and 22 single extensive primers for SNP like:c.256A G loci ( 9.210, P 0.01). On the contrary, the 13 other loci were in Hardy–Weinberg equilibrium genotypes were designed using the PRIMER-BLAST and PRIMER (v2 < 5.991, P > 0.05). Meanwhile, 16 loci showed low PREMIER 5 programs. The primer sequences, fragment sizes < and annealing temperatures (Tm) mentioned above are polymorphism levels (PIC 0.25), and six loci showed a < < shown in Tables S3 and S4. medium level of polymorphism (0.25 PIC 0.50) Polymerase chain reaction (PCR) was conducted using a (Table 1). This phenomenon might be a result of the Veriti thermal cycler (Applied Biosystems, Inc.) in a total recruitment system used for police dogs. volume of 50 ll that included 4 ll of 100 ng of genomic We also observed that the olfactory sensitivity of dogs varied with certain genotypes. The biggest difference DNA, 25 ll of Premix Taq, 1 lM (final concentration) of > each specific primer and up to 50 ll of water. The following occurred relating to the OR4C11-like:c.511T G and > conditions for the reaction were applied: 10 s at 98 °C, 30 s OR4C11-like:c.692G A loci, as dogs with a TT and GG at 53 °Cor58°C, 30 s at 72 °C and 32 amplification genotype respectively were scored at 26.85 for the detection cycles. PCR products were loaded onto a 2% agarose gel stained with GeneFinder (Biov), and DNA was extracted from the gel. SNP–primer extension reaction was performed Table 1 Genetic polymorphism analysis of SNPs. in a 5-ll reaction system that consisted 2 ll of Premix, 2 ll 2 Num Loci v He PIC of the purified PCR products and 1 ll(1lM) of primer, starting with 25 cycles of denaturation at 96 °C for 10 s, 1 OR10H1-like:c.632C>T 0.087 0.202 0.183 OR10H1-like: > annealing at 50 °C for 10 s and extension at 72 °C for 30 s, 2 c.770A T 0.087 0.202 0.183 3 OR52J3:c.469G>A 42.013 0.278 0.239 followed by an extra extension at 72 °C for 5 min. A total 4 OR2K2-like:c.172>C/T – 00 of 4.25 ll of product digestion was initiated by adding 5 OR2K2-like:c.518G>A 41.245 0.249 0.217 0.5 llof109 buffer and 0.25 llof1U/ll SAP (TaKaRa) 6 OR8S1:c.658T>C 1.732 0.329 0.275 for 1 h in a 37 °C water bath and for 15 min in a 80 °C 7 OR1L4:c.214G>C 0.167 0.457 0.353 OR51I2-like: > water bath. One half microliter (0.5 ll) of the product was 8 c.710T G 0.034 0.186 0.169 9 OR51I2-like:c.770T>C 0.034 0.186 0.169 added to 0.5 ll of Size Standard 80 and 39 ll of sample 10 OR51I2-like:c.778T>A 0.034 0.186 0.169 loading solution. The capillary electrophoresis was ana- 11 OR49-like:c.147A>G 0.088 0.152 0.141 lyzed on a Beckman GeXP Genetic Analysis System 12 cOR52E17:c.214G>A 1.732 0.329 0.275 according to GenomeLab SNP-Start Primer Extension Kit 13 cOR52E17:c.791G>A – 00 OR2M5: > – (Beckman).