Journal of Heredity Advance Access originally published online on October 26, 2005
Journal of Heredity 2005 96(7):812-816; doi:10.1093/jhered/esi113
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Olfactory Receptor Sequence Polymorphism Within and Between Breeds of Dogs
From UMR 6061,Génétique et Développement CNRS-Université de Rennes 1, France
Address correspondence to Francis Galibert at the address above, or e-mail: galibert{at}univ-rennes1.fr.
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Abstract |
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Olfactory receptors, to which odorant molecules specifically bind, are encoded by the largest gene family yet identified in the mammalian genome. We investigated additional polymorphism due to the possible existence of multiple alleles dispersed in different dog breeds by carrying out a survey of the sequences of 16 olfactory receptor genes in a sample of 95 dogs of 20 different breeds. The level of polymorphism was high—all genes were found to have allelic variants—leading to amino acid changes and pseudogenization of some alleles in a number of cases. This preliminary study also revealed that some alleles are breed specific (or rare in the dog population), with some representing the major allele in the breeds concerned.
Dogs, which were domesticated from the wolf around 13,000 BCE, have been the subject of intensive breeding programs (Clutton-Brock 1995). More than 350 different breeds exist, offering a spectrum of phenotypic polymorphism that cannot be matched by any other mammal. This intense selection process has led to the creation of breeds for specific purposes, such as hunting. Despite the development of sophisticated electronic devices, dogs, with their keen sense of smell, remain the best option for finding people buried in avalanches or hidden objects, drugs, and illicit substances.
The detection and recognition of an odorant volatile molecule is a complex mechanism. It begins in the nasal epithelium, where odorant molecules bind specifically to olfactory receptors (ORs) expressed by the olfactory neurons (Buck 2000; Buck and Axel 1991; Firestein 2001; Mombaerts 1999), which send signals to the olfactory bulb and then to various areas of the cortex. The complexity of the biological system governing the detection and recognition of odorant molecules makes it difficult to understand the workings of this sophisticated sense in dogs and the reasons why some animals are more gifted than others in this domain. Recently, a draft dog genome sequence was released (Canfam1.0) by which we retrieved 1,094 OR genes, 20.3% of which are pseudogenes (Quignon et al. 2005). Because ORs are the first element activated in this cascade of reactions, we questioned how important ORs were in individual or breed-specific sensing capabilities. To this end, we investigated the level of genetic polymorphism of ORs within and between breeds and report here the sequences of 16 OR genes, analyzed in 95 dogs of 20 breeds. A high level of allelic polymorphism was observed, with up to 11 single nucleotide polymorphism (SNP) sites between two alleles, whereas some alleles appeared to be breed specific.
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Materials and Methods |
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Animals and DNA Samples
Blood samples of each dog were collected by veterinarians, as 5 ml of fresh blood on EDTA tubes. DNA was extracted with Nucleon kit (Amersham Biosciences, Piscataway, NJ) using standard protocols.
Primer Design
We used the Primer 3 program (Rozen and Skaletsky 2000; http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) to design pairs of specific primers for the amplification of each complete ORF and for sequencing. We also used a third primer binding within the ORF to ensure high-quality sequencing and accurate SNP identification.
Polymerase Chain Reaction Amplification, Sequencing, and Analysis
OR sequences were amplified in a final volume of 10 µl containing dog DNA (10 ng), 0.5 U AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA), 0.15 µM of each specific primer, 0.25 mM of each dNTP (Amersham Biosciences), 1x buffer, and 2 mM MgCl2. Polymerase chain reaction was carried out as follows: 7 min at 94°C, followed by 20 cycles of 30 s at 94°C, 30 s at annealing temperature (beginning at 61°C and decreasing by 0.5°C per cycle), 1 min at 72°C and then 15 cycles of 30 s at 94°C, 30 s at 51°C, 1 min at 72°C, and a final extension phase of 3 min at 72°C.
Sequencing reactions were carried out with Big Dye chemistry method, with migration on an ABI PRISM 3100 sequencer (Applied Biosystems). Sequence traces were analyzed with PhredPhrap/Consed/Polyphred softwares (Ewing et al. 1998; Ewing and Green 1998; Gordon et al. 1998; Nickerson et al. 1997). SNPs were identified on the basis of sequence mismatch alignments identified by the program, with visual checking in each case.
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Results |
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Sixteen dog OR genes were selected from the two classes and various families and subfamilies composing the OR repertoire (Ben-Arie et al. 1994; Freitag et al. 1998; Quignon et al. 2005). Their sequences were compared in a panel of 95 unrelated dogs of 20 breeds representing the 10 Federation Cynologique Internationale groups but the Teckel group (group IV; see Table 1).
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A total of 98 SNPs and four insertions/deletions (indels) were detected, and all genes were found to be polymorphic but to different degrees. For three genes (CfOR0011, CfOR0123, and CfOR0184) up to 11 SNPs per gene were detected, whereas for one gene only (CfOR0500), we detected only two SNPs (Table 2).
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This frequency of nucleotide polymorphism corresponds to one nucleotide difference per 920 sequenced nucleotides, taking into account the number of genes analyzed (16), the mean size of the open reading frame (ORF) (940 bp), the number of animals (95 x 2 alleles) and the total occurrence of the minor alleles (3,099; 16 x 940 x 95 x 2 / 3,099 = 920). In parallel to this study, we analyzed the sequence polymorphism of 153 noncoding sequences with a mean length of 500 bp. These noncoding sequences were selected at random from throughout the dog genome and sequenced after polymerase chain reaction amplification from a sample of 20 dogs representing 20 breeds. The alignment of these sequences led the identification of 134 SNPs with a total occurrence of 533 for the minor alleles. The level of polymorphism, calculated as described here, gave one change per 5,741 sequenced nucleotides.
Interestingly, 55 of the SNPs found in OR genes induced a change in amino acid sequence, with 30 involving an amino acid of a different chemical group: changing a proline into a serine (CfOR0011 at position 844) or a glutamic acid into a glycine (CfOR0044 at position 122). Amino acid changes occurred in all parts of the protein: 4 in the N-terminal region, 25 in the transmembrane domains, 5 in the C-terminal region, 10 in intracellular loops, and 11 in extracellular loops (Figure 1). These allelic polymorphisms predominantly (48/55SNPs) affected amino acids identified as being variable and highly variable within the OR genes (Quignon et al. 2005) with the exception of two residues corresponding to highly conserved positions in transmembrane III and transmembrane VII (Buck and Axel 1991).
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Five of the 16 genes analyzed in this study (CfOR0115, 0184, 0237, 0423, 0432) had an allele with an interrupted ORF due to an SNP introducing a stop codon or due to a short indel. These five pseudogenized alleles are found in the panel of 95 dogs at both heterozygous (17 dogs) and homozygous states (9 dogs). However, in all five boxers the two alleles of CfOR0184 had an interrupted ORF, indicating that this gene might be a pseudogene in this breed.
The observed frequencies of minor alleles were highly variable (from 0.5% to 50%), and 35 had a frequency less than 5% (table available on the website: http://idefix.univ-rennes1.fr:8080/Dogs/ORpolymorphism.html). As 25 of the 35 SNPs with a minor allele frequency less than 5% corresponded to alleles present in only one breed, we recruited three to eight additional animals from the breed concerned for further sequencing. It confirmed that six SNPs corresponded to a major allele, with the others having a frequency of 0.038 to 0.333 (Table 3). Moreover, four of those six SNP induced an amino acid change in the OR protein.
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Discussion |
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Sequence surveys were carried out to analyze the allelic polymorphism of 16 OR genes from different subfamilies. Although the sample size was small, this preliminary study showed that OR genes are highly polymorphic, as already suggested for human leukocyte antigen-linked OR genes (Ehlers et al. 2000; Eklund et al. 2000). Each analyzed gene had multiple alleles, and we not only detected up to 11 SNPs per allele but observed, on average, one nucleotide difference per 920 nucleotides—a much higher figure than that found for coding sequences (Masuda et al. 2004) or even in noncoding sequences that appeared six times less polymorphic (this work).
Minor allele frequencies varied between 0.5% and 50%. Interestingly, 25 of the SNPs with frequencies less than 5% were found to be present in only one breed, and six of these were found to correspond to the major allele in the breed concerned. More than half—55 out of the 98 SNPs—induced an amino acid change, in some cases resulting in the insertion of an amino acid from a different chemical group. The potential impact of these amino acid changes on the function of the OR is unclear, but some may contribute to the differences in odorant-sensing capabilities observed between animals or breeds, either by modulating ligand-binding affinities or via intracellular responses and signal processing.
Our first estimate of the number of pseudogenes, based on analysis of the sequences derived from the 1.5x shotgun of a DNA poodle sample, was 18% (Quignon et al. 2003). With the 7.5x shotgun sequence derived from a boxer DNA sample, we obtained a value of 20.3% (Quignon et al. 2005). Of the genes common to these two analyses, 17 OR genes were pseudogenes in the poodle genome but not in the boxer genome, whereas the reverse was true for 22 genes. Some of these differences might be attributed to sequencing errors; however, we clearly showed that some SNPs do induce pseudogenization. Among the 16 genes analyzed, 5 had an SNP introducing a stop codon or a frameshift. In particular, all five boxers were homozygous for an allele of CfOR0184 that has an interrupted ORF; therefore, none of the boxers has a functional CfOR0184 gene. Even with the small number of genes analyzed, it is evident that different animals or breeds may have a different subset of pseudogenes, as has already been noticed for different human populations (Gilad and Lancet 2003; Menashe et al. 2002, 2003). However, it is tempting to link the higher percentage of pseudogenes (21%) found in the DNA of the boxer than in that of the poodle with the current view that boxers have a less acute sense of smell than that of poodles. More extensive studies are needed to explore this possibility as well as the impact of domestication, if any, by analyzing the same set of genes in wolves.
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Acknowledgments |
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We would like to thank the Centre National Recherche Scientifique, the Université de Rennes 1, the Conseil Régional de Bretagne, and the Technical Support Working Group for grants to Francis Galibert and for encouragement. We want to acknowledge Dr. Gilles Chaudieu, Dr. Philippe Pilorge, Dr. Catherine Lefevre, Dr. Hervé Lefevre, and Dr. Vincent Biourges (Royal Canin) for canine blood samples, as well as Dr. Sandrine Paget. This article was presented at the 2nd International Conference on the "Advances in Canine and Feline Genomics: Comparative Genome Anatomy and Genetic Disease," Universiteit Utrecht, Utrecht, The Netherlands, October 14–16, 2004.
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Footnotes |
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Corresponding Editor: Bernard van Oost
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References |
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