Breed Differences in Dopamine Receptor D4 Gene (DRD4) inHorses
Yusuke HORI
Takatoshi OZAKI
Yoshimitsu YAMADA
Teruaki TOZAKI
Heui-Soo KIM
Ayaka TAKIMOTO
Maiko ENDO
Noboru MANABE
Miho INOUE-MURAYAMA
Kazuo FUJITA
Corresponding author. e-mail:hori.yuusuke.74z@st.kyoto-u.ac.jp
Accepted 2013 Jun 14; Issue date 2013.
This is an open-access article distributed under the terms of the CreativeCommons Attribution Non-Commercial No Derivatives (by-nc-nd) License.
Abstract
Genetic polymorphisms in genes related to neurotransmitters or hormones affectpersonality or behavioral traits in many animal species including humans. In domesticanimals, the allele frequency of such genes has been reported to be different among breedsand it may account for breed differences in behavior. In this study, we investigated breeddifferences in horses in the dopamine receptor D4 gene (DRD4), which has been reported toaffect horse personality. We collected samples from seven horse breeds including thosenative to Japan and Korea, and compared the sequence of the DRD4 exon3 region among thesebreeds. We found that there were two types of polymorphisms (VNTR and SNPs) in the exon3region, and some of them seemed to be breed-specific. In addition, we found that theallele frequency of G292A, reported to be associated with horse personality, differedgreatly between native Japanese horses and Thoroughbred horses. The frequency of the Aallele which is associated with low curiosity and high vigilance, was much lower in nativeJapanese horses (Hokkaido, 0.03; Taishu, 0.08) than in Thoroughbreds (0.62). Thisdifference may account for breed differences in personality or behavioral traits. Furtherstudies of the function of these polymorphisms and their effect on behavior areindicated.
Keywords: behavioral trait, breed difference, dopamine receptor D4 gene, native Japanese horse, SNP
Selective breeding by humans has resulted in various breeds of domestic animals. Greatdiversity between different breeds has been created not only in body size and coat color, butalso in behavioral traits and personality. For example, dogs of different breeds differgreatly in social behavioral traits [9,15]. Differences between breeds in personality, as measuredby questionnaire surveys, are also reported in horses [11]. Breed differences in behaviors are especially important in companion animalssuch as dogs or horses. Scientific evaluation of behavioral differences between breeds shouldbe useful for the development of methods of management or training better suited to eachbreed.
Variability of behavioral traits between breeds may be partly affected by genes. Recentstudies have shown that genetic polymorphisms in genes related to neurotransmitters orhormones influence individual differences in personality [7]. The dopamine receptor D4 gene (DRD4) encodes one of thesubtypes of dopamine receptor, and is one of the candidate genes underlying individualdifferences in personality in many species including humans. In humanDRD4there is a variable number of tandem repeats (VNTR), which is reported to be associated with a“novelty-seeking” trait [1,2].
In dogs, breed differences in the allele frequency ofDRD4 have beenreported. For example, a VNTR in the canineDRD4 exon3 region is reported tobe associated with aggressiveness and activity/impulsivity [5]. The allele frequency of this polymorphism is different among breeds [8], particularly between Asian breeds and European breeds[10].
In horses,DRD4 is located on chromosome 12 [13], and two types of polymorphisms have been found. They are VNTR consisting of 18base pairs (6 amino acids) and some single nucleotide polymorphisms (SNPs) in the exon3 region[3]. One of these SNPs, G292A, was reported to beassociated with horse personality scores measured by a questionnaire survey. TheA allele in G292A is associated with low curiosity and high vigilance inThoroughbred horses [12].
If there are breed differences in personality related genes such asDRD4, itmay be possible to predict the behavioral traits of breeds based on the genotype. A previousstudy reported that one Kiso horse, a native Japanese horse breed, had shorter repeats in theVNTR region than Thoroughbred horses [3]. However,whether the allele frequency of this polymorphism differs between breeds has not yet beenverified. Samples from more breeds are needed to validate the breed differences ofDRD4 in horses.
In this study, we investigated breed differences in equineDRD4 usingsamples from more breeds than previously studied. We collected samples from two breeds ofEuropean origin (Thoroughbred and Selle Français), one South American breed (Criollo), threenative Japanese breeds (Hokkaido, Taishu and Yonaguni) and one native Korean breed. Weexplored the polymorphisms inDRD4 and compared its allele frequency amongbreeds.
Materials and Methods
In total, we collected 70 horse samples including two European-origin breeds (Thoroughbred;n=4, Selle Français; n=1), one South American-origin breed (Criollo; n=5), three nativeJapanese breeds, and a native Korean breed. The Japanese samples were from Hokkaido (n=31),Taishu (Tsushima; n=20), and Yonaguni (n=1). The Korean samples were from Jeju in Korea(n=8). We excluded offspring or full-siblings from our samples when estimating the allelefrequency of each breed.
We extracted genomic DNA from blood, buccal swabs, or hair using a QIAamp blood and tissuekit (QIAGEN, Valencia, CA, USA). For some hair samples, an InstaGeneTM Matrix(Bio-Rad Laboratories, Hercules, CA, USA) was used.
We amplified 504 base pairs of the exon3 region in equineDRD4 (GenBank:AB080626.1) by polymerase chain reaction (PCR). We used the primer pairs and PCR conditionsdescribed in a previous study [12]. PCR products werepurified using a High Pure PCR Product Purification Kit (Roche, Basel, Switzerland), andtheir nucleotide sequences were determined by cycle sequencing using a Big Dye Terminatorv3. 1 Cycle Sequencing Kit and an Applied Biosystems 3130xl GeneticAnalyzer (Applied Biosystems, Foster City, CA, USA). All determined sequences were checkedvisually using FinchTV software, version 1. 4. 0. Polymorphisms in the sequences (SNPs andVNTR) were detected by the alignment using MEGA software, version 5.
For SNPs found in more than one breed, the allele frequencies of each breed werestatistically compared using Fisher’s exact test.
Results
In equineDRD4 exon3 region we found two types of polymorphisms, VNTR andSNPs. The sequence of the exon3 region and locations of polymorphisms we found are shown inFig. 1. VNTR was found only in Criollo horses; these horses had a nine-repeat allele and aten-repeat allele (Fig. 1). The ten-repeat allelewas longer than previously reported alleles (nine and eight repeats), and its frequency wasapproximately 0.30. No length polymorphisms were found in the other breeds (all of them hadthe nine-repeat allele).
Fig. 1.
Sequences of theDRD4 exon3 region in four breeds (Criollo,Hokkaido, Taishu and Thoroughbred). Locations of SNPs are marked by squares. VNTRregions are marked by lines and bold numbers indicate the number of repeats. Thenative Korean breed had G292A and G445A polymorphisms. Novel sequences determined inthis study (Criollo and Taishu) were deposited in the DDBJ database (Accession Nos.AB817803 andAB817804 ).
We also found six SNPs, three of which (C199T, C415T and G445A) have not previously beenreported. Information about these SNPs is summarized inTable 1. The allele frequencies of each SNP in the five breeds (Criollo, Hokkaido,Korean, Taishu and Thoroughbred) are shown inTable2.
Table 1. SNPs found in equineDRD4.
Table 2. The allele frequencies of SNPs in five breeds.
| Criollo | Hokkaido | Korean | Taishu | Thoroughbred | ||
|---|---|---|---|---|---|---|
| n | 5 | 31 | 8 | 20 | 4 | |
| VNTR | 9 | 0.70 | 1.00 | 1.00 | 1.00 | 1.00 |
| 10 | 0.30 | 0.00 | 0.00 | 0.00 | 0.00 | |
| He | 0.47 | 0.00 | 0.00 | 0.00 | 0.00 | |
| C147T | C | 1.00 | 0.97 | 1.00 | 1.00 | 0.88 |
| T | 0.00 | 0.03 | 0.00 | 0.00 | 0.12 | |
| He | 0.00 | 0.06 | 0.00 | 0.00 | 0.25 | |
| C199T | C | 0.70 | 1.00 | 1.00 | 1.00 | 1.00 |
| T | 0.30 | 0.00 | 0.00 | 0.00 | 0.00 | |
| He | 0.47 | 0.00 | 0.00 | 0.00 | 0.00 | |
| G292A | G | 0.70 | 0.97 | 0.62 | 0.92 | 0.38 |
| A | 0.30 | 0.03 | 0.38 | 0.08 | 0.62 | |
| He | 0.47 | 0.06 | 0.50 | 0.14 | 0.54 | |
| C318A | C | 1.00 | 1.00 | 1.00 | 0.72 | 1.00 |
| A | 0.00 | 0.00 | 0.00 | 0.28 | 0.00 | |
| He | 0.00 | 0.00 | 0.00 | 0.41 | 0.00 | |
| C415T | C | 1.00 | 1.00 | 1.00 | 0.65 | 1.00 |
| T | 0.00 | 0.00 | 0.00 | 0.35 | 0.00 | |
| He | 0.00 | 0.00 | 0.00 | 0.47 | 0.00 | |
| G445A | G | 0.90 | 0.81 | 0.88 | 0.90 | 1.00 |
| A | 0.10 | 0.19 | 0.12 | 0.10 | 0.00 | |
| He | 0.20 | 0.32 | 0.23 | 0.18 | 0.00 |
He: expected heterozygosity.
Among the SNPs we identified, C147T and C318A are synonymous SNPs, which cause no aminoacid changes. We found the C147T polymorphism only in Hokkaido and Thoroughbred horses, andthe C318A polymorphism only in Taishu horses. The nucleotide substitution reported at theC318A location in a previous study [3] was fromcytosine to thymine, but the substitution we found in Taishu horses was from cytosine toadenine. In both cases, these substitutions cause no amino acid change.
The other four SNPs (C199T, G292A, C415T and G445A) are non-synonymous SNPs, which causeamino acid changes. C199T was unique to Criollo horses and C415T was unique to Taishuhorses.
G292A is a non-synonymous SNP that causes an amino acid change from asparagine to asparticacid, and it was linked to horse personality in a previous study [12]. TheA allele of G292A is associated with lowcuriosity and high vigilance scores. We compared the allele frequency of G292A among fivebreeds (Hokkaido, Taishu, Korean, Criollo and Thoroughbred) and found a significantdifference among breeds (Fisher’s exact test, P=4.4 × 10–6). In native Japanesebreeds (Hokkaido and Taishu) the frequency of theA allele was very low(approximately 0.03 for Hokkaido, 0.08 for Taishu). In contrast, the frequency of theA allele was considerably higher (approximately 0.62) in Thoroughbreds.One Selle Français horse and one Yonaguni horse were heterozygous (their genotypes wereA/G).
G445A is a novel SNP that causes an amino acid change from alanine to threonine. Thispolymorphism was found in four breeds (Hokkaido, Taishu, Korean and Criollo), but the allelefrequencies did not differ significantly among these breeds (Fisher’s exact test,P=0.65).
The novel sequence data determined in this study was registered in the DDBJ nucleotidedatabase with the accession numbersAB817803 (Criollo) andAB817804 (Taishu).
Discussion
In this study we found several novel polymorphisms in equineDRD4 exon3region. Some of these seem to be breed-specific. Criollo horses had a unique non-synonymousSNP (C199T) and longer alleles (ten-repeat) in the VNTR region than other breeds. Taishuhorses also had a unique non-synonymous SNP (C415T). These polymorphisms may change theamino acid sequence of the protein and modify its function. Functional analysis of thesepolymorphisms is now needed.
We also compared the allele frequencies of polymorphisms among different breeds. Althoughsamples from unrelated individuals are needed to estimate the allele frequency moreaccurately, it is difficult to collect completely unrelated samples in the case of rarenative breeds. So, we excluded offspring or full-siblings from our samples when estimatingthe allele frequency. We found that the allele frequency of G292A, which has been reportedto be associated with horse personality, was different among breeds. In particular, nativeJapanese horses had quite different allele frequencies of G292A from Thoroughbred horses.Although our sample size of Thoroughbreds was relatively small, theAallele seems to be a major allele in these horses, because the frequency of this allele wasalso high in a previous study [12] (approximately0.54). This allele was in the minority in all of the other breeds tested. This suggests thattheA allele is a minor allele of horses in general, except forThoroughbreds. It may be a byproduct of the special breeding history of Thoroughbred horsesfor racing performance. The low frequency of theA allele, linked with lowcuriosity and high vigilance, suggests that Japanese horses should be more curious and lessvigilant than Thoroughbreds. In fact, a questionnaire-based behavioral evaluation of Kisohorses revealed these animals are curious and friendly with humans [14].
The differences of theA allele frequency of G292A may be used as apredictor of behavioral traits of horse breed. Also polymorphisms within each breed may beuseful for management or training suited to each individual trait. However, the associationbetween G292A and behavioral traits requires further confirmation using more objectivebehavioral tests. Behavioral tests used in canine studies showing the association betweenDRD4 and behavioral traits [4,6] may be useful, following modification, for usewith horses.
In conclusion, this study showed that there are differences inDRD4polymorphisms among breeds of horses. These differences may be a result of differentbreeding histories or of geographical variation. They may affect the variance of behavioraltraits or personality among the breeds. Further studies of both functional aspects of genesand behavioral variations are needed to validate the effect of these polymorphisms.
Acknowledgments
This study was supported financially by the Japan Society for the Promotion of Sciences(JSPS) with Grants-in-aid for Scientific Research (#21310150 to MI-M and #20220004 to KF)and the Asia and Africa Science Platform Program. This study was supported by theCooperation Research Program of the Wildlife Research Center, Kyoto University. The authorswould like to thank Dr. Shin’ichi Ito for cooperation with the collection of samples andsuggestions useful to this research. We also would like to thank Ms. Tomoko Yabe (Ueno Zoo),Mr. Eiichiro Fujiwara (Tsurui Dosanko Farm), and the students of Shimabara Agricultural HighSchool for their cooperation with the collection of samples.
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