in total , we collected 70 horse samples including two european - origin breeds ( thoroughbred ; 
 n=4 , selle franais ; n=1 ) , one south american - origin breed ( criollo ; n=5 ) , three native 
 japanese breeds , and a native korean breed .
the japanese samples were from hokkaido ( n=31 ) , 
 taishu ( tsushima ; n=20 ) , and yonaguni ( n=1 ) .
we excluded offspring or full - siblings from our samples when estimating the allele 
 frequency of each breed .
we extracted genomic dna from blood , buccal swabs , or hair using a qiaamp blood and tissue 
 kit ( qiagen , valencia , ca , usa ) .
for some hair samples , an instagene matrix 
 ( bio - rad laboratories , hercules , ca , usa ) was used .
we amplified 504 base pairs of the exon3 region in equine drd4 ( genbank : 
 ab080626.1 ) by polymerase chain reaction ( pcr ) .
pcr products were 
 purified using a high pure pcr product purification kit ( roche , basel , switzerland ) , and 
 their nucleotide sequences were determined by cycle sequencing using a big dye terminator 
 v3 . 1 cycle sequencing kit and an applied biosystems 3130xl genetic 
 analyzer ( applied biosystems , foster city , ca , usa ) .
polymorphisms in the sequences ( snps and 
 vntr ) were detected by the alignment using mega software , version 5 .
for snps found in more than one breed , the allele frequencies of each breed were 
 statistically compared using fisher s exact test .
in equine drd4 exon3 region we found two types of polymorphisms , vntr and 
 snps .
the sequence of the exon3 region and locations of polymorphisms we found are shown in 
 fig .
1.sequences of the drd4 exon3 region in four breeds ( criollo , 
 hokkaido , taishu and thoroughbred ) .
vntr 
 regions are marked by lines and bold numbers indicate the number of repeats .
novel sequences determined in 
 this study ( criollo and taishu ) were deposited in the ddbj database ( accession nos . 

ab817803 and ab817804 ) .. vntr was found only in criollo horses ; these horses had a nine - repeat allele and a 
 ten - repeat allele ( fig .
the ten - repeat allele 
 was longer than previously reported alleles ( nine and eight repeats ) , and its frequency was 
 approximately 0.30 .
no length polymorphisms were found in the other breeds ( all of them had 
 the nine - repeat allele ) .
sequences of the drd4 exon3 region in four breeds ( criollo , 
 hokkaido , taishu and thoroughbred ) . locations of snps are marked by squares .
vntr 
 regions are marked by lines and bold numbers indicate the number of repeats .
novel sequences determined in 
 this study ( criollo and taishu ) were deposited in the ddbj database ( accession nos . 

we also found six snps , three of which ( c199 t , c415 t and g445a ) have not previously been 
 reported .
the allele frequencies of each snp in the five breeds ( criollo , hokkaido , 
 korean , taishu and thoroughbred ) are shown in table 
 2table 2 .
the allele frequencies of snps in five breedscriollohokkaidokoreantaishuthoroughbredn5318204vntr90.701.001.001.001.00100.300.000.000.000.00he0.470.000.000.000.00c147tc1.000.971.001.000.88t0.000.030.000.000.12he0.000.060.000.000.25c199tc0.701.001.001.001.00t0.300.000.000.000.00he0.470.000.000.000.00g292ag0.700.970.620.920.38a0.300.030.380.080.62he0.470.060.500.140.54c318ac1.001.001.000.721.00a0.000.000.000.280.00he0.000.000.000.410.00c415tc1.001.001.000.651.00t0.000.000.000.350.00he0.000.000.000.470.00g445ag0.900.810.880.901.00a0.100.190.120.100.00he0.200.320.230.180.00he : expected heterozygosity .. he : expected heterozygosity . among the snps we identified , c147 t and c318a are synonymous snps , which cause no amino 
 acid changes .
we found the c147 t polymorphism only in hokkaido and thoroughbred horses , and 
 the c318a polymorphism only in taishu horses . the nucleotide substitution reported at the 
 c318a location in a previous study   was from 
 cytosine to thymine , but the substitution we found in taishu horses was from cytosine to 
 adenine . in both cases , these substitutions cause no amino acid change .
the other four snps ( c199 t , g292a , c415 t and g445a ) are non - synonymous snps , which cause 
 amino acid changes . c199 t was unique to criollo horses and c415 t was unique to taishu 
 horses .
g292a is a non - synonymous snp that causes an amino acid change from asparagine to aspartic 
 acid , and it was linked to horse personality in a previous study .
the a allele of g292a is associated with low 
 curiosity and high vigilance scores .
we compared the allele frequency of g292a among five 
 breeds ( hokkaido , taishu , korean , criollo and thoroughbred ) and found a significant 
 difference among breeds ( fisher s exact test , p=4.4  10 ) . in native japanese 
 breeds ( hokkaido and taishu ) the frequency of the a allele was very low 
 ( approximately 0.03 for hokkaido , 0.08 for taishu ) .
in contrast , the frequency of the 
 a allele was considerably higher ( approximately 0.62 ) in thoroughbreds . 
 one selle franais horse and one yonaguni horse were heterozygous ( their genotypes were 
 a / g ) .
g445a is a novel snp that causes an amino acid change from alanine to threonine .
this 
 polymorphism was found in four breeds ( hokkaido , taishu , korean and criollo ) , but the allele 
 frequencies 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 nucleotide 
 database with the accession numbers ab817803 ( criollo ) and ab817804 ( taishu ) .
in this study we found several novel polymorphisms in equine drd4 exon3 
 region . some of these seem to be breed - specific .
criollo horses had a unique non - synonymous 
 snp ( c199 t ) and longer alleles ( ten - repeat ) in the vntr region than other breeds .
taishu 
 horses also had a unique non - synonymous snp ( c415 t ) .
these polymorphisms may change the 
 amino acid sequence of the protein and modify its function .
we also compared the allele frequencies of polymorphisms among different breeds . although 
 samples from unrelated individuals are needed to estimate the allele frequency more 
 accurately , it is difficult to collect completely unrelated samples in the case of rare 
 native breeds .
so , we excluded offspring or full - siblings from our samples when estimating 
 the allele frequency .
we found that the allele frequency of g292a , which has been reported 
 to be associated with horse personality , was different among breeds .
in particular , native 
 japanese horses had quite different allele frequencies of g292a from thoroughbred horses . 

although our sample size of thoroughbreds was relatively small , the a 
 allele seems to be a major allele in these horses , because the frequency of this allele was 
 also high in a previous study   ( approximately 
 0.54 ) .
this suggests that 
 the a allele is a minor allele of horses in general , except for 
 thoroughbreds .
it may be a byproduct of the special breeding history of thoroughbred horses 
 for racing performance .
the low frequency of the a allele , linked with low 
 curiosity and high vigilance , suggests that japanese horses should be more curious and less 
 vigilant than thoroughbreds .
in fact , a questionnaire - based behavioral evaluation of kiso 
 horses revealed these animals are curious and friendly with humans . the differences of the a allele frequency of g292a may be used as a 
 predictor of behavioral traits of horse breed . also polymorphisms within each breed may be 
 useful for management or training suited to each individual trait .
however , the association 
 between g292a and behavioral traits requires further confirmation using more objective 
 behavioral tests .
behavioral tests used in canine studies showing the association between 
 drd4 and behavioral traits [ 4 , 
 6 ] may be useful , following modification , for use 
 with horses . in conclusion , this study showed that there are differences in drd4 
 polymorphisms among breeds of horses .
these differences may be a result of different 
 breeding histories or of geographical variation .
further studies of both functional aspects of genes 
 and behavioral variations are needed to validate the effect of these polymorphisms .
 genetic polymorphisms in genes related to neurotransmitters or hormones affect 
 personality or behavioral traits in many animal species including humans . in domestic 
 animals , the allele frequency of such genes 
 has been reported to be different among breeds 
 and it may account for breed differences in behavior . in this study , we investigated breed 
 differences in horses in the dopamine receptor d4 gene ( drd4 ) , which has been reported to 
 affect horse personality . 
 we collected samples from seven horse breeds including those 
 native to japan and korea , and compared the sequence of the drd4 exon3 region among these 
 breeds . 
 we found that there were two types of polymorphisms ( vntr and snps ) in the exon3 
 region , and some of them seemed to be breed - specific . 
 in addition , we found that the 
 allele frequency of g292a , reported to be associated with horse personality , differed 
 greatly between native japanese horses and thoroughbred horses . 
 the frequency of the a 
 allele which is associated with low curiosity and high vigilance , was much lower in native 
 japanese horses ( hokkaido , 0.03 ; taishu , 0.08 ) than in thoroughbreds ( 0.62 ) . 
 this 
 difference may account for breed differences in personality or behavioral traits . 
 further 
 studies of the function of these polymorphisms and their effect on behavior are 
 indicated . 