Studies reported over the past decade indicate that polymorphisms in the Drd4 gene may be associated with variation in measures of novelty-seeking behaviour in humans (reviewed in Kluger et al. 2002; Schinka et al. 2002; Reif & Lesch 2003; van Gestel & Van Broeckhoven 2003; Savitz & Ramesar 2004; Ebstein 2006). However, culture and environment play such a large role in determining individual human behaviours that it is to be expected, that genetic contributions to interindividual behavioural differences will be relatively modest. The importance of learned behaviour, along with the complexities of human population genetic structuring, has been evoked to account for inconsistencies and contradictions in the many replicate studies of associations between Drd4 gene polymorphisms and human personality parameters (Kluger et al. 2002; Schinka et al. 2002; Reif & Lesch 2003; van Gestel & Van Broeckhoven 2003; Savitz & Ramesar 2004; Ebstein 2006).

Therefore, non-human vertebrates may prove useful subjects for genotype–personality phenotype association studies as, in these species, ‘cultural’ determinants of behaviour are not expected to be as strong as among humans. Different breeds of domesticated animals (chickens (Gallus gallus), Sugiyama et al. 2004; dogs (Canis familiaris), Ito et al. 2004) have been found to have differing frequencies of Drd4 alleles, while studies of both domesticated horses (Equus caballus) and captive monkeys (Cercopithecus aethiops) have reported associations between personality parameters and Drd4 gene polymorphisms (Momozawa et al. 2005; Bailey et al. 2007). Furthermore, Drd4 gene polymorphisms have been reported for free-living species (Mogensen et al. 2006), but studies associating this variation with behaviour are absent.

Notwithstanding the publication of a draft jungle fowl (Gallus gallus) genomic sequence (International Chicken Genome Sequencing Consortium 2004), avian genomic studies are in their infancy when compared with those of mammals, particularly humans. Our present knowledge of polymorphic variation in potentially behaviour-related genes of birds, other than the domestic chicken (Keeling et al. 2004; Natt et al. 2007), is very limited. However, the well-established advantages of birds as subjects for ethological and behavioural ecological research indicate that these avian genomic challenges are worth confronting.

Previous studies have reported that polymorphisms in the exon encoding the DRD4 receptor third intracellular loop are associated with personality variation among humans, monkeys and horses. Therefore, we examined the corresponding coding region in the P. major Drd4 cDNA sequence (nucleotides 647–947 of DQ006802) for sequence polymorphisms. We directly sequenced PCR products amplified from genomic DNA from 27 individuals from two P. major lines selected over four generations for divergent levels of EEB (fast, i.e. having high EEB scores and slow, i.e. having low EEB scores; Drent et al. 2003). Only one single nucleotide polymorphism (SNP) was identified, corresponding to either C or T at position 830 of the P. major Drd4 cDNA sequence (DQ006802). Frequencies of the three Drd4 SNP830 genotypes differed significantly between birds of the slow (mean EEB score=0.81±1.40s.d., n=21) and fast EEB (mean EEB score=14.55±3.31s.d., n=29) lines, with the slow EEB line having a lower SNP830T allele frequency than the fast line (figure 1a). Furthermore, the SNP830 genotype frequencies in the slow EEB line, but not in the fast EEB line, differed significantly from genotype frequencies in unselected birds taken from a Dutch natural P. major population, with the SNP830T/T and SNP830C/T genotypes being scarcer in the slow EEB line than among the unselected birds (table 1). These results are consistent with the EEB phenotype-based selection regime having selected against the SNP830T allele in the slow EEB selection line. However, the relative scarcity of the SNP830T allele in the slow EEB selection line could also be a consequence of simple random genetic effects, such as founder effects and genetic drift, within the small populations forming the EEB-selected lines (Drent et al. 2003). To address this problem, we investigated the association between SNP830 genotypes and EEB score among unselected birds taken from a natural population.

Among the unselected birds, differences in EEB scores were associated with differences in Drd4 SNP830 genotype: the mean EEB score of SNP830C/C homozygotes was significantly lower than the means of both SNP830C/T heterozygotes and SNP830T/T homozygotes (figure 2). This finding suggests a dominance effect of the SNP830T allele, which is consistent with the SNP830 genotype frequencies found in the EEB-selected lines. Thus, selecting for the slow EEB phenotype may have resulted in selection against both SNP830C/T heterozygotes and SNP830T/T homozygotes, leading to the observed lower frequency of the SNP830T allele in the slow EEB line (figure 1a).

These results indicate a statistically significant association between the P. major Drd4 SNP830 genotype and EEB scores. Such correlational findings, however, do not prove any functional link between the analysed locus and phenotype. Indeed, the SNP830 polymorphism, located at the third base of codon 249, is synonymous, with both alleles encoding Ala₂₄₉. Examination of an avian (G. gallus) codon usage table (http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=Gallus+gallus+[gbvrt]) indicated that the alternative Ala₂₄₉ codons would be translated with similar efficiency. Although a phenotypic effect through the protein structure of the Drd4 SNP830 polymorphism can be excluded, it is noteworthy that synonymous polymorphisms have been shown to affect transcription, splicing, mRNA stability and translation, in general (Chamary et al. 2006; Goymer 2007), and in dopamine receptors, in particular (Duan et al. 2003).

The association between SNP830 genotype and EEB phenotype might be explained by linkage disequilibrium between SNP830 variants and one or more functionally significant polymorphism(s) which could possibly be located within the Drd4 gene itself. In a search for such additional polymorphisms, P. major Drd4 genomic sequences were obtained from a minimum of two SNP830C/C and two SNP830T/T homozygous genomes with a representative (SNP830C) sequence deposited on GenBank (10897bp, acc. no. DQ006801; figure 2 in electronic supplementary material). Alignment of the Drd4 genomic (DQ006801) and cDNA (DQ006802) sequences allowed definitive identification of exon/intron boundaries, while a possible transcription initiation region was tentatively assigned as being adjacent to the 5′ end of the 5′-RACE products (i.e. more than 1763 of DQ006801) (figure 3 and figure 2 in electronic supplementary material).

The P. major Drd4 gene, like its human orthologue, is clearly highly polymorphic (figure 3; Cravchik & Goldman 2000). By comparing the Drd4 SNP830C and SNP830T genomic sequences, we identified 73 polymorphisms: 66 SNPs and 7 indels (figure 3), which were grouped for declining degrees of association with the SNP830 polymorphisms: category-1, positions where the alternative sequences were associated exclusively with either the SNP830C or SNP830T allele; category-2, positions where either the SNP830C or the SNP830T sequences, but not both, were heterogeneous; and category-3, positions where both the SNP830C and the SNP830T allelic sequences were heterogeneous for the polymorphism. No additional polymorphisms were found within the Drd4-coding region and a single C/T SNP in the 5′UTR showed no evidence of an association with either the SNP830C or SNP830T polymorphism (i.e. category-3; figure 3). All 21 category-1 polymorphisms were SNPs located within introns and, on the basis of their nature and position, functional significance could not be attributed to these variants. Among the 47 polymorphisms in category-2, we identified an indel of possible functional significance. A 15bp indel, located 1036bp from the tentatively assigned transcription initiation site (coordinates 713–727 of DQ006801; ID15, figure 3), was predicted by transcription factor-binding site identification software (Tfsearch) to potentially result in the presence or absence of two putative transcription factor-binding sites. As polymorphisms in the Drd4 promoter region had previously been associated with variation in human personality traits (Kluger et al. 2002; Schinka et al. 2002; Reif & Lesch 2003; van Gestel & Van Broeckhoven 2003; Savitz & Ramesar 2004; Ebstein 2006), we decided to determine if there was any evidence for an association between the P. major Drd4 ID15 polymorphism and EEB score.

Drd4 ID15 genotypes of both EEB-selected and unselected birds were determined on the basis of the size of amplification products generated using primers flanking the ID15 polymorphism. The slow and fast EEB lines did not differ significantly in the frequencies of the three ID15 genotypes (figure 1b). However, the slow EEB line did differ significantly in ID15 genotype frequencies when compared with the unselected birds (table 1). It is not surprising that the slow EEB line differed significantly from the unselected birds in both the SNP830 and ID15 genotype frequencies, because the two polymorphic sites are separated by only 9359bp and selection was only over four generations. Therefore, any allelic associations between polymorphisms (i.e. haplotype combinations) in the selection line founder birds are likely to remain among their descendents.