- Top of page
- Material and methods
A growth-related QTL on chicken chromosome 1 has previously been shown to influence domestication behaviour in chickens. In this study, we used Red Junglefowl (RJF) and White Leghorn (WL) as well as the intercross between them to investigate whether stress affects the way birds allocate their time between familiar and unfamiliar conspecifics in a social preference test (‘social support seeking’), and how this is related to genotype at specific loci within the growth QTL. Red Junglefowl males spent more time with unfamiliar chickens before the stressful event compared to the other birds, whereas all birds except WL males tended to spend less time with unfamiliar ones after stress. A significant QTL locus was found to influence both social preference under undisturbed circumstances and social support seeking. The WL allele at this QTL was associated not only with a preference for unfamiliar individuals but also with a shift towards familiar ones in response to stress (social support seeking). A second, suggestive QTL also affected social support seeking, but in the opposite direction; the WL allele was associated with increased time spent with unfamiliar individuals. The region contains several possible candidate genes, and gene expression analysis of a number of them showed differential expression between RJF and WL of AVPR2 (receptor for vasotocin), and possibly AVPR1a (another vasotocin receptor) and NRCAM (involved in neural development) in the lower frontal lobes of the brains of RJF and WL animals. These three genes continue to be interesting candidates for the observed behavioural effects.
The chicken is an excellent model species for studying evolution in the form of animal domestication. Domestication changes the physiology and behaviour of animals, and these changes can be interpreted as a process of adaptation to the captive environment with its specific selection pressures (Price 1998). Compared to the wild, captivity is signified by a lack of predators, higher numbers of animals on a smaller space and a continuous presence of humans. In addition, humans have introduced new selection pressures, often for production traits such as high milk yield, egg production and growth rate. Given these simultaneous changes in multiple selection pressures, it is not unexpected that domestic animals often are less fearful of predators and more tolerant to unfamiliar conspecifics and humans (Price 1998) and at the same time have more favourable production traits. However, domestication experiments (e.g. the classic silver fox experiment; Trut et al. 2009) have shown that selecting animals for only one trait (e.g. tameness) can yield a correlated response in others, such as earlier sexual maturation, altered coat colour and later onset of fear response. This reoccurrence of a set of correlated traits in domestic species has been called the ‘domestic phenotype’ (Price 2002). This phenomenon may be explained by either pleiotropy or linkage of several genes affecting different traits.
We have earlier reported that a growth-related quantitative trait locus (QTL) on chromosome 1 of an intercross line between the domestic White Leghorn (WL) layer and the Red Junglefowl (RJF, main ancestor of domestic chickens) simultaneously affects emotionality and social behaviours (sociality and tolerance of social novelty). White Leghorn genotypes in this locus are associated with less exploration of novel environments and more time spent with conspecifics (Väisänen 2005; Väisänen et al. 2005; Wirén and Jensen 2011; Wirén et al. 2009). The region has also been found to be involved in fear reactions, for example, tonic immobility and open field behaviour (Schütz et al. 2001). A region spanning 8 MB around the QTL includes behaviourally potent genes such as AVPR1a, AVPR2, NRCAM and Contactin-1. However, whether pleiotropy or linkage is responsible for the correlation between traits and which parts of the QTL region that affect which traits remains unknown.
Stress is the response to a challenging situation. This response can be alleviated by the presence of familiar conspecifics (Kaiser et al. 2003; Kirschbaum et al. 1995), so called social support. Because of the greater tolerance of domestic birds to unfamiliar individuals (Wirén and Jensen 2011) we hypothesized that WL chickens depend less on social support from familiar individuals, than RJF. To test this hypothesis and elucidate the genetic basis for such a difference, we subjected purebred WL and RJF as well as animals from an RJF × WL intercross line to a social preference test, where birds had a choice of spending time with familiar or unfamiliar stimulus birds before and after a stressful episode of physical restraint. The two pure breeds were chosen, since they have previously been extensively studied with respect to genetic mapping of behavioural traits, and because the intercross line is based on precisely the two lines used here. We then performed a refined QTL study using markers limited to the region of the growth QTL. In addition, we examined differential expression of a number of genes in the region in brain tissue from purebred birds.
- Top of page
- Material and methods
Our results show that social preference of familiar over unfamiliar birds may have been modified by domestication in chickens, and the breed effect was most clear in males. Furthermore, the tendency to associate with conspecifics, and to seek social support after a stressful event is affected by loci within a major growth QTL on chromosome 1. This indicates that the QTL region may contribute to domestication effects on social behaviour and stress coping, in addition, to its already documented effect on growth and reproduction (Schütz et al. 2004).
The social preference test showed that undisturbed birds preferred to stay close to familiar conspecifics, as has been demonstrated earlier (Väisänen and Jensen 2003), and a brief stress experience made this tendency stronger, particularly in females. However, purebred RJF males associated more with unfamiliar birds, which may be related to territory defence rather than social affiliations. Seeking of social support is also a well-known response to fear and stressful stimuli in other species (Kaiser et al. 2003), and the present results may, therefore, be closely linked to the previously shown effect on fear reactions in this cross (Schütz et al. 2001).
After a brief period of restraint stress, this preference increased, in line with the known importance of social support (Kaiser et al. 2003; Kawachi and Berkman 2001; Kirschbaum et al. 1995). The notable exception was the WL males, which appeared not to be socially affected by the stress experience. Among the intercross birds, there was a large individual variation in the preference for familiar over unfamiliar birds for this social support, which made it possible to investigate the role of the growth QTL on chromosome 1 in this respect. Although no heritability estimates are known for the exact phenotypes measured here, we have previously reported moderate to high heritability in social reinstatement tendency in chickens, which is a closely related behavioural response (Agnvall et al. 2012).
The present level of resolution does not allow us to distinguish whether we are dealing with one single locus or two separate QTL within the examined region, as one of the peaks is only suggestive. This could eventually be resolved with a larger animal material and higher marker density. However, opposite direction of effect of the two QTL peaks for Diff-U suggests the presence of two separate loci. Assuming that this is correct, a QTL peaking at marker 1_36652477 affected the tendency to spend more time with familiar conspecifics before restraint, which may indicate that this locus is related to the preference for social familiarity under undisturbed circumstances. After a stressful episode of restraint, birds with at least one RJF allele at this locus did not change this preference for social familiarity – if they sought social support from conspecifics, they did so from familiar individuals. However, birds with two WL alleles shifted their previous preference for social novelty (which may also be interpreted as high social tolerance) to a preference for familiar birds. It, therefore, appears that this locus affects social preference as well as support seeking.
The QTL locus at 69–70 cM, however, affected the shift in social preference in response to restraint in the opposite direction: if birds homozygous for a WL allele preferred social novelty under undisturbed circumstances, they did so even more after an episode of stress. This indicates that one or more genes within the C.I. make WL genotype birds more interested in social novelty and more socially tolerant than RJF genotype birds.
Comparing the additive and dominance effects (a and d) at the two QTL loci affecting Diff-U and Diff-F (the shift in social preference in response to stress), the locus at 149 cM had a greater effect than that the one at 70 cM. If there were little recombination between these two loci, there would still be a small net effect on the behavioural outcome. In the case of WL alleles, this net effect would be to make birds shift their social preference towards familiar birds in response to stress. Hence, if birds are selected for being more tolerant to unfamiliar individuals under non-stressed circumstances (which could be considered adaptive in a captive environment), they would get a small net tendency to shift social preference towards familiar individuals when stressed.
It is interesting to note that the locus peaking at 149 cM was associated with the tendency to seek social contact both before and after stress, whereas the one at 70 cM only affected social behaviour after restraint stress. Hence, the 149 cM locus may be a general sociality associated locus, whereas the 70 cM locus may be more related to stress coping ability and stress recovery.
The QTL region investigated here has been shown to affect several domestication-related traits in chickens, e.g growth (Kerje et al. 2003), fearfulness (Schütz et al. 2004), reproduction (Carlborg et al. 2003) sociality (Väisänen and Jensen 2003) and comb size (Wright et al. 2010). It has been argued that the locus represents an ancient selection signature, perhaps from the early ages of domestication, as the effects of this locus are much less pronounced in QTL crosses involving different strains of domesticated chickens (Schütz 2002). Our present results show that the locus also affects social tolerance and supports seeking following stress, and regardless of which of the QTL-related phenotypes that were the target of early selection, genetic linkage would cause a correlated response in the rest. Hence, together with earlier studies of the effects of this locus, this shows that a complex of domesticated phenotypes may emerge due to genetic architecture.
The effect of individual loci within the QTL region is not directly reflected in the phenotypic differences between breeds (purebred RJF males spend more time with unfamiliar individuals than other birds do). One possibility is that this is because of spurious QTL effects in a small sample size. However, it could also be that additional fixed loci affect the overall phenotype in RJF and WL and mask variation in the specific region examined in this study – variation that has been freed by several generations of intercrossing.
It is still only possible to speculate about the genes causing the individual QTL found in this study. Some candidates may be identified by visual inspection of gene content in the chromosome region, e.g. NRCAM, which is associated with autism in humans (Marui et al. 2009) and impaired sociability in mice (Moy et al. 2009), Contactin-1, which may be involved in neuronal development (Chung et al. 2008; Stoeckli 2010; Suter et al. 1995) and AVPR2, a homologue of the Arginine vasotocin receptor. In order to provide a first examination of which genes that may be involved in the observed behavioural difference between RJF and WL, gene expression analysis was performed on a number of tentative candidates in the QTL region. AVPR2 was significantly differentially expressed, and a tendency was found for AVPR1a and NRCAM. It should be remembered that the gene expression analysis was performed on a fairly large part of the brain, and this could easily ‘dilute’ a signal originating from differential expression in a smaller area or cell population. These three genes, therefore, remain candidates for causing the observed effects on behaviour, but other possible candidates need to be examined as well in future experiments.
Arginine vasotocin receptor density in this brain region has previously been found to correlate with gregariousness in a comparison between several species of finches (Goodson et al. 2006). Another vasotocin receptor homologue, AVPR1a, is known to affect social behaviour in many other species (Bielsky et al. 2005; Goodson et al. 2006; Walum et al. 2008). It is interesting to note that AVPR1a is positioned in the overlapping C.I.s of the two QTL regions discovered here, and therefore could be an interesting candidate for both QTL effects.
At this point, it is not possible to assess with certainty whether the two loci are driven by a single polymorphism or by two or more. One possibility is that a single mutation affects the expression of several genes in the interval, but it is equally likely that there are at least two different mutations, appearing in each of the QTL regions. This could be addressed by marker-assisted selection in a further advanced intercross, where recombination break-up would be even more intense, or by breeding of introgression lines, where small regions of the QTL locus are bred into a background of either WL or RJF.
The genes in the QTL region may have been selected independently, or they may be hitch-hiking on some unknown mutation in one of them, or in non-coding parts of the QTL. With the present resolution, it is not possible to resolve which of these suggestions are more likely. The expression differences could indicate that non-coding regulatory regions have been selected, but the sex differences complicate this suggestion. They may result from different selection pressures in the two sexes, or by genetic effects from translocated loci (e.g. on the sex chromosomes). However, the overexpression of AVPR1a in males is in line with what has been observed in other species (Bielsky et al. 2005; Walum et al. 2008). Phenotypically, we have shown earlier that stress reactions differ between the sexes, where males often have a stronger fear and stress reaction, and this may be an effect of the genetic differences (Campler et al. 2009).
In conclusion, the present experiment revealed two loci with significant effects on social preference and social support seeking following stress. As the QTL region has earlier been found to affect a range of domestication-related traits, the results indicate that changes in social behaviour may be genetically linked to these. AVPR1a, AVPR2 and NRCAM are three possible candidate genes for the observed QTL effects.