The regulation of methylation on the Z chromosome and the identification of multiple novel Male Hyper-Methylated regions in the chicken.

DNA methylation is a key regulator of eukaryote genomes, and is of particular relevance in the regulation of gene expression on the sex chromosomes, with a key role in dosage compensation in mammalian XY systems. In the case of birds, dosage compensation is largely absent, with it being restricted to two small Male Hyper-Methylated (MHM) regions on the Z chromosome. To investigate how variation in DNA methylation is regulated on the Z chromosome we utilised a wild x domestic advanced intercross in the chicken, with both hypothalamic methylomes and transcriptomes assayed in 124 individuals. The relatively large numbers of individuals allowed us to identify additional genomic MHM regions on the Z chromosome that were significantly differentially methylated between the sexes. These regions appear to down-regulate local gene expression in males, but not remove it entirely (unlike the lncRNAs identified in the initial MHM regions). These MHM regions were further tested and the most balanced genes appear to show decreased expression in males, whilst methylation appeared to be far more correlated with gene expression in the less balanced, as compared to the most balanced genes. In addition, quantitative trait loci (QTL) that regulate variation in methylation on the Z chromosome, and those loci that regulate methylation on the autosomes that derive from the Z chromosome were mapped. Trans-effect hotspots were also identified that were based on the autosomes but affected the Z, and also one that was based on the Z chromosome but that affected both autosomal and sex chromosome DNA methylation regulation. We show that both cis and trans loci that originate from the Z chromosome never exhibit an interaction with sex, whereas trans loci originating from the autosomes but affecting the Z chromosome always display such an interaction. Our results highlight how additional MHM regions are actually present on the Z chromosome, and they appear to have smaller-scale effects on gene expression in males. Quantitative variation in methylation is also regulated both from the autosomes to the Z chromosome, and from the Z chromosome to the autosomes.

A Comparison of Telencephalon Composition among Chickens, Junglefowl, and Wild Galliforms.

INTRODUCTION: Domestication is the process of modifying animals for human benefit through selective breeding in captivity. One of the traits that often diverges is the size of the brain and its constituent regions; almost all domesticated species have relatively smaller brains and brain regions than their wild ancestors. Although the effects of domestication on the brain have been investigated across a range of both mammal and bird species, almost nothing is known about the neuroanatomical effects of domestication on the world's most common bird: the chicken (Gallus gallus). METHODS: We compared the quantitative neuroanatomy of the telencephalon of white leghorn chickens with red junglefowl, their wild counterpart, and several wild galliform species. We focused specifically on the telencephalon because telencephalic regions typically exhibit the biggest differences in size in domesticate-wild comparisons. RESULTS: Relative telencephalon size was larger in chickens than in junglefowl and ruffed grouse (Bonasa umbellus). The relative size of telencephalic regions did not differ between chickens and junglefowl, but did differ in comparison with ruffed grouse. Ruffed grouse had larger hyperpallia and smaller entopallial, nidopallial, and striatal volumes than chickens and junglefowl. Multivariate analyses that included an additional three wild grouse species corroborated these findings: chicken and junglefowl have relatively larger nidopallial and striatal volumes than grouse. Conversely, the mesopallial and hyperpallial volumes tended to be relatively smaller in chickens and junglefowl. CONCLUSION: From this suite of comparisons, we conclude that chickens do not follow a pattern of widespread decreases in telencephalic region sizes that is often viewed as typical of domestication. Instead, chickens have undergone a mosaic of changes with some regions increasing and others decreasing in size, and there are few differences between chickens and junglefowl.

Population structure and hybridisation in a population of Hawaiian feral chickens.

Chickens are believed to have inhabited the Hawaiian island of Kauai since the first human migrations around 1200AD, but numbers have peaked since the tropical storms Iniki and Iwa in the 1980s and 1990s that destroyed almost all the chicken coops on the island and released large numbers of domestic chickens into the wild. Previous studies have shown these now feral chickens are an admixed population between Red Junglefowl (RJF) and domestic chickens. Here, using genetic haplotypic data, we estimate the time of the admixture event between the feral population on the island and the RJF to 1981 (1976-1995), coinciding with the timings of storm Iwa and Iniki. Analysis of genetic structure reveals a greater similarity between individuals inhabiting the northern and western part of the island to RJF than individuals from the eastern part of the island. These results point to the possibility of introgression events between feral chickens and the wild chickens in areas surrounding the Koke'e State Park and the Alaka'i plateau, posited as two of the major RJF reservoirs in the island. Furthermore, we have inferred haplotype blocks from pooled data to determine the most plausible source of the feral population. We identify a clear contribution from RJF and layer chickens of the White Leghorn (WL) breed. This work provides independent confirmation of the traditional hypothesis surrounding the origin of the feral populations and draws attention to the possibility of introgression of domestic alleles into the wild reservoir.

Neuronal and non-neuronal scaling across brain regions within an intercross of domestic and wild chickens.

The allometric scaling of the brain size and neuron number across species has been extensively studied in recent years. With the exception of primates, parrots, and songbirds, larger brains have more neurons but relatively lower neuronal densities than smaller brains. Conversely, when considering within-population variability, it has been shown that mice with larger brains do not necessarily have more neurons but rather more neurons in the brain reflect higher neuronal density. To what extent this intraspecific allometric scaling pattern of the brain applies to individuals from other species remains to be explored. Here, we investigate the allometric relationships among the sizes of the body, brain, telencephalon, cerebellum, and optic tectum, and the numbers of neurons and non-neuronal cells of the telencephalon, cerebellum, and optic tectum across 66 individuals originated from an intercross between wild and domestic chickens. Our intercross of chickens generates a population with high variation in brain size, making it an excellent model to determine the allometric scaling of the brain within population. Our results show that larger chickens have larger brains with moderately more neurons and non-neuronal cells. Yet, absolute number of neurons and non-neuronal cells correlated strongly and positively with the density of neurons and non-neuronal cells, respectively. As previously shown in mice, this scaling pattern is in stark contrast with what has been found across different species. Our findings suggest that neuronal scaling rules across species are not a simple extension of the neuronal scaling rules that apply within a species, with important implications for the evolutionary developmental origins of brain diversity.

Genomic and gene expression associations to morphology of a sexual ornament in the chicken.

How sexual selection affects the genome ultimately relies on the strength and type of selection, and the genetic architecture of the involved traits. While associating genotype with phenotype often utilizes standard trait morphology, trait representations in morphospace using geometric morphometric approaches receive less focus in this regard. Here, we identify genetic associations to a sexual ornament, the comb, in the chicken system (Gallus gallus). Our approach combined genome-wide genotype and gene expression data (>30k genes) with different aspects of comb morphology in an advanced intercross line (F8) generated by crossing a wild-type Red Junglefowl with a domestic breed of chicken (White Leghorn). In total, 10 quantitative trait loci were found associated to various aspects of comb shape and size, while 1,184 expression QTL were found associated to gene expression patterns, among which 98 had overlapping confidence intervals with those of quantitative trait loci. Our results highlight both known genomic regions confirming previous records of a large effect quantitative trait loci associated to comb size, and novel quantitative trait loci associated to comb shape. Genes were considered candidates affecting comb morphology if they were found within both confidence intervals of the underlying quantitative trait loci and eQTL. Overlaps between quantitative trait loci and genome-wide selective sweeps identified in a previous study revealed that only loci associated to comb size may be experiencing on-going selection under domestication.

Proportional Cerebellum Size Predicts Fear Habituation in Chickens.

The cerebellum has a highly conserved neural structure across species but varies widely in size. The wide variation in cerebellar size (both absolute and in proportion to the rest of the brain) among species and populations suggests that functional specialization is linked to its size. There is increasing recognition that the cerebellum contributes to cognitive processing and emotional control in addition to its role in motor coordination. However, to what extent cerebellum size reflects variation in these behavioral processes within species remains largely unknown. By using a unique intercross chicken population based on parental lines with high divergence in cerebellum size, we compared the behavior of individuals repeatedly exposed to the same fear test (emergence test) early in life and after sexual maturity (eight trials per age group) with proportional cerebellum size and cerebellum neural density. While proportional cerebellum size did not predict the initial fear response of the individuals (trial 1), it did increasingly predict adult individuals response as the trials progressed. Our results suggest that proportional cerebellum size does not necessarily predict an individual's fear response, but rather the habituation process to a fearful stimulus. Cerebellum neuronal density did not predict fear behavior in the individuals which suggests that these effects do not result from changes in neuronal density but due to other variables linked to proportional cerebellum size which might underlie fear habituation.

The cerebellar anatomy of red junglefowl and white leghorn chickens: insights into the effects of domestication on the cerebellum.

Domestication is the process by which wild organisms become adapted for human use. Many phenotypic changes are associated with animal domestication, including decreases in brain and brain region sizes. In contrast with this general pattern, the chicken has a larger cerebellum compared with the wild red junglefowl, but what neuroanatomical changes are responsible for this difference have yet to be investigated. Here, we quantified cell layer volumes, neuron numbers and neuron sizes in the cerebella of chickens and junglefowl. Chickens have larger, more folded cerebella with more and larger granule cells than junglefowl, but neuron numbers and cerebellar folding were proportional to cerebellum size. However, chickens do have relatively larger granule cell layer volumes and relatively larger granule cells than junglefowl. Thus, the chicken cerebellum can be considered a scaled-up version of the junglefowl cerebellum, but with enlarged granule cells. The combination of scaling neuron number and disproportionate enlargement of cell bodies partially supports a recent theory that domestication does not affect neuronal density within brain regions. Whether the neuroanatomical changes we observed are typical of domestication or not requires similar quantitative analyses in other domesticated species and across multiple brain regions.

The genomics of phenotypically differentiated Asellus aquaticus cave, surface stream and lake ecotypes.

Organisms well suited for the study of ecotype formation have wide distribution ranges, where they adapt to multiple drastically different habitats repeatedly over space and time. Here we study such ecotypes in a Crustacean model, Asellus aquaticus, a commonly occurring isopod found in freshwater habitats as diverse as streams, caves and lakes. Previous studies focusing on cave vs. surface ecotypes have attributed depigmentation, eye loss and prolonged antennae to several south European cave systems. Likewise, surveys across multiple Swedish lakes have identified the presence of dark-pigmented "reed" and light-pigmented "stonewort" ecotypes, which can be found within the same lake. In this study, we sequenced the first draft genome of A. aquaticus, and subsequently use this to map reads and call variants in surface stream, cave and two lake ecotypes. In addition, the draft genome was combined with a RADseq approach to perform a quantitative trait locus (QTL) mapping study using a laboratory bred F2 and F4 cave × surface intercross. We identified genomic regions associated with body pigmentation, antennae length and body size. Furthermore, we compared genome-wide differentiation between natural populations and found several genes potentially associated with these habitats. The assessment of the cave QTL regions in the light-dark comparison of lake populations suggests that the regions associated with cave adaptation are also involved with genomic differentiation in the lake ecotypes. These demonstrate how troglomorphic adaptations can be used as a model for related ecotype formation.

Cerebellum size is related to fear memory and domestication of chickens.

Red Junglefowl (Gallus gallus) were selected for divergent levels of fear of humans during eight generations, causing the selection lines to differ in fear levels as well as in the proportional brain and cerebellum masses. Birds from the two lines were then crossed to obtain an F3 intercross in order to study the correlations between brain mass and fear learning. We exposed 105 F3-animals individually to a fear habituation and memory test at 8 days of age, where the reactions to repeated light flashes were assessed on 2 consecutive days. After culling, the absolute and relative sizes of each of four brain regions were measured. Stepwise regression was used to analyse the effects of the size of each brain region on habituation and memory. There were no effects of any brain region on the habituation on day one. However, birds with a larger absolute size of cerebellum had significantly reduced reactions to the fearful stimuli on day two, indicating a better memory of the stimuli. No other regions had significant effects. We conclude that increased cerebellum size may have been important in facilitating chicken domestication, allowing them to adapt to a life with humans.

Effect of contact incubation on stress, behavior and body composition in the precocial Red jungle fowl.

Birds use contact incubation to warm their eggs above ambient temperature required for embryonic development. In contrast, birds in the industry as well as many birds in breeding programs and scientific studies are incubated in conventional incubators that warm eggs via circulating warm air. This means that contact incubated eggs have different thermal properties than eggs incubated in a conventional incubator. In light of previous studies showing that small differences in incubation temperature can affect chicks post-hatching phenotype, we investigated the consequences of incubating Red jungle fowl eggs at the same temperature (37 °C) either via contact incubation or warm air incubation. We found that contact incubated chicks had a more robust body composition, were more explorative and had a higher temperature preference early in life, as well as a sex dependent difference in plasma Corticosterone levels pre-hatch (measured in down-feathers) and post-hatch (measured in plasma) compared to chicks incubated in a conventional warm air incubator. While previous studies have demonstrated that embryonic development and post-hatch phenotype is sensitive to small variations in temperature, our study demonstrates for the first time that the way heat is distributed to the egg has a similar magnitude of effect on post-hatch phenotype and highlights the sensitivity of the incubation period in shaping birds post-hatch phenotype.

Intra-Individual Behavioural Variability: A Trait under Genetic Control.

When individuals are measured more than once in the same context they do not behave in exactly the same way each time. The degree of predictability differs between individuals, with some individuals showing low levels of variation around their behavioural mean while others show high levels of variation. This intra-individual variability in behaviour has received much less attention than between-individual variability in behaviour, and very little is known about the underlying mechanisms that affect this potentially large but understudied component of behavioural variation. In this study, we combine standardized behavioural tests in a chicken intercross to estimate intra-individual behavioural variability with a large-scale genomics analysis to identify genes affecting intra-individual behavioural variability in an avian population. We used a variety of different anxiety-related behavioural phenotypes for this purpose. Our study shows that intra-individual variability in behaviour has a direct genetic basis that is largely unique compared to the genetic architecture for the standard behavioural measures they are based on (at least in the detected quantitative trait locus). We identify six suggestive candidate genes that may underpin differences in intra-individual behavioural variability, with several of these candidates having previously been linked to behaviour and mental health. These findings demonstrate that intra-individual variability in behaviour appears to be a heritable trait in and of itself on which evolution can act.

The methylation landscape and its role in domestication and gene regulation in the chicken.

Domestication is one of the strongest examples of artificial selection and has produced some of the most extreme within-species phenotypic variation known. In the case of the chicken, it has been hypothesized that DNA methylation may play a mechanistic role in the domestication response. By inter-crossing wild-derived red junglefowl with domestic chickens, we mapped quantitative trait loci for hypothalamic methylation (methQTL), gene expression (eQTL) and behaviour. We find large, stable methylation differences, with 6,179 cis and 2,973 trans methQTL identified. Over 46% of the trans effects were genotypically controlled by five loci, mainly associated with increased methylation in the junglefowl genotype. In a third of eQTL, we find that there is a correlation between gene expression and methylation, while statistical causality analysis reveals multiple instances where methylation is driving gene expression, as well as the reverse. We also show that methylation is correlated with some aspects of behavioural variation in the inter-cross. In conclusion, our data suggest a role for methylation in the regulation of gene expression underlying the domesticated phenotype of the chicken.

The genetic regulation of size variation in the transcriptome of the cerebrum in the chicken and its role in domestication and brain size evolution.

BACKGROUND: Large difference in cerebrum size exist between avian species and populations of the same species and is believed to reflect differences in processing power, i.e. in the speed and efficiency of processing information in this brain region. During domestication chickens developed a larger cerebrum compared to their wild progenitor, the Red jungle fowl. The underlying mechanisms that control cerebrum size and the extent to which genetic regulation is similar across brain regions is not well understood. In this study, we combine measurement of cerebrum size with genome-wide genetical genomics analysis to identify the genetic architecture of the cerebrum, as well as compare the regulation of gene expression in this brain region with gene expression in other regions of the brain (the hypothalamus) and somatic tissue (liver). RESULTS: We identify one candidate gene that putatively regulates cerebrum size (MTF2) as well as a large number of eQTL that regulate the transcriptome in cerebrum tissue, with the majority of these eQTL being trans-acting. The overall regulation of gene expression variation in the cerebrum was markedly different to the hypothalamus, with relatively few eQTL in common. In comparison, the cerebrum tissue shared more eQTL with a distant tissue (liver) than with a neighboring tissue (hypothalamus). CONCLUSION: The candidate gene for cerebrum size (MTF2) has previously been linked to brain development making it a good candidate for further investigation as a regulator of inter-population variation in cerebrum size. The lack of shared eQTL between the two brain regions implies that genetic regulation of gene expression appears to be relatively independent between the two brain regions and suggest that coevolution between these two brain regions might be more functionally driven than developmental. These findings have relevance for current brain size evolution theories.

Getting Back to Nature: Feralization in Animals and Plants.

Formerly domesticated organisms and artificially selected genes often escape controlled cultivation, but their subsequent evolution is not well studied. In this review, we examine plant and animal feralization through an evolutionary lens, including how natural selection, artificial selection, and gene flow shape feral genomes, traits, and fitness. Available evidence shows that feralization is not a mere reversal of domestication. Instead, it is shaped by the varied and complex histories of feral populations, and by novel selection pressures. To stimulate further insight we outline several future directions. These include testing how 'domestication genes' act in wild settings, studying the brains and behaviors of feral animals, and comparative analyses of feral populations and taxa. This work offers feasible and exciting research opportunities with both theoretical and practical applications.

Maladaptation in feral and domesticated animals.

Selection regimes and population structures can be powerfully changed by domestication and feralization, and these changes can modulate animal fitness in both captive and natural environments. In this review, we synthesize recent studies of these two processes and consider their impacts on organismal and population fitness. Domestication and feralization offer multiple windows into the forms and mechanisms of maladaptation. Firstly, domestic and feral organisms that exhibit suboptimal traits or fitness allow us to identify their underlying causes within tractable research systems. This has facilitated significant progress in our general understandings of genotype-phenotype relationships, fitness trade-offs, and the roles of population structure and artificial selection in shaping domestic and formerly domestic organisms. Additionally, feralization of artificially selected gene variants and organisms can reveal or produce maladaptation in other inhabitants of an invaded biotic community. In these instances, feral animals often show similar fitness advantages to other invasive species, but they are also unique in their capacities to modify natural ecosystems through introductions of artificially selected traits. We conclude with a brief consideration of how emerging technologies such as genome editing could change the tempos, trajectories, and ecological consequences of both domestication and feralization. In addition to providing basic evolutionary insights, our growing understanding of mechanisms through which artificial selection can modulate fitness has diverse and important applications-from enhancing the welfare, sustainability, and efficiency of agroindustry, to mitigating biotic invasions.

Genetical Genomics of Tonic Immobility in the Chicken.

Identifying the molecular mechanisms of animal behaviour is an enduring goal for researchers. Gaining insight into these mechanisms enables us to gain a greater understanding of behaviour and their genetic control. In this paper, we perform Quantitative Trait Loci (QTL) mapping of tonic immobility behaviour in an advanced intercross line between wild and domestic chickens. Genes located within the QTL interval were further investigated using global expression QTL (eQTL) mapping from hypothalamus tissue, as well as causality analysis. This identified five candidate genes, with the genes PRDX4 and ACOT9 emerging as the best supported candidates. In addition, we also investigated the connection between tonic immobility, meat pH and struggling behaviour, as the two candidate genes PRDX4 and ACOT9 have previously been implicated in controlling muscle pH at slaughter. We did not find any phenotypic correlations between tonic immobility, struggling behaviour and muscle pH in a smaller additional cohort, despite these behaviours being repeatable within-test.

Genetics and Genomics of Social Behavior in a Chicken Model.

The identification of genes affecting sociality can give insights into the maintenance and development of sociality and personality. In this study, we used the combination of an advanced intercross between wild and domestic chickens with a combined QTL and eQTL genetical genomics approach to identify genes for social reinstatement, a social and anxiety-related behavior. A total of 24 social reinstatement QTL were identified and overlaid with over 600 eQTL obtained from the same birds using hypothalamic tissue. Correlations between overlapping QTL and eQTL indicated five strong candidate genes, with the gene TTRAP being strongly significantly correlated with multiple aspects of social reinstatement behavior, as well as possessing a highly significant eQTL.

Genetical genomics of growth in a chicken model.

BACKGROUND: The genetics underlying body mass and growth are key to understanding a wide range of topics in biology, both evolutionary and developmental. Body mass and growth traits are affected by many genetic variants of small effect. This complicates genetic mapping of growth and body mass. Experimental intercrosses between individuals from divergent populations allows us to map naturally occurring genetic variants for selected traits, such as body mass by linkage mapping. By simultaneously measuring traits and intermediary molecular phenotypes, such as gene expression, one can use integrative genomics to search for potential causative genes. RESULTS: In this study, we use linkage mapping approach to map growth traits (N = 471) and liver gene expression (N = 130) in an advanced intercross of wild Red Junglefowl and domestic White Leghorn layer chickens. We find 16 loci for growth traits, and 1463 loci for liver gene expression, as measured by microarrays. Of these, the genes TRAK1, OSBPL8, YEATS4, CEP55, and PIP4K2B are identified as strong candidates for growth loci in the chicken. We also show a high degree of sex-specific gene-regulation, with almost every gene expression locus exhibiting sex-interactions. Finally, several trans-regulatory hotspots were found, one of which coincides with a major growth locus. CONCLUSIONS: These findings not only serve to identify several strong candidates affecting growth, but also show how sex-specificity and local gene-regulation affect growth regulation in the chicken.