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.

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.

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.

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.

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.

Epigenetics and early domestication: differences in hypothalamic DNA methylation between red junglefowl divergently selected for high or low fear of humans.

BACKGROUND: Domestication of animals leads to large phenotypic alterations within a short evolutionary time-period. Such alterations are caused by genomic variations, yet the prevalence of modified traits is higher than expected if they were caused only by classical genetics and mutations. Epigenetic mechanisms may also be important in driving domesticated phenotypes such as behavior traits. Gene expression can be modulated epigenetically by mechanisms such as DNA methylation, resulting in modifications that are not only variable and susceptible to environmental stimuli, but also sometimes transgenerationally stable. To study such mechanisms in early domestication, we used as model two selected lines of red junglefowl (ancestors of modern chickens) that were bred for either high or low fear of humans over five generations, and investigated differences in hypothalamic DNA methylation between the two populations. RESULTS: Twenty-two 1-kb windows were differentially methylated between the two selected lines at p < 0.05 after false discovery rate correction. The annotated functions of the genes within these windows indicated epigenetic regulation of metabolic and signaling pathways, which agrees with the changes in gene expression that were previously reported for the same tissue and animals. CONCLUSIONS: Our results show that selection for an important domestication-related behavioral trait such as tameness can cause divergent epigenetic patterns within only five generations, and that these changes could have an important role in chicken domestication.

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.

The evolution of Sex-linked barring alleles in chickens involves both regulatory and coding changes in CDKN2A.

Sex-linked barring is a fascinating plumage pattern in chickens recently shown to be associated with two non-coding and two missense mutations affecting the ARF transcript at the CDKN2A tumor suppressor locus. It however remained a mystery whether all four mutations are indeed causative and how they contribute to the barring phenotype. Here, we show that Sex-linked barring is genetically heterogeneous, and that the mutations form three functionally different variant alleles. The B0 allele carries only the two non-coding changes and is associated with the most dilute barring pattern, whereas the B1 and B2 alleles carry both the two non-coding changes and one each of the two missense mutations causing the Sex-linked barring and Sex-linked dilution phenotypes, respectively. The data are consistent with evolution of alleles where the non-coding changes occurred first followed by the two missense mutations that resulted in a phenotype more appealing to humans. We show that one or both of the non-coding changes are cis-regulatory mutations causing a higher CDKN2A expression, whereas the missense mutations reduce the ability of ARF to interact with MDM2. Caspase assays for all genotypes revealed no apoptotic events and our results are consistent with a recent study indicating that the loss of melanocyte progenitors in Sex-linked barring in chicken is caused by premature differentiation and not apoptosis. Our results show that CDKN2A is a major locus driving the differentiation of avian melanocytes in a temporal and spatial manner.

Characterization of the GPI-anchored lipid transfer proteins in the moss Physcomitrella patens.

The non-specific lipid transfer proteins (nsLTPs) are characterized by a compact structure with a central hydrophobic cavity very suitable for binding hydrophobic ligands, such as lipids. The nsLTPs are encoded by large gene families in all land plant lineages, but seem to be absent from green algae. The nsLTPs are classified to different types based on molecular weight, sequence similarity, intron position or spacing between the cysteine residues. The Type G nsLTPs (LTPGs) have a GPI-anchor in the C-terminal region which may attach the protein to the exterior side of the plasma membrane. Here, we present the first characterization of nsLTPs from an early diverged plant, the moss Physcomitrella patens. Moss LTPGs were heterologously produced and purified from Pichia pastoris. The purified moss LTPGs were found to be extremely heat stable and showed a binding preference for unsaturated fatty acids. Structural modeling implied that high alanine content could be important for the heat stability. Lipid profiling revealed that cutin monomers, such as C16 and C18 mono- and di-hydroxylated fatty acids, could be identified in P. patens. Expression of a moss LTPG-YFP fusion revealed localization to the plasma membrane. The expressions of many of the moss LTPGs were found to be upregulated during drought and cold treatments.