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Leif Andersson and colleagues report the genome sequence of the ruff, a bird species with three male morphs with different reproductive strategies. Satellite and faeder morphs differ from the common independent morph by a 4.5-Mb inversion that occurred approximately 3.8 million years ago, and multiple genetic changes within this inverted region are associated with the satellite and faeder morphs. The ruff is a Palearctic wader with a spectacular lekking behavior where highly ornamented males compete for females1,2,3,4. This bird has one of the most remarkable mating systems in the animal kingdom, comprising three different male morphs (independents, satellites and faeders) that differ in behavior, plumage color and body size. Remarkably, the satellite and faeder morphs are controlled by dominant alleles5,6. Here we have used whole-genome sequencing and resolved the enigma of how such complex phenotypic differences can have a simple genetic basis. The Satellite and Faeder alleles are both associated with a 4.5-Mb inversion that occurred about 3.8 million years ago. We propose an evolutionary scenario where the Satellite chromosome arose by a rare recombination event about 500,000 years ago. The ruff mating system is the result of an evolutionary process in which multiple genetic changes contributing to phenotypic differences between morphs have accumulated within the inverted region.

Onset of sexual maturation is a trait of extreme importance both evolutionarily and economically. Unsurprisingly therefore, domestication has acted to reduce the time to sexual maturation in a variety of animals, including the chicken. In comparison with wild progenitor chickens [the Red Junglefowl (RJF)], domestic layer hens attain maturity approximately 20% earlier. In addition, domestic layers also possess larger combs (a sexual ornament), produce more eggs and have denser bones. A large quantitative trait loci (QTL) analysis (n=377) was performed using an F(2) intercross between a White Leghorn layer breed and a RJF population, with onset of sexual maturity measured and mapped to three separate loci. This cross has already been analysed for comb mass, egg production and bone allocation. Onset of sexual maturity significantly correlated with comb mass, whilst the genetic architecture for sexual maturity and comb mass overlapped at all three loci. For two of these loci, the QTL for sexual maturity and comb mass were statistically indistinguishable from pleiotropy, suggesting that the alleles that increase comb mass also decrease onset of sexual maturity.

Autoimmune diseases are endemic, but the disease mechanisms are poorly understood. A way to better understand these are to find disease-regulating genes. However, this is difficult as the diseases are complex, with several genes as well as environmental factors influencing the development of disease. A way to facilitate the search for genes responsible for the diseases is to use comparative genomic studies. Animal models are relatively easy to analyze since control of environment and breeding are obtained.The University of California at Davies – line 200 (UCD-200) chickens have a hereditary disease that is similar to systemic sclerosis. Using a backcross between UCD-200 chickens and red junglefowl (RJF) chickens we identified three loci linked to the disease. The loci contained immune-regulatory genes suggested to be involved in systemic sclerosis in humans, as well as a previously unidentified linkage between systemic sclerosis in UCD-200 chickens and IGFBP3.The Dark brown (Db) gene enhances red pheomelanin and restricts expression of eumelanin in chickens. The Db phenotype is regulated by an 8 kb deletion upstream of SOX10. Pigmentation studies are potentially useful when trying to identify pathogenic mechanisms and candidate genes in vitiligoThe Obese strain (OS) of chickens spontaneously develops an autoimmune thyroiditis which closely resembles human Hashimoto’s thyroiditis. By using an intercross between OS chickens and RJF chickens, we found several disease phenotypes that can be used in an ongoing linkage analysis with the goal to find candidate genes for autoimmune disease. An important phenotype to record and add to the linkage analysis is autoantibodies against thyroid peroxidase, since this phenotype is a key feature in Hashimoto’s thyroiditis. Previous attempts to measure these titres in OS chickens have failed, hence an assay was developed for this purpose.

Sex-linked barring, a common plumage colour found in chickens, is characterized by black and white barred feathers. Previous studies have indicated that the white bands are caused by an absence of melanocytes in the feather follicle during the growth of this region. Here, we show that Sex-linked barring is controlled by the CDKN2A/B locus, which encodes the INK4b and ARF transcripts. We identified two non-coding mutations in CDKN2A that showed near complete association with the phenotype. In addition, two missense mutations were identified at highly conserved sites, V9D and R10C, and every bird tested with a confirmed Sex-linked barring phenotype carried one of these missense mutations. Further work is required to determine if one of these or a combined effect of two or more CDKN2A mutations is causing Sex-linked barring. This novel finding provides the first evidence that the tumour suppressor locus CDKN2A/B can affect pigmentation phenotypes and sheds new light on the functional significance of this gene.

Understanding the evolution of sexual ornaments, and particularly that of female sexual ornaments, is an enduring challenge in evolutionary biology. Key to this challenge are establishing the relationship between ornament expression and female reproductive investment, and determining the genetic basis underpinning such relationship. Advances in genomics provide unprecedented opportunities to study the genetic architecture of sexual ornaments in model species. Here, we present a quantitative trait locus (QTL) analysis of a female sexual ornament, the comb of the fowl, Gallus gallus, using a large-scale intercross between red junglefowl and a domestic line, selected for egg production. First, we demonstrate that female somatic investment in comb reflects female reproductive investment. Despite a trade-off between reproductive and skeletal investment mediated by the mobilization of skeletal minerals for egg production, females with proportionally large combs also had relatively high skeletal investment. Second, we identify a major QTL for bisexual expression of comb mass and several QTL specific to female comb mass. Importantly, QTL for comb mass were nonrandomly clustered with QTL for female reproductive and skeletal investment on chromosomes one and three. Together, these results shed light onto the physiological and genetic architecture of a female ornament.

Hyaluronan is a negatively charged polydisperse polysaccharide where both its size and tissue concentration play an important role in many physiological and pathological processes. The various functions of hyaluronan depend on its molecular size. Up to now, it has been difficult to study the role of hyaluronan in diseases with pathological changes in the extracellular matrix where availability is low or tissue samples are small. Difficulty to obtain large enough biopsies from human diseased tissue or tissue from animal models has also restricted the study of hyaluronan. In this paper, we demonstrate that gas-phase electrophoretic molecular mobility analyzer (GEMMA) can be used to estimate the distribution of hyaluronan molecular sizes in biological samples with a limited amount of hyaluronan. The low detection level of the GEMMA method allows for estimation of hyaluronan molecular sizes from different parts of small organs. Hence, the GEMMA method opens opportunity to attain a profile over the distribution of hyaluronan molecular sizes and estimate changes caused by disease or experimental conditions that has not been possible to obtain before.

An F 5 generation of an advanced inter-cross between red junglefowl (wild-type) and White Leghorn (domesticated) was used to investigate earlier findings suggesting that a mutation in the plumage color gene PMEL17 protects against victimization to feather pecking (FP). F 4 parents were selected according to genotype to produce PMEL17 homozygous offspring (i/i and I/I respectively). Birds were raised and their behavior recorded in groups of either two wild-type i/i (dark colored) and one white I/I, or two I/I and one i/i. In addition each bird was tested for feather preference, reaction to novelty, open-field activity, fear for humans, and tonic-immobility. In the home-pens, i/i birds were more feather pecked and had poorer feather condition than I/I birds. No pecking preference for immobile dark colored feathers was observed. In the open-field test i/i birds vocalized more and earlier than I/I birds, and in the fear-for-human test I/I birds had higher activity at 21 weeks of age. No other behavior differences were observed, but clearly, genotypes of PMEL17 affected some aspects of behavior. Such behavioral differences might be important aspects of the mechanism which predispose i/i individuals for being victims of FP.

Dominant white, Dun, and Smoky are alleles at the Dominant white locus, which is one of the major loci affecting plumage color in the domestic chicken. Both Dominant white and Dun inhibit the expression of black eumelanin. Smoky arose in a White Leghorn homozygous for Dominant white and partially restores pigmentation. PMEL17 encodes a melanocyte-specific protein and was identified as a positional candidate gene due to its role in the development of eumelanosomes. Linkage analysis of PMEL17 and Dominant white using a red jungle fowl/White Leghorn intercross revealed no recombination between these loci. Sequence analysis showed that the Dominant white allele was exclusively associated with a 9-bp insertion in exon 10, leading to an insertion of three amino acids in the PMEL17 transmembrane region. Similarly, a deletion of five amino acids in the transmembrane region occurs in the protein encoded by Dun. The Smoky allele shared the 9-bp insertion in exon 10 with Dominant white, as expected from its origin, but also had a deletion of 12 nucleotides in exon 6, eliminating four amino acids from the mature protein. These mutations are, together with the recessive silver mutation in the mouse, the only PMEL17 mutations with phenotypic effects that have been described so far in any species.

The aim of this work was to study fear responses and their relation to production traits in red junglefowl ( Gallus gallus spp.), White Leghorn ( Gallus domesticus ), and their F2-progeny. Quantitative trait locus (QTL) analyses were performed for behavioral traits to gain information about possible genetic links between fear-related behaviors and production. Four behavioral tests were performed that induce different levels of acute fear (open field [OF], exposure to a novel object, tonic immobility, and restraint). Production traits, that is, egg production, sexual maturity (in females), food intake, and growth, were measured individually. A genome scan using 105 microsatellite markers was carried out to identify QTLs controlling the traits studied. In the OF and novel object tests (NO), Leghorns showed less fear behavior than junglefowl, whereas junglefowl behaved less fearfully in the tonic immobility test (TI) and were more active in the restraint test. In the F2 progeny, only weak phenotypic associations were found between production traits and fear behavior. A significant QTL for TI duration was found on chromosome 1 that coincided with a QTL for egg weight and growth in the same animals. Another QTL for NO in males coincided with another major growth QTL. These two known growth QTLs affected a wide range of reactions in different tests. Several other significant and suggestive QTLs for behavioral traits related to fear were found. These QTLs did not coincide with QTLs for production traits, indicating that these fear variables may not be genetically linked to the production traits we measured here. The results show that loci affecting important production traits are located in the same chromosomal region as loci affecting different fear-related behaviors.

Behaviors with high energetic costs may decrease in frequency in domestic animals as a response to selection for increased production. The aim of this study was to quantify production traits, foraging behavior, and social motivation in F2 progeny from a White Leghorn x red junglefowl intercross (n = 751-1046) and to perform QTL analyses on the behavioral traits. A foraging-social maze was used for behavioral testing, which consisted of four identical arms and a central box. In two arms there was ad libitum access to the birds' usual food, and in the other two there was novel food (sunflower seeds) mixed with cat litter. In one arm with each of the two food sources, social stimuli were simulated by the presence of a mirror. Each bird could therefore feed on novel or well known food either alone or in the perceived company of a conspecific. Egg production, sexual maturity (females), food intake, and growth were measured individually, and residual food intake and metabolic body weight were estimated using standard methods. A genome scan using 104 microsatellite markers was carried out to identify QTLs affecting behavioral traits. Phenotypic growth rates at different ages showed weak associations in both sexes. Sexual maturity and egg weight were not strongly correlated to growth, indicating that these traits are not genetically linked. Time spent in each arm and in the central part of the maze was analyzed using principal component analyses. Four principal components (PC) were extracted, each reflecting a pattern of behavior in the maze. Females with early onset of sexual maturity scored higher on the PC1 reflecting preference for free food without social stimuli, and females with higher egg production scored higher on the PC2 reflecting exploration. Males with an overall higher growth rate and higher residual food intake scored higher on the PC3, which possibly reflected fear of the test situation, and tended to score higher on the PC4 reflecting low contrafreeloading. Significant QTLs were found for PC1 and PC4 scores on chromosomes 27 and 7, respectively. The location of the QTLs coincided with known QTLs for growth rate and body weight. The results suggest a trade-off between energy-demanding behavior and high production and that some of this may be caused by genetic linkage or pleiotropic gene effects.

Systemic sclerosis (SSc) or scleroderma is a rare, autoimmune, multi-factorial disease characterized by early microvascular alterations, inflammation, and fibrosis. Chickens from the UCD-200 line develop a hereditary SSc-like disease, showing all the hallmarks of the human disorder, which makes this line a promising model to study genetic factors underlying the disease. A backcross was generated between UCD-200 chickens and its wild ancestor – the red jungle fowl and a genome-scan was performed to identify loci affecting early (21 days of age) and late (175 days of age) ischemic lesions of the comb. A significant difference in frequency of disease was observed between sexes in the BC population, where the homogametic males were more affected than females, and there was evidence for a protective W chromosome effect. Three suggestive disease predisposing loci were mapped to chromosomes 2, 12 and 14. Three orthologues of genes implicated in human SSc are located in the QTL region on chromosome 2, TGFRB1 , EXOC2-IRF4 and COL1A2 , as well as CCR8 , which is more generally related to immune function. IGFBP3 is also located within the QTL on chromosome 2 and earlier studies have showed increased IGFBP3 serum levels in SSc patients. To our knowledge, this study is the first to reveal a potential genetic association between IGFBP3 and SSc. Another gene with an immunological function, SOCS1 , is located in the QTL region on chromosome 14. These results illustrate the usefulness of the UCD-200 chicken as a model of human SSc and motivate further in-depth functional studies of the implicated candidate genes.

PMEL is a pigment cell-specific protein that forms physiological amyloid fibrils upon which melanins ultimately deposit in the lumen of the pigment organelle, the melanosome. Whereas hypomorphic PMEL mutations in several species result in a mild pigment dilution that is inherited in a recessive manner, PMEL alleles found in the Dominant white (DW) chicken and Silver horse (HoSi)—which bear mutations that alter the PMEL transmembrane domain (TMD) and that are thus outside the amyloid core—are associated with a striking loss of pigmentation that is inherited in a dominant fashion. Here we show that the DW and HoSi mutations alter PMEL TMD oligomerization and/or association with membranes, with consequent formation of aberrantly packed fibrils. The aberrant fibrils are associated with a loss of pigmentation in cultured melanocytes, suggesting that they inhibit melanin production and/or melanosome integrity. A secondary mutation in the Smoky chicken, which reverts the dominant DW phenotype, prevents the accumulation of PMEL in fibrillogenic compartments and thus averts DW–associated pigment loss; a secondary mutation found in the Dun chicken likely dampens a HoSi–like dominant mutation in a similar manner. We propose that the DW and HoSi mutations alter the normally benign amyloid to a pathogenic form that antagonizes melanosome function, and that the secondary mutations found in the Smoky and Dun chickens revert or dampen pathogenicity by functioning as null alleles, thus preventing the formation of aberrant fibrils. We speculate that PMEL mutations can model the conversion between physiological and pathological amyloid., Author Summary Amyloid is a protein fold that is normally associated with pathology, such as neurodegeneration in Alzheimer, Parkinson, and Creutzfeldt–Jakob diseases. The amyloid fold has also been exploited by nature for functional purposes; for example, proteolytic fragments of the pigment cell-specific integral membrane protein, PMEL, form amyloid fibrils upon which melanin pigments polymerize within subcellular organelles called melanosomes. Whereas animal models that entirely lack PMEL expression have modest pigment loss, chickens or horses with small in-frame mutations that alter a non-amyloidogenic region of PMEL have severe pigment loss that is thought to be associated with pigment cell death. We show here that these mutations alter the capacity of this region to self-associate, likely changing the conformation of full-length PMEL oligomers. While these changes do not affect the intrinsic ability of PMEL to form amyloid fibrils, they alter either the fibrils themselves or the kinetics of fibril formation such that they form more compact structures and inhibit melanin formation when expressed in melanocytes in culture. Based on our results, we speculate that minor alterations in pre-amyloid assembly of an amyloidogenic protein influences entry into either a benign or a pathogenic amyloid pathway.

Chickens homozygous for the Dominant white or wild-type allele of PMEL17 were subjected to a broad phenotyping in order to detect consistent differences between genotypes. To exclude feather pecking, the chickens were individually housed without physical contact, from the day of hatching, and tested for social, aggressive, fear and exploratory behaviors, and corticosterone and testosterone levels were assessed. In a principal component analysis, 53.2% of the behavior variation was explained by two factors. Factor one was an activity and social factor, and there was a significant effect of genotype on the factor scores. On factor two, related to aggressive behavior, there were significant effects of genotype, sex and their interaction. There were no genotype effects on hormone levels or any other measured non-behavioral phenotypes. Hence, differences in behavior between PMEL17 genotypes remained when negative social experiences were excluded, indicating a direct pleiotropic effect of the gene on behavior. The original publication is available at www.springerlink.com:Anna-Carin Karlsson, Pierre Mormede, Susanne Kerje and Per Jensen, Genotype on the Pigmentation Regulating PMEL17 Gene Affects Behavior in Chickens Raised Without Physical Contact with Conspecifics, 2011, BEHAVIOR GENETICS, (41), 2, 312-322.http://dx.doi.org/10.1007/s10519-010-9379-4Copyright: Springer Science Business Mediahttp://www.springerlink.com/

With chicken used as a model species, we used QTL analysis to examine the genetic contribution to bone traits. We report the identification of four QTLs for femoral traits: one for bone strength, one for endosteal circumference, and two affecting mineral density of noncortical bone. Introduction: BMD is a highly heritable phenotype, governed by elements at numerous loci. In studies examining the genetic contribution to bone traits, many loci have been identified in humans and in other species. The goal of this study was to identify quantitative trait loci (QTLs) controlling BMD and bone strength in an intercross between wildtype and domestic chickens. Materials and Methods: A set of 164 markers, covering 30 chromosomes (chr.), were used to genotype 337 F2-individuals from an intercross of domesticated white Leghorn and wildtype red junglefowl chicken. DXA and pQCT were used to measure BMD and bone structure. Three-point bending tests and torsional strength tests were performed to determine the biomechanical strength of the bone. QTLs were mapped using forward selection for loci with significant marginal effects. Results: Four QTLs for femoral bone traits were identified in QTL analysis with body weight included as a covariate. A QTL on chr. 1 affected female noncortical BMD (LOD 4.6) and is syntenic to human 12q21–12q23. Also located on chr. 1, a locus with synteny to human 12q13–14 affected endosteal circumference (LOD 4.6). On chr. 2, a QTL corresponding to human 5p13-p15, 7p12, 18q12, 18q21, and 9q22–9q31 affected BMD in females; noncortical (LOD 4.0) and metaphyseal (LOD 7.0) BMD by pQCT and BMD by DXA (LOD 5.9). A QTL located on chr. 20 (LOD 5.2) affected bone biomechanical strength and had sex-dependent effects. In addition to the significant QTLs, 10 further loci with suggestive linkage to bone traits were identified. Conclusions: Four QTLs were identified: two for noncortical BMD, one for endosteal circumference, and one affecting bone biomechanical strength. The future identification of genes responsible for these QTLs will increase the understanding of vertebrate skeletal biology.

Dominant white, Dun, and Smoky are alleles at the Dominant white locus, which is one of the major loci affecting plumage color in the domestic chicken. Both Dominant white and Dun inhibit the expression of black eumelanin. Smoky arose in a White Leghorn homozygous for Dominant white and partially restores pigmentation. PMEL17 encodes a melanocyte-specific protein and was identified as a positional candidate gene due to its role in the development of eumelanosomes. Linkage analysis of PMEL17 and Dominant white using a red jungle fowl/White Leghorn intercross revealed no recombination between these loci. Sequence analysis showed that the Dominant white allele was exclusively associated with a 9-bp insertion in exon 10, leading to an insertion of three amino acids in the PMEL17 transmembrane region. Similarly, a deletion of five amino acids in the transmembrane region occurs in the protein encoded by Dun. The Smoky allele shared the 9-bp insertion in exon 10 with Dominant white, as expected from its origin, but also had a deletion of 12 nucleotides in exon 6, eliminating four amino acids from the mature protein. These mutations are, together with the recessive silver mutation in the mouse, the only PMEL17 mutations with phenotypic effects that have been described so far in any species.

Feather-pecking in domestic birds is associated with cannibalism and severe welfare problems. It is a dramatic example of a spiteful behaviour in which the victim's fitness is reduced for no immediate direct benefit to the perpetrator and its evolution is unexplained. Here we show that the plumage pigmentation of a chicken may predispose it to become a victim: birds suffer more drastic feather-pecking when the colour of their plumage is due to the expression of a wild recessive allele at PMEL17, a gene that controls plumage melanization, and when these birds are relatively common in a flock. These findings, obtained using an intercross between a domestic fowl and its wild ancestor, have implications for the welfare of domestic species and offer insight into the genetic changes associated with the evolution of feather-pecking during the early stages of domestication.

Feather-pecking in domestic birds is associated with cannibalism and severe welfare problems. It is a dramatic example of a spiteful behaviour in which the victim's fitness is reduced for no immediate direct benefit to the perpetrator and its evolution is unexplained. Here we show that the plumage pigmentation of a chicken may predispose it to become a victim: birds suffer more drastic feather-pecking when the colour of their plumage is due to the expression of a wild recessive allele at PMEL17, a gene that controls plumage melanization, and when these birds are relatively common in a flock. These findings, obtained using an intercross between a domestic fowl and its wild ancestor, have implications for the welfare of domestic species and offer insight into the genetic changes associated with the evolution of feather-pecking during the early stages of domestication.

The aim of this study was to investigate methods for detecting QTL in outbred commercial pig populations. Several QTL for back fat and growth rate, previously detected in experimental resource populations, were examined for segregation in 10 different populations. Two hundred trait-by-population-by-chromosome tests were performed, resulting in 20 tests being significant at the 5% level. In addition, 53 QTL tests for 11 meat quality traits were declared significant, using a subset of the populations. These results show that a considerable amount of phenotypic variance observed in these populations can be explained by major alleles segregating at several of the loci described. Thus, despite a relatively strong selection pressure for growth and back fat traits in these populations, these alleles have not yet reached fixation. The approaches used here demonstrate that it is possible to verify segregation of QTL in commercial populations by limited genotyping of a selection of informative animals. Such verified QTL may be directly exploited in marker-assisted selection (MAS) programs in commercial populations and their molecular basis may be revealed by positional candidate cloning.

We have identified quantitative trait loci (QTL) explaining a large proportion of the variation in body weights at different ages and growth between chronological ages in an F2 intercross between red junglefowl and White Leghorn chickens. QTL were mapped using forward selection for loci with significant marginal genetic effects and with a simultaneous search for epistatic QTL pairs. We found 22 significant loci contributing to these traits, nine of these were only found by the simultaneous two-dimensional search, which demonstrates the power of this approach for detecting loci affecting complex traits. We have also estimated the relative contribution of additive, dominance, and epistasis effects to growth and the contribution of epistasis was more pronounced prior to 46 days of age, whereas additive genetic effects explained the major portion of the genetic variance later in life. Several of the detected loci affected either early or late growth but not both. Very few loci affected the entire growth process, which points out that early and late growth, at least to some extent, have different genetic regulation.[Supplemental material is available online at www.genome.org.]

Behaviors with high energetic costs may decrease in frequency in domestic animals as a response to selection for increased production. The aim of this study was to quantify production traits, foraging behavior, and social motivation in F2 progeny from a White Leghorn x red junglefowl intercross (n = 751-1046) and to perform QTL analyses on the behavioral traits. A foraging-social maze was used for behavioral testing, which consisted of four identical arms and a central box. In two arms there was ad libitum access to the birds' usual food, and in the other two there was novel food (sunflower seeds) mixed with cat litter. In one arm with each of the two food sources, social stimuli were simulated by the presence of a mirror. Each bird could therefore feed on novel or well known food either alone or in the perceived company of a conspecific. Egg production, sexual maturity (females), food intake, and growth were measured individually, and residual food intake and metabolic body weight were estimated using standard methods. A genome scan using 104 microsatellite markers was carried out to identify QTLs affecting behavioral traits. Phenotypic growth rates at different ages showed weak associations in both sexes. Sexual maturity and egg weight were not strongly correlated to growth, indicating that these traits are not genetically linked. Time spent in each arm and in the central part of the maze was analyzed using principal component analyses. Four principal components (PC) were extracted, each reflecting a pattern of behavior in the maze. Females with early onset of sexual maturity scored higher on the PC1 reflecting preference for free food without social stimuli, and females with higher egg production scored higher on the PC2 reflecting exploration. Males with an overall higher growth rate and higher residual food intake scored higher on the PC3, which possibly reflected fear of the test situation, and tended to score higher on the PC4 reflecting low contrafreeloading. Significant QTLs were found for PC1 and PC4 scores on chromosomes 27 and 7, respectively. The location of the QTLs coincided with known QTLs for growth rate and body weight. The results suggest a trade-off between energy-demanding behavior and high production and that some of this may be caused by genetic linkage or pleiotropic gene effects.

Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden Department of Medical Sciences, Uppsala University, Uppsala, Sweden Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden Division of Experimental Pathophysiology and Immunology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Central Laboratory Facilities, Innsbruck Medical University, Innsbruck, Austria Department of Rheumatology and Inflammation Research, The Sahlgrenska Academy, Gothenburg, Sweden Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden Department of Clinical Sciences, Division of Computational Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden

Domestication is one of the strongest forms of short-term, directional selection. Although selection is typically only exerted on one or a few target traits, domestication can lead to numerous changes in many seemingly unrelated phenotypes. It is unknown whether such correlated responses are due to pleiotropy or linkage between separate genetic architectures. Using three separate intercrosses between wild and domestic chickens, a locus affecting comb mass (a sexual ornament in the chicken) and several fitness traits (primarily medullary bone allocation and fecundity) was identified. This locus contains two tightly-linked genes, BMP2 and HAO1, which together produce the range of pleiotropic effects seen. This study demonstrates the importance of pleiotropy (or extremely close linkage) in domestication. The nature of this pleiotropy also provides insights into how this sexual ornament could be maintained in wild populations., Author Summary The genetic analysis of phenotypes and the identification of the causative underlying genes remain central to molecular and evolutionary biology. By utilizing the domestication process, it is possible to exploit the large differences between domesticated animals and their wild counterparts to study both this and the mechanism of domestication itself. Domestication has been central to the advent of modern civilization; and yet, despite domesticated animals displaying similar adaptations in morphology, physiology, and behaviour, the genetic basis of these changes are unknown. In addition, though sexual selection theory has been the subject of a vast amount of study, very little is known about which genes are underpinning such traits. We have generated multiple intercrosses and advanced intercrosses based on wild-derived and domestic chickens to fine-map genomic regions affecting a sexual ornament. These regions have been over-laid with putative selective sweeps identified in domestic chickens and found to be significantly associated with them. By using expression QTL analysis, we show that two genes in one region, HAO1 and BMP2, are controlling multiple aspects of the domestication phenotype, from a sexual ornament to multiple life history traits. This demonstrates the importance of pleiotropy (or extremely close linkage) in controlling these genetic changes.

PMEL is an amyloidogenic protein that appears to be exclusively expressed in pigment cells and forms intralumenal fibrils within early stage melanosomes upon which eumelanins deposit in later stages. PMEL is well conserved among vertebrates, and allelic variants in several species are associated with reduced levels of eumelanin in epidermal tissues. However, in most of these cases it is not clear whether the allelic variants reflect gain-of-function or loss-of-function, and no complete PMEL loss-of-function has been reported in a mammal. Here, we have created a mouse line in which the Pmel gene has been inactivated (Pmel −/−). These mice are fully viable, fertile, and display no obvious developmental defects. Melanosomes within Pmel −/− melanocytes are spherical in contrast to the oblong shape present in wild-type animals. This feature was documented in primary cultures of skin-derived melanocytes as well as in retinal pigment epithelium cells and in uveal melanocytes. Inactivation of Pmel has only a mild effect on the coat color phenotype in four different genetic backgrounds, with the clearest effect in mice also carrying the brown/Tyrp1 mutation. This phenotype, which is similar to that observed with the spontaneous silver mutation in mice, strongly suggests that other previously described alleles in vertebrates with more striking effects on pigmentation are dominant-negative mutations. Despite a mild effect on visible pigmentation, inactivation of Pmel led to a substantial reduction in eumelanin content in hair, which demonstrates that PMEL has a critical role for maintaining efficient epidermal pigmentation., Author Summary Pigmentation has since long constituted a prime model to study how genes act and interact. The PMEL gene encodes a protein exclusively found in the melanosomes of pigment-producing cells. Mutations in PMEL underlie some spectacular color phenotypes in animals including Dominant white color in chickens, Silver in horses, and Merle in dogs, but no spontaneous mutation causing a complete inactivation of this gene has yet been found in mammals. We have now developed a PMEL knockout mouse to further study the function of this protein. We show that mice lacking PMEL have almost normal visible pigmentation. However, loss of PMEL has a dramatic effect on the morphology of the melanosomes in skin, hair, and eye, such that the normally rod-shaped melanosomes in wild-type animals are spherical in the knockout mice. The knockout animals also have a substantial reduction in the content of black pigment in hair. The study establishes that PMEL has a critical role for maintaining normal pigment production.

Domestic animals have been selected by humans for thousands of years, which have drastically altered their genetic constitution and phenotypes. In this thesis, several of the most important genes causing pigmentation differences between the wild red junglefowl (Gallus gallus) and domestic chickens have been identified. Pigmentation phenotypes are easily scored, and the genes underlying these phenotypes are valuable models to study gene function and gene interaction. Dominant white colour is widespread among domestic chickens. The Dominant white allele specifically inhibits the expression of black (eumelanin) pigment and we identified several insertion/deletion mutations in the PMEL17 gene causing the different phenotypes controlled by this locus. The Silver allele on the other hand inhibits the expression of red (pheomelanin) colour and is a genetic variant of the SLC45A2 gene. Silver is the first pheomelanin-specific mutation(s) reported for this gene. An 8 kb deletion, including a conserved enhancer element, 14 kb upstream of the transcription factor SOX10 is causing the Dark brown phenotype. This phenotype restricts the expression of eumelanin and enhances red pheomelanin in specific parts of the plumage. These three gene identifications have extended the knowledge about genes affecting melanocyte function. Carotenoid-based pigmentation is of utmost importance in birds and other animals. The yellow skin allele in chicken allows deposition of carotenoids in skin and explains why most domestic chickens have yellow legs. We demonstrated that the yellow skin phenotype is caused by a tissue specific regulatory mutation in the gene for the enzyme beta-caroten dioxygenase 2 (BCDO2). This was the first identification of a specific gene underlying carotenoid-based pigmentation. Interestingly, the yellow skin haplotype was shown to originate from the grey junglefowl (Gallus sonneratii) and not the red junglefowl as expected, thus presenting the first conclusive evidence for a hybrid origin of the domestic chicken.

Background Long-term selection (> 45 generations) for low or high juvenile body weight from a common founder population of White Plymouth Rock chickens has generated two extremely divergent lines, the LWS and HWS lines. In addition to a > 9-fold difference between lines for the selected trait, large behavioural and metabolic differences between the two lines evolved during the course of the selection. We recently compared gene expression in brain tissue from birds representing these lines using a global cDNA array analysis and the results showed multiple but small expression differences in protein coding genes. The main differentially expressed transcripts were endogenous retroviral sequences identified as avian leucosis virus subgroup-E (ALVE). Results In this work we confirm the differential ALVE expression and analysed expression and number of proviral integrations in the two parental lines as well as in F9 individuals from an advanced intercross of the lines. Correlation analysis between expression, proviral integrations and body weight showed that high ALVE levels in the LWS line were inherited and that more ALVE integrations were detected in LWS than HWS birds. Conclusion We conclude that only a few of the integrations contribute to the high expression levels seen in the LWS line and that high ALVE expression was significantly correlated with lower body weights for the females but not males. The conserved correlation between high expression and low body weight in females after 9 generations of intercrosses, indicated that ALVE loci conferring high expression directly affects growth or are very closely linked to loci regulating growth.