The wild species genome ancestry of domestic chickens.

BACKGROUND: Hybridisation and introgression play key roles in the evolutionary history of animal species. They are commonly observed within several orders in wild birds. The domestic chicken Gallus gallus domesticus is the most common livestock species. More than 65 billion chickens are raised annually to produce meat and 80 million metric tons of egg for global human consumption by the commercial sector. Unravelling the origin of its genetic diversity has major application for sustainable breeding improvement programmes. RESULTS: In this study, we report genome-wide analyses for signatures of introgression between indigenous domestic village chicken and the four wild Gallus species. We first assess the genome-wide phylogeny and divergence time across the genus Gallus. Genome-wide sequence divergence analysis supports a sister relationship between the Grey junglefowl G. sonneratii and Ceylon junglefowl G. lafayettii. Both species form a clade that is sister to the Red junglefowl G. gallus, with the Green junglefowl G. varius the most ancient lineage within the genus. We reveal extensive bidirectional introgression between the Grey junglefowl and the domestic chicken and to a much lesser extent with the Ceylon junglefowl. We identify a single case of Green junglefowl introgression. These introgressed regions include genes with biological functions related to development and immune system. CONCLUSIONS: Our study shows that while the Red junglefowl is the main ancestral species, introgressive hybridisation episodes have impacted the genome and contributed to the diversity of the domestic chicken, although likely at different levels across its geographic range.

Transcriptional profile of breast muscle in heat stressed layers is similar to that of broiler chickens at control temperature.

BACKGROUND: In recent years, the commercial importance of changes in muscle function of broiler chickens and of the corresponding effects on meat quality has increased. Furthermore, broilers are more sensitive to heat stress during transport and at high ambient temperatures than smaller egg-laying chickens. We hypothesised that heat stress would amplify muscle damage and expression of genes that are involved in such changes and, thus, lead to the identification of pathways and networks associated with broiler muscle and meat quality traits. Broiler and layer chickens were exposed to control or high ambient temperatures to characterise differences in gene expression between the two genotypes and the two environments. RESULTS: Whole-genome expression studies in breast muscles of broiler and layer chickens were conducted before and after heat stress; 2213 differentially-expressed genes were detected based on a significant (P < 0.05) genotype × treatment interaction. This gene set was analysed with the BioLayout Express3D and Ingenuity Pathway Analysis software and relevant biological pathways and networks were identified. Genes involved in functions related to inflammatory reactions, cell death, oxidative stress and tissue damage were upregulated in control broilers compared with control and heat-stressed layers. Expression of these genes was further increased in heat-stressed broilers. CONCLUSIONS: Differences in gene expression between broiler and layer chickens under control and heat stress conditions suggest that damage of breast muscles in broilers at normal ambient temperatures is similar to that in heat-stressed layers and is amplified when broilers are exposed to heat stress. The patterns of gene expression of the two genotypes under heat stress were almost the polar opposite of each other, which is consistent with the conclusion that broiler chickens were not able to cope with heat stress by dissipating their body heat. The differentially expressed gene networks and pathways were consistent with the pathological changes that are observed in the breast muscle of heat-stressed broilers.

Higher heritabilities for gait components than for overall gait scores may improve mobility in ducks.

BACKGROUND: Genetic progress in selection for greater body mass and meat yield in poultry has been associated with an increase in gait problems which are detrimental to productivity and welfare. The incidence of suboptimal gait in breeding flocks is controlled through the use of a visual gait score, which is a subjective assessment of walking ability of each bird. The subjective nature of the visual gait score has led to concerns over its effectiveness in reducing the incidence of suboptimal gait in poultry through breeding. The aims of this study were to assess the reliability of the current visual gait scoring system in ducks and to develop a more objective method to select for better gait. RESULTS: Experienced gait scorers assessed short video clips of walking ducks to estimate the reliability of the current visual gait scoring system. Kendall's coefficients of concordance between and within observers were estimated at 0.49 and 0.75, respectively. In order to develop a more objective scoring system, gait components were visually scored on more than 4000 pedigreed Pekin ducks and genetic parameters were estimated for these components. Gait components, which are a more objective measure, had heritabilities that were as good as, or better than, those of the overall visual gait score. CONCLUSIONS: Measurement of gait components is simpler and therefore more objective than the standard visual gait score. The recording of gait components can potentially be automated, which may increase accuracy further and may improve heritability estimates. Genetic correlations were generally low, which suggests that it is possible to use gait components to select for an overall improvement in both economic traits and gait as part of a balanced breeding programme.

A new look at the LTR retrotransposon content of the chicken genome.

BACKGROUND: LTR retrotransposons contribute approximately 10 % of the mammalian genome, but it has been previously reported that there is a deficit of these elements in the chicken relative to both mammals and other birds. A novel LTR retrotransposon classification pipeline, LocaTR, was developed and subsequently utilised to re-examine the chicken LTR retrotransposon annotation, and determine if the proposed chicken deficit is biologically accurate or simply a technical artefact. RESULTS: Using LocaTR 3.01 % of the chicken galGal4 genome assembly was annotated as LTR retrotransposon-derived elements (nearly double the previous annotation), including 1,073 that were structurally intact. Element distribution is significantly correlated with chromosome size and is non-random within each chromosome. Elements are significantly depleted within coding regions and enriched in gene sparse areas of the genome. Over 40 % of intact elements are found in clusters, unrelated by age or genera, generally in poorly recombining regions. The transcription of most LTR retrotransposons were suppressed or incomplete, but individual domain and full length retroviral transcripts were produced in some cases, although mostly with regularly interspersed stop codons in all reading frames. Furthermore, RNAseq data from 23 diverse tissues enabled greater characterisation of the co-opted endogenous retrovirus Ovex1. This gene was shown to be expressed ubiquitously but at variable levels across different tissues. LTR retrotransposon content was found to be very variable across the avian lineage and did not correlate with either genome size or phylogenetic position. However, the extent of previous, species-specific LTR retrotransposon annotation appears to be a confounding factor. CONCLUSIONS: Use of the novel LocaTR pipeline has nearly doubled the annotated LTR retrotransposon content of the chicken genome compared to previous estimates. Further analysis has described element distribution, clustering patterns and degree of expression in a variety of adult tissues, as well as in three embryonic stages. This study also enabled better characterisation of the co-opted gamma retroviral envelope gene Ovex1. Additionally, this work suggests that there is no deficit of LTR retrotransposons within the Galliformes relative to other birds, or to mammalian genomes when scaled for the three-fold difference in genome size.

Differences in hindlimb morphology of ducks and chickens: effects of domestication and selection.

BACKGROUND: Poultry account for the most numerous species farmed for meat and have been subject to intense selection over approximately 60 generations. To assess morphological changes which have occurred in the avian leg due to selection for rapid growth and high meat yields, divergent lines of chicken (Gallus gallus) and duck (Anas platyrhynchos) were studied between 3 and 7 weeks of age. For each line, femoral and tibiotarsal morphology was recorded using computed tomography scanning and tibiotarsal bone quality measures (stiffness, bending stress and porosity) were assessed. RESULTS: In chicken and duck, divergence in hindlimb morphology has occurred in the commercial meat lines compared to their lighter conspecifics. As expected, the differences were largest between species. Leg development nears completion much earlier in ducks than in chickens. Duck tibiotarsi showed a large degree of lateral curvature, which is expected to affect foot position during swimming and walking, and thus to influence gait. All lines have adapted their tibiotarsal morphology to suit the loading forces they experience; however bone quality was found to be poorer in chickens. CONCLUSIONS: We demonstrate that intensive selection for growth rate in both chickens and ducks has resulted in leg morphology changes, which are likely to influence gait. Ducks represent an interesting compromise of adaptation for efficient locomotion in two media-on land and in water. Some aspects of bone morphology in the duck, such as lateral curvature of the tibiotarsus, may result from adaptation to swimming, which potentially imposes limitations on terrestrial locomotion.

Spectral domain optical coherence tomography imaging of the posterior segment of the eye in the retinal dysplasia and degeneration chicken, an animal model of inherited retinal degeneration.

OBJECTIVE: To investigate qualitative and quantitative differences in the structure of the posterior segment of the eye in 1-day post-hatch and 12-month-old retinal dysplasia and degeneration (rdd) and wild-type chickens. ANIMAL STUDIED: Retinal dysplasia and degeneration and wild-type chickens. PROCEDURE: Using a commercially available spectral domain optical coherence tomography (OCT) system, 15° horizontal line scans were performed in both eyes of 24 live birds. Qualitative differences in retinal lamination and choroidal structure were investigated, and retinal and choroidal thickness were measured. RESULTS: Progressive retinal thinning with loss of outer retinal lamination and changes in the appearance of the choroid were seen in the rdd birds. Mean total retinal thickness was 202 µm (SD 7.8) and 251 µm (SD 8.8) in the rdd and wild-type chicks and 154 µm (SD 18) and 280 µm (SD 10.8) in the adult birds. Much of the difference was the result of loss of outer retinal lamination and thickness in the rdd birds. Mean choroidal thickness was 76 µm (SD 19.6) and 112 µm (SD 36.9) in the rdd and wild-type chicks and 85 µm (SD 23.7) and 228 µm (SD 44.1) in the rdd and wild-type adult birds, respectively. CONCLUSIONS: Differences in retinal and choroidal structure and thickness between rdd and wild-type birds were evident on spectral domain OCT imaging at 1-day post-hatch and more marked at 1 year. Spectral domain OCT may provide a reliable end point for therapeutic intervention in this animal model of inherited retinal degeneration.

An x-ray scattering study into the structural basis of corneal refractive function in an avian model.

Avian vision diseases in which eye growth is compromised are helping to define what governs corneal shape and ultrastructural organization. The highly specific collagen architecture of the main corneal layer, the stroma, is believed to be important for the maintenance of corneal curvature and hence visual quality. Blindness enlarged globe (beg) is a recessively inherited condition of chickens characterized by retinal dystrophy and blindness at hatch, with secondary globe enlargement and loss of corneal curvature by 3-4 months. Here we define corneal ultrastructural changes as the beg eye develops posthatch, using wide-angle x-ray scattering to map collagen fibril orientation across affected corneas at three posthatch time points. The results disclosed alterations in the bulk alignment of corneal collagen in beg chicks compared with age-matched controls. These changes accompanied the eye globe enlargement and corneal flattening observed in affected birds, and were manifested as a progressive loss of circumferential collagen alignment in the peripheral cornea and limbus in birds older than 1 month. Progressive remodeling of peripheral stromal collagen in beg birds posthatch may relate to the morphometric changes exhibited by the disease, likely as an extension of myopia-like scleral remodeling triggered by deprivation of a retinal image.

Development of a high density 600K SNP genotyping array for chicken.

BACKGROUND: High density (HD) SNP genotyping arrays are an important tool for genetic analyses of animals and plants. Although the chicken is one of the most important farm animals, no HD array is yet available for high resolution genetic analysis of this species. RESULTS: We report here the development of a 600 K Affymetrix® Axiom® HD genotyping array designed using SNPs segregating in a wide variety of chicken populations. In order to generate a large catalogue of segregating SNPs, we re-sequenced 243 chickens from 24 chicken lines derived from diverse sources (experimental, commercial broiler and layer lines) by pooling 10-15 samples within each line. About 139 million (M) putative SNPs were detected by mapping sequence reads to the new reference genome (Gallus_gallus_4.0) of which ~78 M appeared to be segregating in different lines. Using criteria such as high SNP-quality score, acceptable design scores predicting high conversion performance in the final array and uniformity of distribution across the genome, we selected ~1.8 M SNPs for validation through genotyping on an independent set of samples (n = 282). About 64% of the SNPs were polymorphic with high call rates (>98%), good cluster separation and stable Mendelian inheritance. Polymorphic SNPs were further analysed for their population characteristics and genomic effects. SNPs with extreme breach of Hardy-Weinberg equilibrium (P < 0.00001) were excluded from the panel. The final array, designed on the basis of these analyses, consists of 580,954 SNPs and includes 21,534 coding variants. SNPs were selected to achieve an essentially uniform distribution based on genetic map distance for both broiler and layer lines. Due to a lower extent of LD in broilers compared to layers, as reported in previous studies, the ratio of broiler and layer SNPs in the array was kept as 3:2. The final panel was shown to genotype a wide range of samples including broilers and layers with over 100 K to 450 K informative SNPs per line. A principal component analysis was used to demonstrate the ability of the array to detect the expected population structure which is an important pre-investigation step for many genome-wide analyses. CONCLUSIONS: This Affymetrix® Axiom® array is the first SNP genotyping array for chicken that has been made commercially available to the public as a product. This array is expected to find widespread usage both in research and commercial application such as in genomic selection, genome-wide association studies, selection signature analyses, fine mapping of QTLs and detection of copy number variants.

Decreased expression of the satiety signal receptor CCKAR is responsible for increased growth and body weight during the domestication of chickens.

Animal domestication has resulted in changes in growth and size. It has been suggested that this may have involved selection for differences in appetite. Divergent growth between chickens selected for egg laying or meat production is one such example. The neurons expressing AGRP and POMC in the basal hypothalamus are important components of appetite regulation, as are the satiety feedback pathways that carry information from the intestine, including CCK and its receptor CCKAR (CCK1 receptor). Using 16 generations of a cross between a fast and a relatively slow growing strain of chicken has identified a region on chromosome 4 downstream of the CCKAR gene, which is responsible for up to a 19% difference in body weight at 12 wk of age. Animals possessing the high-growth haplotype at the locus have lower expression of mRNA and immunoreactive CCKAR in the brain, intestine, and exocrine organs, which is correlated with increased levels of orexigenic AGRP in the hypothalamus. Animals with the high-growth haplotype are resistant to the anorectic effect of exogenously administered CCK, suggesting that their satiety set point has been altered. Comparison with traditional breeds shows that the high-growth haplotype has been present in the founders of modern meat-type strains and may have been selected early in domestication. This is the first dissection of the physiological consequences of a genetic locus for a quantitative trait that alters appetite and gives us an insight into the domestication of animals. This will allow elucidation of how differences in appetite occur in birds and also mammals.

Comparative analysis of quantitative trait loci for body weight, growth rate and growth curve parameters from 3 to 72 weeks of age in female chickens of a broiler-layer cross.

BACKGROUND: Comparisons of quantitative trait loci (QTL) for growth and parameters of growth curves assist in understanding the genetics and ultimately the physiology of growth. Records of body weight at 3, 6, 12, 24, 48 and 72 weeks of age and growth rate between successive age intervals of about 500 F2 female chickens of the Roslin broiler-layer cross were available for analysis. These data were analysed to detect and compare QTL for body weight, growth rate and parameters of the Gompertz growth function. RESULTS: Over 50 QTL were identified for body weight at specific ages and most were also detected in the nearest preceding and/or subsequent growth stage. The sum of the significant and suggestive additive effects for bodyweight at specific ages accounted for 23-43% of the phenotypic variation. A single QTL for body weight on chromosome 4 at 48 weeks of age had the largest additive effect (550.4 ± 68.0 g, 11.5% of the phenotypic variation) and a QTL at a similar position accounted 14.5% of the phenotypic variation at 12 weeks of age. Age specific QTL for growth rate were detected suggesting that there are specific genes that affect developmental processes during the different stages of growth. Relatively few QTL influencing Gompertz growth curve parameters were detected and overlapped with loci affecting growth rate. Dominance effects were generally not significant but from 12 weeks of age they exceeded the additive effect in a few cases. No evidence for epistatic QTL pairs was found. CONCLUSIONS: The results confirm the location for body weight and body weight gain during growth that were identified in previous studies and were consistent with QTL for the parameters of the Gompertz growth function. Chromosome 4 explained a relatively large proportion of the observed growth variation across the different ages, and also harboured most of the detected QTL for Gompertz parameters, confirming its importance in controlling growth. Very few QTL were detected for body weight or gain at 48 and 72 weeks of age, probably reflecting the effect of differences in reproduction and random environmental effects.

Identification of novel candidate genes for follicle selection in the broiler breeder ovary.

BACKGROUND: Broiler breeders fed ad libitum are characterised by multiple ovulation, which leads to poor shell quality and egg production. Multiple ovulation is controlled by food restriction in commercial flocks. However, the level of food restriction raises welfare concerns, including that of severe hunger. Reducing the rate of multiple ovulation by genetic selection would facilitate progress towards developing a growth profile for optimum animal welfare. RESULTS: The study utilised 3 models of ovarian follicle development; laying hens fed ad libitum (experiment 2) and broiler breeders fed ad libitum or a restricted diet (experiments 1 & 3). This allowed us to investigate gene candidates for follicular development by comparing normal, abnormal and "controlled" follicle hierarchies at different stages of development. Several candidate genes for multiple ovulation were identified by combining microarray analysis of restricted vs. ad libitum feeding, literature searches and QPCR expression profiling throughout follicle development. Three candidate genes were confirmed by QPCR as showing significant differential expression between restricted and ad libitum feeding: FSHR, GDF9 and PDGFRL. PDGFRL, a candidate for steroidogenesis, showed significantly up-regulated expression in 6-8 mm follicles of ad libitum fed broiler breeders (P = 0.016), the period at which follicle recruitment occurs. CONCLUSIONS: Gene candidates have been identified and evidence provided to support a possible role in regulation of ovarian function and follicle number. Further characterisation of these genes will be required to assess their potential for inclusion into breeding programmes to improve the regulation of follicle selection and reduce the need for feed restriction.

Systems analysis of immune responses in Marek's disease virus-infected chickens identifies a gene involved in susceptibility and highlights a possible novel pathogenicity mechanism.

Marek's disease virus (MDV) is a highly contagious oncogenic alphaherpesvirus that causes disease that is both a cancer model and a continuing threat to the world's poultry industry. This comprehensive gene expression study analyzes the host response to infection in both resistant and susceptible lines of chickens and inherent expression differences between the two lines following the infection of the host. A novel pathogenicity mechanism, involving the downregulation of genes containing HIC1 transcription factor binding sites as early as 4 days postinfection, was suggested from this analysis. HIC1 drives antitumor mechanisms, suggesting that MDV infection switches off genes involved in antitumor regulation several days before the expression of the MDV oncogene meq. The comparison of the gene expression data to previous QTL data identified several genes as candidates for involvement in resistance to MD. One of these genes, IRG1, was confirmed by single nucleotide polymorphism analysis to be involved in susceptibility. Its precise mechanism remains to be elucidated, although the analysis of gene expression data suggests it has a role in apoptosis. Understanding which genes are involved in susceptibility/resistance to MD and defining the pathological mechanisms of the disease gives us a much greater ability to try to reduce the incidence of this virus, which is costly to the poultry industry in terms of both animal welfare and economics.

Pre-treatment choroidal thickness is not predictive of susceptibility to form-deprivation myopia in chickens.

PURPOSE: Chicks developing experimentally-induced myopia show profound thinning of the choroid. We observed a wide range of choroidal thicknesses in a sample of normal chicks prior to their use in a pedigree-based study of form-deprivation myopia. Hence, we tested whether pre-treatment choroidal thickness predicted susceptibility to myopia. METHODS: Retinal, choroidal and scleral thickness were measured using A-scan ultrasonography in normal White Leghorn chicks (n= 891) aged 4 days old, and again (n=498) after 4 days of monocular form-deprivation at age 8 days of age. Refractive error was assessed by retinoscopy. Relationships between pre-treatment choroidal thickness and other variables were investigated using general linear models and variance components analysis. RESULTS: Untreated 4 day-old male chicks had choroids approximately 10% thinner than females (p<0.001), but sex explained <2% of the overall variability in choroidal thickness. Axial eye length in these untreated chicks was not significantly associated with choroidal thickness (p=0.25). Moreover, pre-treatment choroidal thickness was not predictive of susceptibility to form-deprivation myopia (p=0.89). Heritability analysis suggested that at least 50% of the variation in pre-treatment choroidal thickness was determined by additive genetic effects (p<0.001). CONCLUSIONS: Parental choroidal thickness is the major determinant of choroidal thickness in untreated 4-day old chicks. Despite choroidal thickness potentially being indicative of ongoing emmetropisation to innate refractive errors, in this study it was not predictive of subsequent susceptibility to form-deprivation myopia.

Quantitative trait loci and transcriptome signatures associated with avian heritable resistance to Campylobacter.

Campylobacter is the leading cause of bacterial foodborne gastroenteritis worldwide. Handling or consumption of contaminated poultry meat is a key risk factor for human campylobacteriosis. One potential control strategy is to select poultry with increased resistance to Campylobacter. We associated high-density genome-wide genotypes (600K single nucleotide polymorphisms) of 3000 commercial broilers with Campylobacter load in their caeca. Trait heritability was modest but significant (h2 = 0.11 ± 0.03). Results confirmed quantitative trait loci (QTL) on chromosomes 14 and 16 previously identified in inbred chicken lines, and detected two additional QTLs on chromosomes 19 and 26. RNA-Seq analysis of broilers at the extremes of colonisation phenotype identified differentially transcribed genes within the QTL on chromosome 16 and proximal to the major histocompatibility complex (MHC) locus. We identified strong cis-QTLs located within MHC suggesting the presence of cis-acting variation in MHC class I and II and BG genes. Pathway and network analyses implicated cooperative functional pathways and networks in colonisation, including those related to antigen presentation, innate and adaptive immune responses, calcium, and renin-angiotensin signalling. While co-selection for enhanced resistance and other breeding goals is feasible, the frequency of resistance-associated alleles was high in the population studied and non-genetic factors significantly influenced Campylobacter colonisation.

Proteomic profiling of the retinal dysplasia and degeneration chick retina.

PURPOSE: In our previous paper we undertook proteomic analysis of the normal developing chick retina to identify proteins that were differentially expressed during retinal development. In the present paper we use the same proteomic approach to analyze the development and onset of degeneration in the retinal dysplasia and degeneration (rdd) chick. The pathology displayed by the rdd chick resembles that observed in some of the more severe forms of human retinitis pigmentosa. METHODS: Two-dimensional gel electrophoresis (pH 4-7), gel image analysis, and mass spectrometry were used to profile the developing and degenerating retina of the rdd and wild-type (wt) chick retina. RESULTS: Several proteins were identified by mass spectrometry that displayed differential expression between normal and rdd retina between embryonic day 12 (E12) and post-hatch day 1 (P1). Secernin 1 displayed the most significant variation in expression between rdd and wt retina; this may be due to differential phosphorylation in the rdd retina. Secernin 1 has dipeptidase activity and has been demonstrated to play a role in exocytosis; it has been shown to be overexpressed in certain types of cancer and has also been suggested as a potential neurotoxicologically relevant target. Its role in the retina and in particular its differential expression in the degenerate rdd retina remains unknown and will require further investigation. Other proteins that were differentially expressed in the rdd retina included valosin-containing protein, beta-synuclein, stathmin 1, nucleoside diphosphate kinase, histidine triad nucleotide-binding protein, and 40S ribosomal protein S12. These proteins are reported to be involved in several cellular processes, including the ubiquitin proteasome pathway, neuroprotection, metastatic suppression, transcriptional and translational regulation, and regulation of microtubule dynamics. CONCLUSIONS: This proteomic study is the first such investigation of the rdd retina and represents a unique data set that has revealed several proteins that are differentially expressed during retinal degeneration in the rdd chick. Secernin 1 showed the most significant differences in expression during this degeneration period. Further investigation of the proteins identified may provide insight into the complex events underlying retinal degeneration in this animal model.

Ex vivo magnetic resonance imaging of crystalline lens dimensions in chicken.

PURPOSE: A reduction in the power of the crystalline lens during childhood is thought to be important in the emmetropization of the maturing eye. However, in humans and model organisms, little is known about the factors that determine the dimensions of the crystalline lens and in particular whether these different parameters (axial thickness, surface curvatures, equatorial diameter, and volume) are under a common source of control or regulated independently of other aspects of eye size and shape. METHODS: Using chickens from a broiler-layer experimental cross as a model system, three-dimensional magnetic resonance imaging (MRI) scans were obtained at 115-microm isotropic resolution for one eye of 501 individuals aged 3-weeks old. After fixation with paraformaldehyde, the excised eyes were scanned overnight (16 h) in groups of 16 arranged in a 2x2x4 array. Lens dimensions were calculated from each image by fitting a three-dimensional mesh model to the lens, using the semi-automated analysis program mri3dX. The lens dimensions were compared to measures of eye and body size obtained in vivo using techniques that included keratometry and A-scan ultrasonography. RESULTS: A striking finding was that axial lens thickness measured using ex vivo MRI was only weakly correlated with lens thickness measured in vivo by ultrasonography (r=0.19, p<0.001). In addition, the MRI lens thickness estimates had a lower mean value and much higher variance. Indeed, about one-third of crystalline lenses showed a kidney-shaped appearance instead of the typical biconvex shape. Since repeat MRI scans of the same eye showed a high degree of reproducibility for the scanning and mri3dX analysis steps (the correlation in repeat lens thickness measurements was r=0.95, p<0.001) and a recent report has shown that paraformaldehyde fixation induces a loss of water from the human crystalline lens, it is likely that the tissue fixation step caused a variable degree of shrinkage and a change in shape to the lenses examined here. Despite this serious source of imprecision, we found significant correlations between lens volume and eye/body size (p<0.001) and between lens equatorial diameter and eye/body size (p<0.001) in these chickens. CONCLUSIONS: Our results suggest that certain aspects of lens size (specifically, lens volume and equatorial diameter) are controlled by factors that also regulate the size of the eye and body (presumably, predominantly genetic factors). However, since it has been shown previously that axial lens thickness is regulated almost independently of eye and body size, these results suggest that different systems might operate to control lens volume/diameter and lens thickness in normal chickens.

Identification and characterisation of endogenous Avian Leukosis Virus subgroup E (ALVE) insertions in chicken whole genome sequencing data.

BACKGROUND: Endogenous retroviruses (ERVs) are the remnants of retroviral infections which can elicit prolonged genomic and immunological stress on their host organism. In chickens, endogenous Avian Leukosis Virus subgroup E (ALVE) expression has been associated with reductions in muscle growth rate and egg production, as well as providing the potential for novel recombinant viruses. However, ALVEs can remain in commercial stock due to their incomplete identification and association with desirable traits, such as ALVE21 and slow feathering. The availability of whole genome sequencing (WGS) data facilitates high-throughput identification and characterisation of these retroviral remnants. RESULTS: We have developed obsERVer, a new bioinformatic ERV identification pipeline which can identify ALVEs in WGS data without further sequencing. With this pipeline, 20 ALVEs were identified across eight elite layer lines from Hy-Line International, including four novel integrations and characterisation of a fast feathered phenotypic revertant that still contained ALVE21. These bioinformatically detected sites were subsequently validated using new high-throughput KASP assays, which showed that obsERVer was highly precise and exhibited a 0% false discovery rate. A further fifty-seven diverse chicken WGS datasets were analysed for their ALVE content, identifying a total of 322 integration sites, over 80% of which were novel. Like exogenous ALV, ALVEs show site preference for proximity to protein-coding genes, but also exhibit signs of selection against deleterious integrations within genes. CONCLUSIONS: obsERVer is a highly precise and broadly applicable pipeline for identifying retroviral integrations in WGS data. ALVE identification in commercial layers has aided development of high-throughput diagnostic assays which will aid ALVE management, with the aim to eventually eradicate ALVEs from high performance lines. Analysis of non-commercial chicken datasets with obsERVer has revealed broad ALVE diversity and facilitates the study of the biological effects of these ERVs in wild and domesticated populations.

Ultrastructural changes in the retinopathy, globe enlarged (rge) chick cornea.

In the cornea, the precise organisation of fibrillar collagen and associated proteoglycans comprising the stromal extracellular matrix plays a major role in governing tissue form and function. Recently, abnormal collagen alignment was noted in the misshapen corneas of mature chickens affected by the retinopathy, globe enlarged (rge) mutation. Here we further characterize corneal ultrastructural changes as the rge eye develops post-hatch. Wide-angle X-ray scattering disclosed alteration to dominant collagen lamellae directions in the rge chick cornea, compared to age-matched controls. These changes accompanied eye globe enlargement and corneal flattening in affected birds, manifesting as a progressive loss of circumferential collagen alignment in the peripheral cornea and limbus in birds older than 1 month. Collagen intermolecular separation was unchanged in rge. However, small-angle X-ray scattering results suggest collagen fibril separation and diameter increase more rapidly towards the corneal periphery in rge at 3 months post-hatch compared to controls, although central collagen fibril diameter was unchanged. By transmission electron microscopy utilising cuprolinic blue stain, the morphology and distribution of stromal proteoglycans were unaltered in rge corneas otherwise demonstrating abnormal collagen fibril organisation. From a numerical simulation of tissue mechanics, progressive remodelling of stromal collagen in rge during globe enlargement post-hatch appears to be related to the corneal morphometric changes presented by the disease.

Common determinants of body size and eye size in chickens from an advanced intercross line.

Myopia development is characterised by an increased axial eye length. Therefore, identifying factors that influence eye size may provide new insights into the aetiology of myopia. In humans, axial length is positively correlated to height and weight, and in mice, eye weight is positively correlated with body weight. The purpose of this study was to examine the relationship between eye size and body size in chickens from a genetic cross in which alleles with major effects on eye and body size were segregating. Chickens from a cross between a layer line (small body size and eye size) and a broiler line (large body and eye size) were interbred for 10 generations so that alleles for eye and body size would have the chance to segregate independently. At 3 weeks of age, 510 chicks were assessed using in vivo high resolution A-scan ultrasonography and keratometry. Equatorial eye diameter and eye weight were measured after enucleation. The variations in eye size parameters that could be explained by body weight (BW), body length (BL), head width (HW) and sex were examined using multiple linear regression. It was found that BW, BL and HW and sex together predicted 51-56% of the variation in eye weight, axial length, corneal radius, and equatorial eye diameter. By contrast, the same variables predicted only 22% of the variation in lens thickness. After adjusting for sex, the three body size parameters predicted 45-49% of the variation in eye weight, axial length, corneal radius, and eye diameter, but only 0.4% of the variation in lens thickness. In conclusion, about half of the variation in eye size in the chickens of this broiler-layer advanced intercross line is likely to be determined by pleiotropic genes that also influence body size. Thus, mapping the quantitative trait loci (QTL) that determine body size may be useful in understanding the genetic determination of eye size (a logical inference of this result is that the 20 or more genetic variants that have recently been shown to influence human height may also be found to influence axial eye length). Furthermore, adjusting for body size will be essential in mapping pure eye size QTL in this chicken population, and may also have value in mapping eye size QTL in humans.