Complex genetic architecture of the chicken Growth1 QTL region.

The genetic complexity of polygenic traits represents a captivating and intricate facet of biological inheritance. Unlike Mendelian traits controlled by a single gene, polygenic traits are influenced by multiple genetic loci, each exerting a modest effect on the trait. This cumulative impact of numerous genes, interactions among them, environmental factors, and epigenetic modifications results in a multifaceted architecture of genetic contributions to complex traits. Given the well-characterized genome, diverse traits, and range of genetic resources, chicken (Gallus gallus) was employed as a model organism to dissect the intricate genetic makeup of a previously identified major Quantitative Trait Loci (QTL) for body weight on chromosome 1. A multigenerational advanced intercross line (AIL) of 3215 chickens whose genomes had been sequenced to an average of 0.4x was analyzed using genome-wide association study (GWAS) and variance-heterogeneity GWAS (vGWAS) to identify markers associated with 8-week body weight. Additionally, epistatic interactions were studied using the natural and orthogonal interaction (NOIA) model. Six genetic modules, two from GWAS and four from vGWAS, were strongly associated with the studied trait. We found evidence of both additive- and non-additive interactions between these modules and constructed a putative local epistasis network for the region. Our screens for functional alleles revealed a missense variant in the gene ribonuclease H2 subunit B (RNASEH2B), which has previously been associated with growth-related traits in chickens and Darwin's finches. In addition, one of the most strongly associated SNPs identified is located in a non-coding region upstream of the long non-coding RNA, ENSGALG00000053256, previously suggested as a candidate gene for regulating chicken body weight. By studying large numbers of individuals from a family material using approaches to capture both additive and non-additive effects, this study advances our understanding of genetic complexities in a highly polygenic trait and has practical implications for poultry breeding and agriculture.

Whole-genome selective sweep analyses identifies the region and candidate gene associated with white earlobe color in Mediterranean chickens.

We compared the genomes of multiple domestic chicken breeds with red and white earlobes to identify the differentiated regions between groups of breeds differing in earlobe color. This was done using a selective sweep mapping approach based on whole-genome sequence data. The most significant selective sweep was identified on chromosome 11, where the white earlobe chicken breeds originated from Mediterranean share a common haplotype, and where multiple candidate genes are located. The most plausible functional candidate gene is the Melanocortin 1 Receptor (MC1R), a receptor known to regulate pigmentation in the skin and hair, and it is also the gene with the strongest positional support from the haplotype-based analyses. It, however, still needs to be explored experimentally to identify effects also on chicken earlobe color variation. Our study is the first exploration of the genetic basis of white earlobe color in Mediterranean chickens using a selective sweep mapping method based on whole-genome sequencing data and shows its value for identifying likely functional genes mediating the pigmentation in earlobe. It also indicates a potential novel role of MC1R in birds and exemplifies how selection on fancy traits has influenced the genome during formation of the modern chicken breeds.

Mapping and Functional Dissection of the Rumpless Trait in Piao Chicken Identifies a Causal Loss of Function Mutation in the Novel Gene Rum.

Rumpless chickens exhibit an abnormality in their tail development. The genetics and biology of this trait has been studied for decades to illustrate a broad variation in both the types of inheritance and the severity in the developmental defects of the tail. In this study, we created a backcross pedigree by intercrossing Piao (rumpless) with Xianju (normal) to investigate the genetic mechanisms and molecular basis of the rumpless trait in Piao chicken. Through genome-wide association and linkage analyses, the candidate region was fine-mapped to 798.5 kb (chromosome 2: 86.9 to 87.7 Mb). Whole-genome sequencing analyses identified a single variant, a 4.2 kb deletion, which was completely associated with the rumpless phenotype. Explorations of the expression data identified a novel causative gene, Rum, that produced a long, intronless transcript across the deletion. The expression of Rum is embryo-specific, and it regulates the expression of MSGN1, a key factor in regulating T-box transcription factors required for mesoderm formation and differentiation. These results provide genetic and molecular experimental evidence for a novel mechanism regulating tail development in chicken and report the likely causal mutation for the tail abnormity in the Piao chicken. The novel regulatory gene, Rum, will, due to its role in fundamental embryo development, be of interest for further explorations of a potential role in tail and skeletal development also in other vertebrates.

Researching on the fine structure and admixture of the worldwide chicken population reveal connections between populations and important events in breeding history.

Here, we have evaluated the general genomic structure and diversity and studied the divergence resulting from selection and historical admixture events for a collection of worldwide chicken breeds. In total, 636 genomes (43 populations) were sequenced from chickens of American, Chinese, Indonesian, and European origin. Evaluated populations included wild junglefowl, rural indigenous chickens, breeds that have been widely used to improve modern western poultry populations and current commercial stocks bred for efficient meat and egg production. In-depth characterizations of the genome structure and genomic relationships among these populations were performed, and population admixture events were investigated. In addition, the genomic architectures of several domestication traits and central documented events in the recent breeding history were explored. Our results provide detailed insights into the contributions from population admixture events described in the historical literature to the genomic variation in the domestic chicken. In particular, we find that the genomes of modern chicken stocks used for meat production both in eastern (Asia) and western (Europe/US) agriculture are dominated by contributions from heavy Asian breeds. Further, by exploring the link between genomic selective divergence and pigmentation, connections to functional genes feather coloring were confirmed.

Dissecting the Genetic Regulation of Yeast Growth Plasticity in Response to Environmental Changes.

Variable individual responses to environmental changes, such as phenotype plasticity, are heritable, with some genotypes being robust and others plastic. This variation for plasticity contributes to variance in complex traits as genotype-by-environment interactions (G × E). However, the genetic basis of this variability in responses to the same external stimuli is still largely unknown. In an earlier study of a large haploid segregant yeast population, genotype-by-genotype-by-environment interactions were found to make important contributions to the release of genetic variation in growth responses to alterations of the growth medium. Here, we explore the genetic basis for heritable variation of different measures of phenotype plasticity in the same dataset. We found that the central loci in the environmentally dependent epistatic networks were associated with overall measures of plasticity, while the specific measures of plasticity identified a more diverse set of loci. Based on this, a rapid one-dimensional genome-wide association (GWA) approach to overall plasticity is proposed as a strategy to efficiently identify key epistatic loci contributing to the phenotype plasticity. The study thus provided both analytical strategies and a deeper understanding of the complex genetic regulation of phenotype plasticity in yeast growth.

Multiple ancestral haplotypes harboring regulatory mutations cumulatively contribute to a QTL affecting chicken growth traits.

In depth studies of quantitative trait loci (QTL) can provide insights to the genetic architectures of complex traits. A major effect QTL at the distal end of chicken chromosome 1 has been associated with growth traits in multiple populations. This locus was fine-mapped in a fifteen-generation chicken advanced intercross population including 1119 birds and explored in further detail using 222 sequenced genomes from 10 high/low body weight chicken stocks. We detected this QTL that, in total, contributed 14.4% of the genetic variance for growth. Further, nine mosaic precise intervals (Kb level) which contain ancestral regulatory variants were fine-mapped and we chose one of them to demonstrate the key regulatory role in the duodenum. This is the first study to break down the detail genetic architectures for the well-known QTL in chicken and provides a good example of the fine-mapping of various of quantitative traits in any species.

Haplotype Purging After Relaxation of Selection in Lines of Chickens that Had Undergone Long-Term Selection for High and Low Body Weight.

Bi-directional selection for increased and decreased 56-day body weights (BW56) has been applied to two lines of White Plymouth Rock chickens-the Virginia high (HWS) and low (LWS) body weight lines. Correlated responses have been observed, including negative effects on traits related to fitness. Here, we use high and low body weight as proxies for fitness. On a genome-wide level, relaxed lines (HWR, LWR) bred from HWS and LWS purged some genetic variants in the selected lines. Whole-genome re-sequencing was here used to identify individual loci where alleles that accumulated during directional selection were purged when selection was relaxed. In total, 11 loci with significant purging signals were identified, five in the low (LW) and six in the high (HW) body weight lineages. Associations between purged haplotypes in these loci and BW56 were tested in an advanced intercross line (AIL). Two loci with purging signals and haplotype associations to BW56 are particularly interesting for further functional characterization, one locus on chromosome 6 in the LW covering the sour-taste receptor gene PKD2L1, a functional candidate gene for the decreased appetite observed in the LWS and a locus on chromosome 20 in the HW containing a skeletal muscle hypertrophy gene, DNTTIP1.

Dynamic genetic architecture of yeast response to environmental perturbation shed light on origin of cryptic genetic variation.

Cryptic genetic variation could arise from, for example, Gene-by-Gene (G-by-G) or Gene-by-Environment (G-by-E) interactions. The underlying molecular mechanisms and how they influence allelic effects and the genetic variance of complex traits is largely unclear. Here, we empirically explored the role of environmentally influenced epistasis on the suppression and release of cryptic variation by reanalysing a dataset of 4,390 haploid yeast segregants phenotyped on 20 different media. The focus was on 130 epistatic loci, each contributing to segregant growth in at least one environment and that together explained most (69-100%) of the narrow sense heritability of growth in the individual environments. We revealed that the epistatic growth network reorganised upon environmental changes to alter the estimated marginal (additive) effects of the individual loci, how multi-locus interactions contributed to individual segregant growth and the level of expressed genetic variance in growth. The estimated additive effects varied most across environments for loci that were highly interactive network hubs in some environments but had few or no interactors in other environments, resulting in changes in total genetic variance across environments. This environmentally dependent epistasis was thus an important mechanism for the suppression and release of cryptic variation in this population. Our findings increase the understanding of the complex genetic mechanisms leading to cryptic variation in populations, providing a basis for future studies on the genetic maintenance of trait robustness and development of genetic models for studying and predicting selection responses for quantitative traits in breeding and evolution.

Genotyping by low-coverage whole-genome sequencing in intercross pedigrees from outbred founders: a cost-efficient approach.

BACKGROUND: Experimental intercrosses between outbred founder populations are powerful resources for mapping loci that contribute to complex traits i.e. quantitative trait loci (QTL). Here, we present an approach and its accompanying software for high-resolution reconstruction of founder mosaic genotypes in the intercross offspring from such populations using whole-genome high-coverage sequence data on founder individuals (~ 30×) and very low-coverage sequence data on intercross individuals (< 0.5×). Sets of founder-line informative markers were selected for each full-sib family and used to infer the founder mosaic genotypes of the intercross individuals. The application of this approach and the quality of the estimated genome-wide genotypes are illustrated in a large F2 pedigree between two divergently selected lines of chickens. RESULTS: We describe how we obtained whole-genome genotype data for hundreds of individuals in a cost- and time-efficient manner by using a Tn5-based library preparation protocol and an imputation algorithm that was optimized for this application. In total, 7.6 million markers segregated in this pedigree and, within each full-sib family, between 10.0 and 13.7% of these were fully informative, i.e. fixed for alternative alleles in the founders from the divergent lines, and were used for reconstruction of the offspring mosaic genotypes. The genotypes that were estimated based on the low-coverage sequence data were highly consistent (> 95% agreement) with those obtained using individual single nucleotide polymorphism (SNP) genotyping. The estimated resolution of the inferred recombination breakpoints was relatively high, with 50% of them being defined on regions shorter than 10 kb. CONCLUSIONS: A method and software for inferring founder mosaic genotypes in intercross offspring from low-coverage whole-genome sequencing in pedigrees from heterozygous founders are described. They provide high-quality, high-resolution genotypes in a time- and cost-efficient manner. The software is freely available at https://github.com/CarlborgGenomics/Stripes .

Bidirectional Selection for Body Weight on Standing Genetic Variation in a Chicken Model.

Experimental populations of model organisms provide valuable opportunities to unravel the genomic impact of selection in a controlled system. The Virginia body weight chicken lines represent a unique resource to investigate signatures of selection in a system where long-term, single-trait, bidirectional selection has been carried out for more than 60 generations. At 55 generations of divergent selection, earlier analyses of pooled genome resequencing data from these lines revealed that 14.2% of the genome showed extreme differentiation between the selected lines, contained within 395 genomic regions. Here, we report more detailed analyses of these data exploring the regions displaying within- and between-line genomic signatures of the bidirectional selection applied in these lines. Despite the strict selection regime for opposite extremes in body weight, this did not result in opposite genomic signatures between the lines. The lines often displayed a duality of the sweep signatures, where an extended region of homozygosity in one line, in contrast to mosaic pattern of heterozygosity in the other line. These haplotype mosaics consisted of short, distinct haploblocks of variable between-line divergence, likely the results of a complex demographic history involving bottlenecks, introgressions and moderate inbreeding. We demonstrate this using the example of complex haplotype mosaicism in the growth1 QTL. These mosaics represent the standing genetic variation available at the onset of selection in the founder population. Selection on standing genetic variation can thus result in different signatures depending on the intensity and direction of selection.

A Polygenic Genetic Architecture of Flowering Time in the Worldwide Arabidopsis thaliana Population.

Here, we report an empirical study of the polygenic basis underlying the evolution of complex traits. Flowering time variation measured at 10 and 16°C in the 1,001-genomes Arabidopsis thaliana collection of natural accessions were used as a model. The polygenic architecture of flowering time was defined as the 48 loci that were significantly associated with flowering time-at 10 and/or 16°C and/or their difference-in this population. Contributions from alleles at flowering time associated loci to global and local adaptation were explored by evaluating their distribution across genetically and geographically defined subpopulations across the native range of the species. The dynamics in the genetic architecture of flowering time in response to temperature was evaluated by estimating how the effects of these loci on flowering changed with growth temperature. Overall, the genetic basis of flowering time was stable-about 2/3 of the flowering time loci had similar effects at 10°C and 16°C-but many loci were involved in gene by temperature interactions. Globally present alleles, mostly of moderate effect, contributed to the differences in flowering times between the subpopulations via subtle changes in allele frequencies. More extreme local adaptations were, on several occasions, due to regional alleles with relatively large effects, and their linkage disequilibrium-patterns suggest coevolution of functionally connected alleles within local populations. Overall, these findings provide a significant contribution to our understanding about the possible modes of global and local evolution of a complex adaptive trait in A. thaliana.

On the Relationship Between High-Order Linkage Disequilibrium and Epistasis.

A plausible explanation for statistical epistasis revealed in genome wide association analyses is the presence of high order linkage disequilibrium (LD) between the genotyped markers tested for interactions and unobserved functional polymorphisms. Based on findings in experimental data, it has been suggested that high order LD might be a common explanation for statistical epistasis inferred between local polymorphisms in the same genomic region. Here, we empirically evaluate how prevalent high order LD is between local, as well as distal, polymorphisms in the genome. This could provide insights into whether we should account for this when interpreting results from genome wide scans for statistical epistasis. An extensive and strong genome wide high order LD was revealed between pairs of markers on the high density 250k SNP-chip and individual markers revealed by whole genome sequencing in the Arabidopsis thaliana 1001-genomes collection. The high order LD was found to be more prevalent in smaller populations, but present also in samples including several hundred individuals. An empirical example illustrates that high order LD might be an even greater challenge in cases when the genetic architecture is more complex than the common assumption of bi-allelic loci. The example shows how significant statistical epistasis is detected for a pair of markers in high order LD with a complex multi allelic locus. Overall, our study illustrates the importance of considering also other explanations than functional genetic interactions when genome wide statistical epistasis is detected, in particular when the results are obtained in small populations of inbred individuals.

A multilocus association analysis method integrating phenotype and expression data reveals multiple novel associations to flowering time variation in wild-collected Arabidopsis thaliana.

The adaptation to a new habitat often results in a confounding between genomewide genotype and beneficial alleles. When the confounding is strong, or the allelic effects is weak, it is a major statistical challenge to detect the adaptive polymorphisms. We describe a novel approach to dissect polygenic traits in natural populations. First, candidate adaptive loci are identified by screening for loci directly associated with the adaptive trait or the expression of genes known to affect it. Then, a multilocus genetic architecture is inferred using a backward elimination association analysis across all candidate loci with an adaptive false discovery rate-based threshold. Effects of population stratification are controlled by accounting for genomic kinship in both steps of the analysis and also by simultaneously testing all candidate loci in the multilocus model. We illustrate the method by exploring the polygenic basis of an important adaptive trait, flowering time in Arabidopsis thaliana, using public data from the 1,001 genomes project. We revealed associations between 33 (29) loci and flowering time at 10 (16)°C in this collection of natural accessions, where standard genomewide association analysis methods detected five (3) loci. The 33 (29) loci explained approximately 55.1 (48.7)% of the total phenotypic variance of the respective traits. Our work illustrates how the genetic basis of highly polygenic adaptive traits in natural populations can be explored in much greater detail using new multilocus mapping approaches taking advantage of prior biological information, genome and transcriptome data.

On the relationship between epistasis and genetic variance heterogeneity.

Epistasis and genetic variance heterogeneity are two non-additive genetic inheritance patterns that are often, but not always, related. Here we use theoretical examples and empirical results from earlier analyses of experimental data to illustrate the connection between the two. This includes an introduction to the relationship between epistatic gene action, statistical epistasis, and genetic variance heterogeneity, and a brief discussion about how genetic processes other than epistasis can also give rise to genetic variance heterogeneity.

Artificial Selection Response due to Polygenic Adaptation from a Multilocus, Multiallelic Genetic Architecture.

The ability of a population to adapt to changes in their living conditions, whether in nature or captivity, often depends on polymorphisms in multiple genes across the genome. In-depth studies of such polygenic adaptations are difficult in natural populations, but can be approached using the resources provided by artificial selection experiments. Here, we dissect the genetic mechanisms involved in long-term selection responses of the Virginia chicken lines, populations that after 40 generations of divergent selection for 56-day body weight display a 9-fold difference in the selected trait. In the F15 generation of an intercross between the divergent lines, 20 loci explained >60% of the additive genetic variance for the selected trait. We focused particularly on fine-mapping seven major QTL that replicated in this population and found that only two fine-mapped to single, bi-allelic loci; the other five contained linked loci, multiple alleles or were epistatic. This detailed dissection of the polygenic adaptations in the Virginia lines provides a deeper understanding of the range of different genome-wide mechanisms that have been involved in these long-term selection responses. The results illustrate that the genetic architecture of a highly polygenic trait can involve a broad range of genetic mechanisms, and that this can be the case even in a small population bred from founders with limited genetic diversity.

Accounting for genetic interactions improves modeling of individual quantitative trait phenotypes in yeast.

Experiments in model organisms report abundant genetic interactions underlying biologically important traits, whereas quantitative genetics theory predicts, and data support, the notion that most genetic variance in populations is additive. Here we describe networks of capacitating genetic interactions that contribute to quantitative trait variation in a large yeast intercross population. The additive variance explained by individual loci in a network is highly dependent on the allele frequencies of the interacting loci. Modeling of phenotypes for multilocus genotype classes in the epistatic networks is often improved by accounting for the interactions. We discuss the implications of these results for attempts to dissect genetic architectures and to predict individual phenotypes and long-term responses to selection.

Genome-wide standing variation facilitates long-term response to bidirectional selection for antibody response in chickens.

BACKGROUND: Long-term selection experiments provide a powerful approach to gain empirical insights into adaptation, allowing researchers to uncover the targets of selection and infer their contributions to the mode and tempo of adaptation. Here we implement a pooled genome re-sequencing approach to investigate the consequences of 39 generations of bidirectional selection in White Leghorn chickens on a humoral immune trait: antibody response to sheep red blood cells. RESULTS: We observed wide genome involvement in response to this selection regime. Many genomic regions were highly differentiated resulting from this experimental selection regime, an involvement of up to 20% of the chicken genome (208.8 Mb). While genetic drift has certainly contributed to this, we implement gene ontology, association analysis and population simulations to increase our confidence in candidate selective sweeps. Three strong candidate genes, MHC, SEMA5A and TGFBR2, are also presented. CONCLUSIONS: The extensive genomic changes highlight the polygenic genetic architecture of antibody response in these chicken populations, which are derived from a common founder population, demonstrating the extent of standing immunogenetic variation available at the onset of selection.

Genetics of Interactive Behavior in Silver Foxes (Vulpes vulpes).

Individuals involved in a social interaction exhibit different behavioral traits that, in combination, form the individual's behavioral responses. Selectively bred strains of silver foxes (Vulpes vulpes) demonstrate markedly different behaviors in their response to humans. To identify the genetic basis of these behavioral differences we constructed a large F2 population including 537 individuals by cross-breeding tame and aggressive fox strains. 98 fox behavioral traits were recorded during social interaction with a human experimenter in a standard four-step test. Patterns of fox behaviors during the test were evaluated using principal component (PC) analysis. Genetic mapping identified eight unique significant and suggestive QTL. Mapping results for the PC phenotypes from different test steps showed little overlap suggesting that different QTL are involved in regulation of behaviors exhibited in different behavioral contexts. Many individual behavioral traits mapped to the same genomic regions as PC phenotypes. This provides additional information about specific behaviors regulated by these loci. Further, three pairs of epistatic loci were also identified for PC phenotypes suggesting more complex genetic architecture of the behavioral differences between the two strains than what has previously been observed.

Imputation-Based Fine-Mapping Suggests That Most QTL in an Outbred Chicken Advanced Intercross Body Weight Line Are Due to Multiple, Linked Loci.

The Virginia chicken lines have been divergently selected for juvenile body weight for more than 50 generations. Today, the high- and low-weight lines show a >12-fold difference for the selected trait, 56-d body weight. These lines provide unique opportunities to study the genetic architecture of long-term, single-trait selection. Previously, several quantitative trait loci (QTL) contributing to weight differences between the lines were mapped in an F2-cross between them, and these were later replicated and fine-mapped in a nine-generation advanced intercross of them. Here, we explore the possibility to further increase the fine-mapping resolution of these QTL via a pedigree-based imputation strategy that aims to better capture the genetic diversity in the divergently selected, but outbred, founder lines. The founders of the intercross were high-density genotyped, and then pedigree-based imputation was used to assign genotypes throughout the pedigree. Imputation increased the marker density 20-fold in the selected QTL, providing 6911 markers for the subsequent analysis. Both single-marker association and multi-marker backward-elimination analyses were used to explore regions associated with 56-d body weight. The approach revealed several statistically and population structure independent associations and increased the mapping resolution. Further, most QTL were also found to contain multiple independent associations to markers that were not fixed in the founder populations, implying a complex underlying architecture due to the combined effects of multiple, linked loci perhaps located on independent haplotypes that still segregate in the selected lines.

A Complex Structural Variation on Chromosome 27 Leads to the Ectopic Expression of HOXB8 and the Muffs and Beard Phenotype in Chickens.

Muffs and beard (Mb) is a phenotype in chickens where groups of elongated feathers gather from both sides of the face (muffs) and below the beak (beard). It is an autosomal, incomplete dominant phenotype encoded by the Muffs and beard (Mb) locus. Here we use genome-wide association (GWA) analysis, linkage analysis, Identity-by-Descent (IBD) mapping, array-CGH, genome re-sequencing and expression analysis to show that the Mb allele causing the Mb phenotype is a derived allele where a complex structural variation (SV) on GGA27 leads to an altered expression of the gene HOXB8. This Mb allele was shown to be completely associated with the Mb phenotype in nine other independent Mb chicken breeds. The Mb allele differs from the wild-type mb allele by three duplications, one in tandem and two that are translocated to that of the tandem repeat around 1.70 Mb on GGA27. The duplications contain total seven annotated genes and their expression was tested during distinct stages of Mb morphogenesis. A continuous high ectopic expression of HOXB8 was found in the facial skin of Mb chickens, strongly suggesting that HOXB8 directs this regional feather-development. In conclusion, our results provide an interesting example of how genomic structural rearrangements alter the regulation of genes leading to novel phenotypes. Further, it again illustrates the value of utilizing derived phenotypes in domestic animals to dissect the genetic basis of developmental traits, herein providing novel insights into the likely role of HOXB8 in feather development and differentiation.