Identification and functional characterization of causal genes affecting feather pecking and aggressive pecking in laying hens
Final Report Abstract
Feather pecking (FP) is a serious problem in layers severely impairing animal welfare and leading to economic losses. FP and aggressive pecking are complex traits, which are hard to measure. Although numerous studies have investigated this phenomenon, the causes for this unwanted behavior are still not well understood. The aim of this project was thus to identify genes and pathways affecting the individual propensity to perform FP and to detect genetic variation controlling these pathways. The work was conducted in layer lines divergently selected for FP behavior. Large half-sib families were established from the lines comprising almost 500 hens in total. This population was combined with a previously established F2 design. Approximately 1,400 animals were genotyped with a medium density SNP chip. The genomes of 49 birds including the F2-design founders were sequenced and used as a reference to impute animals up to sequence level. Aiming to identify putatively causative genes, traitassociated variants and selection signatures were mapped for FP and related traits. Genome-wide association studies (GWAS) based on the imputed sequence level genotypes were conducted in the selection lines as well as the F2 design and combined in a meta-analysis. Using this approach, a number of significantly associated genes and enriched gene-sets were identified pointing to mechanisms that have previously been linked to human neuropsychiatric disorders, mainly schizophrenia, and providing evidence for a prominent involvement of GABAergic signaling. In addition, brain transcriptomes of 48 hens were analyzed by RNA-sequencing. An animal experiment was established allowing for the comparison between selection lines before and after triggering actual FP behaviour by light stimulation. Differential gene expression and downstream analyses identified genes and pathways related to cholinergic signalling and immune response. Previous studies found monoamine signalling to play a central role, but also pointed to GABAergic signalling, immune response and gut microbiota. There could be a connection with cholinergic signalling, which in turn projects into monoamine signalling, directly or via GABA or glutamate signalling. Furthermore, a surprisingly low level of transcriptomic response to the light stimulus was found in the line selected for high feather pecking (HFP). Dicer1 is among the top differentially expressed genes (DEGs) in this experiment and about one third of DEGs are non-coding RNAs. Thus, it can be hypothesized that the processing of miRNAs is disturbed in HFP birds due to the absence of Dicer1. In humans, it has been shown that the downregulation of multiple miRNAs affects GABA signalling in neuropsychiatric disorders. Lower expression levels of GABRB2 found in HFP birds might thus be caused by miRNA dysregulation in the GABAergic neurons. GABA is a major inhibitory neurotransmitter, which might explain the low number of DEGs in HFP birds in response to light: A constantly high level of neuronal excitation in the absence of inhibitory GABA signalling may not allow for a response. Other GABA receptor genes are upregulated in the low feather pecker’s response to light supporting this line of argumentation. Based on the results of the differential gene expression analysis, a set of DEGs was selected that were validated in 167 animals from both lines using a Fluidigm BioMark microfluidics system. The relative expression values were used as phenotypes in an expression GWAS revealing a number of significantly associated loci controlling the expression of these genes. The results of downstream analyses point to gene-gene interactions that form the basis for the identification of further regulatory mechanisms. As previous studies highlighted a possible involvement of gut microbiota, the ileum microbiota composition of hens from both lines was analyzed. Although significant differences between the two selection lines were found, the microbiota composition did not explain any variance of FP behavior within lines, i.e. the microbiability was not significant. Thus, it can be concluded, that the ileum microbiota composition is not a driver of FP behavior. Taken together, the results substantially extend the knowledge about genetic causes of FP and contribute to the understanding of pathophysiological mechanisms leading to this behaviour. The results are in line with previous work and beyond that can contribute to integration of existing hypotheses into a clearer mechanistic picture. In this regard, the work regarding differences in the response to light stimuli is important. The current working hypothesis places Dicer1 on top of the list of behaviour modulating genes. We believe that downregulation of Dicer1 leads to a decrease in miRNA production and further downstream to a downregulation of GABAergic signalling, which relates to previous findings regarding monoamine signalling. This can serve as a starting point for functional validation. Nevertheless, the results also clearly confirm the polygenic and complex nature of the trait.
Publications
- (2020): A Novel Model to Explain Extreme Feather Pecking Behavior in Laying Hens. Behavior Genetics 50, 41–50
Iffland, H., R. Wellmann, S. Preuß, J. Tetens, W. Bessei, H.-P. Piepho, J. Bennewitz
(See online at https://doi.org/10.1007/s10519-019-09971-w) - (2020): Analysis of the brain transcriptome in lines of laying hens divergently selected for feather pecking. BMC Genomics 21, 595
Falker-Gieske, C., A. Mott, S. Preuß, S. Franzenburg, W. Bessei, J. Bennewitz, J. Tetens
(See online at https://doi.org/10.1186/s12864-020-07002-1) - (2020): Genomewide Mapping of Selection Signatures and Genes for Extreme Feather Pecking in Two Divergently Selected Laying Hen Lines. Animals 10, 262
Iffland, H., R. Wellmann, M. Schmid, S. Preuß, J. Tetens, W. Bessei, J. Bennewitz
(See online at https://doi.org/10.3390/ani10020262) - (2020): Meta-analyses of genome wide association studies in lines of laying hens divergently selected for feather pecking using imputed sequence level genotypes. BMC Genetics 21, 114
Falker-Gieske, C., H. Iffland, S. Preuß, W. Bessei, C. Drögemüller, J. Bennewitz, J. Tetens
(See online at https://doi.org/10.1186/s12863-020-00920-9) - (2021): Feather pecking behavior in the Hohenheim selection lines of laying hens-a selective review of genomic, transcriptomic and gut microbiota analyses. Züchtungskunde 93, 229–243
Bennewitz, J. und J. Tetens
- (2021): Gut Microbial Composition and Predicted Functions Are Not Associated with Feather Pecking and Antagonistic Behavior in Laying Hens. Life 11, 235
Borda-Molina, D., H. Iffland, M. Schmid, R. Müller, S. Schad, J. Seifert, J. Tetens, W. Bessei, J. Bennewitz, A. Camarinha-Silva
(See online at https://doi.org/10.3390/life11030235) - (2021): Phenotypic and genomic analyses of agonistic interactions in laying hen lines divergently selected for feather pecking. Applied Animal Behaviour Science 234, 105177
Iffland, H., M. Schmid, S. Preuß, W. Bessei, J. Tetens, J. Bennewitz
(See online at https://doi.org/10.1016/j.applanim.2020.105177)
