Feather pecking (FP) is a damaging nonaggressive behavior in laying hens with a heritable component. Its occurrence has been linked to the immune system, the circadian clock, and foraging behavior. Furthermore, dysregulation of miRNA biogenesis, disturbance of the gamma-aminobutyric acid (GABAergic) system, as well as neurodevelopmental deficiencies are currently under debate as factors influencing the propensity for FP behavior. Past studies, which focused on the dissection of the genetic factors involved in FP, relied on single nucleotide polymorphisms (SNPs) and short insertions and deletions < 50 bp (InDels). These variant classes only represent a certain fraction of the genetic variation of an organism. Hence, we reanalyzed whole-genome sequencing data from two experimental populations, which have been divergently selected for FP behavior for over more than 15 generations, performed variant calling for structural variants (SVs) as well as tandem repeats (TRs), and jointly analyzed the data with SNPs and InDels. Genotype imputation and subsequent genome-wide association studies, in combination with expression quantitative trait loci analysis, led to the discovery of multiple variants influencing the GABAergic system. These include a significantly associated TR downstream of the GABA receptor subunit beta-3 (GABRB3) gene, two microRNAs targeting several GABA receptor genes, and dystrophin (DMD), a direct regulator of GABA receptor clustering. Furthermore, we found the transcription factor ETV1 to be associated with the differential expression of 23 genes, which points toward a role of ETV1, together with SMAD4 and KLF14, in the disturbed neurodevelopment of high-feather pecking chickens.
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Introduction
FP in chickens is a behavioral disorder that severely impacts animal welfare and causes significant economic losses. It has been proposed that FP is obsessive–compulsive-like behavior [3]. In the past, the damage has been controlled by beak trimming, which has now been prohibited in many European countries. Numerous studies found an involvement of environmental factors such as light intensity, nutrition, stocking density, and lack of foraging (reviewed by [4] and [5]). Furthermore, evidence has been accumulating that the immune system plays a major role in the development of FP behavior [1, 6‐8]. The gut microbiota has also been proposed to be involved in FP behavior, but this has been disproven in several studies [9, 10]. Since feather pecking is a complex heritable trait (reviewed by [11]), the dissection of the genetic causes is essential for the development of effective breeding strategies to eradicate the causative alleles. To achieve that goal, chickens were divergently selected for FP behavior over more than 15 generations based on their estimated breeding values for the behavior. Breeding of these lines was initiated in Denmark and continued in Hohenheim, Germany. In Hohenheim, two populations were established—an F2 cross and 12 half-sib families (in the preceding text referred to as F2 and HS). A detailed description of the experimental populations and the research conducted with them was reviewed by Bennewitz and Tetens [12]. Based on the results that were acquired by whole-genome and transcriptome sequencing with the Hohenheim selection lines, FP appears to be a disorder of the γ-aminobutyric acid (GABAergic) system in conjunction with a disturbance of embryonic neurodevelopment by a lack of leukocytes in the developing brain. Several variants in or in close proximity to GABA receptor genes were identified in genome-wide association studies (GWAS) conducted on medium-density single nucleotide polymorphism (SNP) array and imputed-sequence level genotypes [2, 13, 14]. Furthermore, brain transcriptome analysis of high and low feather peckers (HFP and LFP) before and after light stimulation revealed that HFP responds with very few changes in gene expression in comparison to LFP, with numerous GABA receptor genes upregulated in LFP. Only GABRB2 (gamma-aminobutyric acid type A receptor subunit beta2) was among differentially expressed genes (DEGs) in HFP, but it was downregulated, instead of upregulated, a pattern that we observed for most DEGs in HFP brains [14]. We attribute this low level of gene expression changes in response to light to a high level of excitation in HFP brains due to the lack of multiple GABA receptors. Since GABA is the major inhibitory neurotransmitter, a lack of its receptors in the brain would lead to a high neuronal excitatory state, which explains the observed hyperactivity and the obsessive–compulsive-like behavior observed in HFP. Since Dicer1 was among the downregulated DEGs, we assume that miRNA processing is disturbed, as is also the case in schizophrenia patients [15], which in turn leads to low GABA receptor expression levels. In the general comparison of brain transcriptomes between HFP and LFP hens, we observed an enrichment of immune system-related DEGs [1], which we could further pinpoint in an expression quantitative trait loci (eQTL) analysis to a small deletion 652 bp downstream of the KLF14 gene. In total, the differential expression of 40 genes between HFP and LFP chickens was significantly associated with this KLF14 variant, a majority of which are involved in leukocyte biology [8]. It has been shown in mice that CD4 T cells are essential for healthy development from the fetal to the adult brain. A defect in CD4 T cell maturation affected synapse development and led to behavioral abnormalities [16]. The evidence suggests that this mechanism is responsible for disturbing embryonic brain development in HFP chickens, which contributes to FP behavior.
One commonality of all the studies that we conducted on the genetics involved in FP is the overlap in associated genes with human psychiatric disorders, most prevalent schizophrenia. SVs play a notable role in human psychiatric disorders [17‐19], which is also the case for tandem repeats (TRs) [20]. Commonly investigated classes of SVs include insertions, deletions, duplications, inversions, and translocations, which arise from various combinations of DNA gains, losses, or rearrangements [21]. Here, we present the first in-depth study on the potential role of SVs and TRs in FP, which led to a deeper understanding of the mechanisms responsible for this behavioral disorder.
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Material and methods
Animals and husbandry
The F2 and HS lines of White Leghorn chickens were divergently selected for feather pecking behavior for over more than 15 generations at Hohenheim University [22, 23]. Animals, which were used for genotyping in the study presented here were as follows: from the F2 design 25 founder (whole-genome sequenced), 89 F1 (SNP chip genotyped), and 817 F2 animals (SNP chip genotyped) and from the HS population, 24 animals were whole-genome sequenced and 494 animals were SNP chip genotyped. RNA from the whole brains of 167 HS chickens was used for Fluidigm gene expression analysis for the eGWAS approach [8]. All experimental procedures [1], rearing and husbandry conditions [24], as well as phenotyping [2], were described in previous studies. Briefly, the phenotypic data were generated by direct observations made by seven independent investigators at approximately 32 weeks of age. Observations were recorded in 20 min sessions in average group sizes of about 42 animals. The phenotypes are expressed as the number of FP bouts actively delivered during a standardized time span. As these count data are heavily distributed from normality, Box–Cox-transformation was applied [2, 13].
Structural variants and short tandem repeats discovery
Illumina whole genome sequencing data were mapped to chicken genome version GRCg6a (GCF_000002315.5 RefSeq assembly) and used to call SNPs and short (< 50 bp) insertions and deletions (InDels) in our previous study [2]. This was achieved with the genome analysis toolkit (GATK) v. 4.0, according to the best practice guideline of the broad institute [25]. SVs were called as described by Blaj et al. [26] with slightly different settings: A high-confidence SV call set was produced from the output of three variant callers: smove v. 0.2.6 (Brent, P. (2018) Smoove. https://brentp.github.io/post/smoove/), DELLY v. 0.7.7 [27], and manta v. 1.6.0 [28]. SURVIROR v. 1.0.7 [29] was used to combine the output of the three variant callers with the following settings: maximum distance between breakpoints of 1000 bp, minimum number of supporting callers 2, SV type and strands were taken into account, and the minimum SV size was set to 30 bp. Variants with a call rate < 0.8 and variants with QUAL < 1000 were removed. TRs were called with GangSTR v. 2.5 [30]. A library of known TRs as input for GangSTR was acquired from the UCSC data repository (https://hgdownload.soe.ucsc.edu/goldenPath/galGal6/bigZips/). TRs were filtered to retain genotypes with a minimum sequence depth (DP) of 10, a quality score (Q) higher than 0.8, and a call rate < 0.8.
Haplotype construction and imputation
To impute medium-density chip genotypes to whole genome-level SNPs and InDels, we employed the same strategy as we previously described [2], with the deviation that all imputation steps were performed with Beagle v. 5.2 [31] and the setting ne = 1000. SVs and TRs were merged with SNPs and InDels from our previous study, which were acquired with the GATK [25]. This merged call set was phased with Beagle v. 5.2 for the estimation of haplotypes and used as a reference panel to impute medium-density chip genotypes to SVs and TRs with the same strategy as for SNPs and InDels. Chip genotypes from HS animals were directly imputed using the WGS reference panel. For the F2 design, we first imputed chip-genotyped F1 animals to the WGS level, merged the output with the initial reference panel, phased the merged dataset, and used it as a reference panel for the imputation of F2 SNP chip-genotyped animals. To remove SNPs and InDels from the imputed SVs/TRs, GATK SelectVariants was utilized.
Detection of quantitative trait loci
Prior to GWAS, multiallelic variants from the SVs/TR dataset were converted to biallelic variants with the norm function from bcftools v. 1.14 [32]. All GWAS were conducted with gcta v. 1.92.3 beta3 [33], applying a mixed linear model association analysis with a leaving-one-chromosome-out (LOCO) approach and a minor allele frequency (MAF) threshold of 0.01. In brief, relatedness between animals and stratification [6] was corrected by including a random genetic term based on a genomic relationship matrix calculated only from SNP-chip data and following a LOCO approach [34]. Briefly, a model of the form \(y=W\alpha +X\beta +u+\epsilon\)is fitted (y = n × 1 vector of phenotyped (n) hens; W = n × c incidence matrix of fixed effects with c being the number of effects; α = vector of corresponding coefficients including the mean; X = n × 1 vector of marker genotypes at the locus tested; β = corresponding effect size; u = vector of random genetic effects, with \(u\sim N(0,{A}^{-}{\sigma }_{g}^{2})\), where \({\sigma }_{g}^{2}\) represents genetic variance and A− is the genomic relationship matrix based on all SNP-chip markers except those on the chromosome currently analyzed; ε = random residual term, with \(\epsilon \sim N(0,I{\sigma }_{\epsilon }^{2})\), where \({\sigma }_{\epsilon }^{2}\) represents the residual variance and I represents an identity matrix). Line effects (HFP and LFP) and hatch were included in the analysis of the HS design; for the F2 design, only the hatch was included as a fixed effect. The phenotype used for GWAS, “feather pecks delivered Box–Cox transformed” (FPD_BC), has been described by Iffland et al. [13]. Phenotypes for expression GWAS (eGWAS) were normalized gene expression data from our previous study [8]. There we analyzed the expression of 86 genes in 167 HS chickens, which we discovered in a transcriptome analysis performed on 48 of the HS birds [1]. Information on the hatch was used as a covariate in all GWAS and eGWAS. Genomic relationship matrices were created from the target 60 k SNP Chip genotypes. Meta-analyses of GWAS results were performed with METAL v. 1.1 [35] using the sample size-based approach with default settings. The proportion of variance in phenotype explained by a given SNP (PVE) was calculated according to the formula Var(Y) = β2Var(X) + σ2 by Shim et al. [36] (Var(Y) = variance in phenotype; β = effect size of genetic variant X; σ2 = remaining variance). To correct for multiple testing, the threshold for genome-wide significance of variants was calculated by Bonferroni correction (\(\frac{\mathrm{number}\;\mathrm{of}\;\mathrm{variants}}{0.05}\)).
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Association weight matrix construction
The AWM was created as described in our previous study [8] by deploying the strategy for AWM construction by Reverter and Fortes [37], followed by the detection of significant gene–gene interactions with their PCIT algorithm [38]. Input variants were chosen as follows: p-value < 1 × 10−4 for the main phenotype (FPD_BC) or p-value < 1 × 10−4 in at least ten of the eGWAS. That way, variants affecting the main phenotype and gene expression were both considered in the analysis. This led to the selection of 57 input variants, 0.16% of all detected SVs and TRs, for the HS population. The R script by Reverter and Fortes was modified by setting the p-value threshold for primary and secondary SNP selection to 1 × 10−4. The gene–gene interaction map was constructed with Cytoscape [39], and gene class information was acquired with PANTHER [40] and UniProt [41].
Transcription factor enrichment
To detect significant binding site enrichment for the transcription factor ETV1, the CiiiDER software (build May 15th, 2020) [42] was employed with frequency matrix MA0761.2 (https://jaspar.genereg.net/matrix/MA0761.2/) and the following settings: p-value threshold for gene coverage enrichment = 0.05; base position upstream scan limit = 1500 bp; base position downstream scan limit = 500 bp. Additional members of the ETS (E twenty-six) family of transcription factors used in the analysis were ETV2 (MA0762.1), ETV3 (MA0763.1), ETV4 (MA0764.2), ETV5 (MA0765.1), ETV6 (MA0645.1), and ETV7 (MA1708.1). Genes that were associated with the ETV1 variant (p-value < 1 × 10−4; Supplementary Information S1) were used as input, and DEGs with a log2 fold change < 0.2 were used as background genes (14,514 genes).
Results
Genome-wide association studies with different classes of genetic variants
In total, 63,824 TRs and 11,098 SVs were discovered in the joint variant calling of whole genome sequenced HS and F0 chickens. The SVs contained 6014 deletions, 2741 inversions, 1334 duplications, and 995 translocations. The variant calling of SNPs and InDels was reported in our previous study and yielded 12,864,421 SNPs and 2,142,539 InDels [2]. To grasp the whole depth of the datasets at hand, we repeated the imputation of SNPs and InDels with the most recent Beagle version (v. 5.2) [31] and set the effective population size to 1000, which is known to improve the imputation accuracy in small populations [43]. GWAS results for the trait FPD_BC were analyzed for both experimental designs, the F2 cross, and the HS population, separately and after combining the results in a meta-analysis. Manhattan plots from GWAS with imputed SNPs/InDels and imputed SVs/TRs are shown in Fig. 1a. Common peaks for both variant classes on GGA1 and GGA2 were observable in the F2 cross, as well as on GGA1 and GGA3 of the HS population. QTL are summarized in Table 1, and lead SVs and TRs with their predicted effects are listed in Table 2.
Fig. 1
a Genome-wide association studies (GWAS) performed with single nucleotide polymorphisms (SNPs) in combination with short (< 50 bp) insertions and deletions (InDels) and structural variants (SVs) in combination with tandem repeats (TRs). All variant classes were investigated in the F2 cross and half-sib (HS) families of chickens divergently selected for feather pecking and subsequently combined in a meta-analysis. b Heatmap of the 20 highest associated genes from each GWAS. The – log10p-values of the highest associated variants for each gene were used
Table 1
Positional ranges in base pairs of QTL that were discovered in both experimental populations (F2 cross and half-sib (HS) families) for both groups of variant classes: single nucleotide polymorphisms (SNPs) and short insertions/deletions (InDels) as well as structural variants (SVs) and tandem repeats (TRs)
F2 SNPs and short InDels
F2 SVs and TRs
HS SNPs and short InDels
HS SVs and TRs
GGA1: 59,032,772–59,548,358
GGA1: 59,138,261–59,216,858
GGA1: 132,578,705–133,803,185
GGA1: 130,621,356–134,077,752
GGA2: 81,060,432–97,072,903
GGA2: 83,493,152–94,489,210
GGA3: 18,434,015–54,601,143
GGA3: 19,120,538–70,385,553
Table 2
Structural variants (inversions (INV) and deletions (DEL)) and tandem repeats (TRs) that showed the highest association in genome wide association studies for feather pecking behavior in an F2 cross and half-sib (HS) families. Variant effects and closest genes were predicted with SnpEff
GWAS
Type
Size (bp)
Position
p-value
Effect
Gene(s)
F2
INV
1403
GGA1: 59,216,858–59,218,260
1.14 × 10−5
intron_variant
BICD1
F2
TR
GGA1: 59,138,261
2.29 × 10−5
intron_variant
BICD1
F2
TR
GGA2: 83,217,700
2.11 × 10−5
intron_variant
FHOD3
F2
DEL
509
GGA2: 83,493,152–83,493,661
2.11 × 10−5
intergenic_region
ENSGALG00000051475-RPRD1A
F2
TR
GGA2: 83,497,153
2.30 × 10−5
intergenic_region
ENSGALG00000051475-RPRD1A
F2
DEL
209
GGA2: 83,497,939–83,498,148
2.30 × 10−5
intergenic_region
ENSGALG00000051475-RPRD1A
F2
TR
GGA2: 89,486,485
6.90 × 10−5
intergenic_region
ENSGALG00000053898-ENSGALG00000052462
F2
TR
GGA2: 94,489,210
7.28 × 10−5
intergenic_region
ENSGALG00000051191-ENSGALG00000032841
HS
TR
GGA1: 132,889,302
7.79 × 10−7
intergenic_region
GABRB3-ENSGALG00000047084
HS
TR
GGA1: 130,621,356
2.64 × 10−6
intergenic_region
CRLF2-SHOX
HS
TR
GGA1: 133,533,130
4.00 × 10−6
intron_variant
INPP4A
HS
TR
GGA1: 133,533,741
4.09 × 10−6
intron_variant
INPP4A
HS
DEL
368
GGA1: 134,077,752–134,077,908
1.37 × 10−5
intron_variant
AFF3
HS
TR
GGA1: 133,606,912
2.30 × 10−5
downstream_gene_variant
COA5
HS
TR
GGA1: 132,889,302
6.42 × 10−5
intergenic_region
GABRB3-ENSGALG00000047084
HS
TR
GGA1: 134,020,467
9.02 × 10−5
intron_variant
AFF3
HS
TR
GGA3: 19,445,704
0.00012
downstream_gene_variant
TGFB2
HS
TR
GGA3: 40,921,527
0.00016
intron_variant
DLL1
HS
TR
GGA3: 19,264,158
0.00022
intergenic_region
LYPLAL1-ENSGALG00000051126
HS
TR
GGA3: 45,338,399
0.00023
intron_variant
AGPAT4
HS
DEL
531
GGA3: 54,243,436–54,244,326
0.00026
intron_variant
REPS1
HS
TR
GGA3: 70,359,487
0.00032
intron_variant
GRIK2
HS
TR
GGA3: 70,385,553
0.00032
intron_variant
GRIK2
HS
TR
GGA3: 19,120,538
0.00049
intergenic_region
ENSGALG00000047985-LYPLAL1
×
The only variant that reached genome-wide significance after Bonferroni correction (−p < 1.41 × 10−6) was a TR in the HS population, 126,821 bp downstream of GABRB3. The proportion of variance in the phenotype FPD_BC that is explained by this variant is 0.047. Given heritability estimates of around 0.15 [22, 44, 45], the amount of phenotypic variance explained by the lead variant is considerable. To visualize overlaps between the six different sets of results, we created a heat map, which shows the − log10p-values of the top 20 associated genes from the different GWAS (Fig. 1b) and summarized all p-values in Supplementary Information S2. Numerous genes showed high association signals (p < 0.01) in the meta-analyses for both variant classes: AFF3, ATP10A, BICD1, CDH19, CDH7, CRLF2, ENSGALG00000016495 (KLHL29), ENSGALG00000032841 (lncRNA), ENSGALG00000034659 (miRNA), ENSGALG00000046900 (lncRNA), ENSGALG00000048825 (lncRNA), ENSGALG00000050654 (lncRNA), ENSGALG00000053149 (miRNA), ENSGALG00000053459 (lncRNA), FHOD3, GALNT1, gga-mir-187, gga-mir-3524b, INPP4A, LPP, PIEZO2, and SHOX. We assume that these genes are burdened with multiple mutations that contribute to the phenotype, which might be a result of the divergent selection for feather-pecking behavior over multiple generations. Predicted targets of the microRNA (miRNA) gga-mir-187 are GABRA1, GABRB2, GABRB3, GABRG1, and GABRG2. GABRB2 is also a predicted target of gga-mir-3524b (www.targetscan.org, accessed January 27, 2022).
Expression quantitative trait loci (eQTL) analyses
To clarify whether the SVs and TRs that we detected influence the expression of transcripts that we identified in a previous study to be differentially expressed between LFP and HFP [1], we performed an eQTL analysis. We employed the same strategy as in our past study [8] and performed eGWAS for 86 genes from 167 chickens (84 HFP and 83 LFP) from the HS population (Manhattan plots are summarized in Supplementary Information S3). A total of 35,571 SVs and TRs were screened for association with gene expression, and we detected 909 genome-wide significant associated signals. SVs and TRs for which we detected genome-wide association with at least 10 DEGs are shown in Table 3. To identify significant gene–gene interactions from those 86 eGWAS, we constructed an association weight matrix [37], followed by the detection of significant correlations with the PCIT algorithm [38]. Central to the gene–gene interaction map is the transcription factor ETV1. We selected DEGs with p-values < 1 × 10−4 for association with the variant, a tandem repeat with the sequence CCCGGCCCG 70 bp upstream of ETV1 (GGA2: 27,337,541). This led to the selection of 23 genes that were associated with this variant, with p-values ranging from 1.41 × 10−6 to 9.89 × 10−5 with the top associated DEG CERS4L almost reaching genome-wide significance (p-value = 1.41 × 10−6). With the 23 selected genes (Supplementary Information S1), we performed a transcription factor binding site enrichment analysis with Ciiider and found a significant enrichment (p-value = 0.013, log2-enrichment = 0.494) of ETV1 binding sites (Fig. 3a) in proximity to those genes (Fig. 3b, Supplementary Information S4). To demonstrate specificity, we included all available PWMs for members of the ETS family of transcription factors: ETV2‐ETV6. The only other member of the TF family for which transcription factor binding site enrichment was detected was ETV4, but the p-value was not significant. The complete results of the analysis are summarized in Supplementary Information S5. Another noteworthy gene, which we discovered in an eQTL study with SNPs and InDels [8], is dystrophin (DMD). The variant at position GGA1: 116,965,973, which is a biallelic intronic TR between exons 7 and 8, was associated with genome-wide significance to the DEGs LOC112531493 (p-value = 1.36 × 10−8), LOC422393 (p-value = 5.57 × 10−6), RASSF8 (p-value = 5.76 × 10−6), and LOC112533169 (p-value = 8.48 × 10−6). The 2 bp DMD intron deletion between exons 1 and 2 we discovered in our previous study (rs735635304, GGA1: 117,179,438) was associated with genome-wide significance to the DEGs LOC112532977 (p-value = 7.34 × 10−10) and LOC107049114 (p-value = 1.60 × 10−9). The two DMD variants are 213 kb apart and not in linkage disequilibrium (R2 = 0.022868).
Table 3
Large deletions (DEL) and tandem repeats (TR) that showed genome-wide significant association (p-values < 1.41 × 10−6) in at least 10 expression genome-wide association studies for genes that are differentially expressed in brains of high and low feather pecking chickens
Type
Size (bp)
Position
Effect
Gene(s)
No. of associations
TR
GGA25: 1,465,942
intergenic_region
ENSGALG00000055092–IQGAP3
53
TR
GGA25: 3,317,155
intron_variant
S100A16
47
TR
GGA17: 6,359,797
intergenic_region
ENSGALG00000035908–ENSGALG00000047338
39
DEL
294
GGA8: 3,889,333–3,889,627
intron_variant
RO60
30
TR
GGA2: 56,803,419
intron_variant
ATP9B
22
TR
GGA2: 56,944,739
intergenic_region
SALL3-ENSGALG00000054175
22
TR
GGA17: 5,355,006
upstream_gene_variant
SLC27A4
15
TR
GGA27: 3,942,783
intron_variant
GPATCH8
11
TR
GGA27: 3,976,916
frameshift_variant
GPATCH8
11
TR
GGA27: 3,977,142
intron_variant
GRN
11
TR
GGA27: 3,988,533
intergenic_region
TMEM98-ARHGAP27
11
TR
GGA27: 4,009,079
intron_variant
PLEKHM1
11
TR
GGA27: 4,022,832
intron_variant
ENSGALG00000047878
11
Discussion
Numerous studies have been conducted to unravel the genetics behind FP behavior in chickens, as reviewed in [12, 46], but none of these focused on the analysis of structural genetic variation. SVs contribute to complex traits to a higher degree than SNPs [21] and have been the focus of studies on neuropsychiatric disorders in recent years [17‐19]. Here, we combined data from two well-described experimental crosses, an F2 design and a HS population, and performed multiple GWAS and eQTL analyses on different classes of genetic variants. By applying conventional GWAS approaches on the two experimental populations with two sets of variant classes (SNPs and InDels as well as SVs and TRs) followed by meta-analysis, we identified strong associations with numerous putative candidate genes for both variant classes. GALNT1 catalyzes glycosylation of target proteins, and aberrant glycosylation of i.e. the GABAA receptor is a pathological hallmark in postmortem brains of patients suffering from schizophrenia [47]. FHOD3 controls dendritic spine morphology [48] and might contribute to FP behavior by causing aberrant neuronal development in the cerebral cortex. Similarly, KLHL29 has already been discussed to be involved in a neurodevelopmental disorder [49]. AFF3 is also a new promising candidate gene that was previously associated with intellectual disability and cellular migration in the cerebral cortex [50, 51]. Regarding its cellular function, PIEZO2 is of considerable interest. It is a mechanically activated cation channel, required for light/touch sensation and proprioception, and is abundantly expressed in dorsal root ganglion and sensory endings of proprioceptors in mice [52]. Since one of the major triggers of FP behavior is light stimulation [14, 53], genetic variation in PIEZO2 might lead to disturbed light perception in HFP.
However, the lead variant from these analyses is a TR 126 kb downstream of the GABRB3 gene, which explains a considerable amount of the observed phenotypic variance. But, since this variant was detected with a sample size of about 500 animals, its contribution to the phenotype should be interpreted with caution. According to the Beavis effect, the results of QTL studies with sample sizes of around 500 individuals are slightly overestimated [54]. Furthermore, miRNA gga-mir-187 is strongly associated with FP behavior in all conducted GWAS (Fig. 1b), and among predicted targets of this regulatory RNA are several GABA receptor genes. Based on whole-brain transcriptome analyses of the light response of HFP, we previously postulated that downregulation or missing upregulation of GABA receptor expression is caused by miRNA dysregulation due to low levels of Dicer1 expression in HFP brains [14]. The findings presented here provide further evidence for a disturbance in miRNA regulation and involvement of the GABAergic system in FP behavior. Since these chickens have been selected for FP behavior based on their estimated breeding values for multiple generations, it is possible that mutations leading to low expression of several GABA receptors have been accumulating. Previous studies with the same experimental population already pointed in that direction [2, 13]. But these are not only limited to GABA receptor genes, miRNAs, or genes encoding miRNA processing proteins. By conducting an eQTL analysis with SVs and TRs, we were able to confirm the involvement of DMD (dystrophin) in the regulation of genes that are differentially expressed between HFP and LFP. We already discovered DMD in an eQTL analysis with SNPs and InDels [8]. Since dystrophin is a direct regulator of GABA receptor clustering [55], this adds another layer to the GABA receptor disturbance in HFP brains. Several other genes that appear in the gene–gene interaction map based on significant AWM correlations (Fig. 2) have been connected to the GABAergic system. These include COQ4 [56], ETV1 [57], NEURL1 [58], SLC25A26, and SLC27A4 [59]. In this regard, ETV1 is of considerable interest since it is the only transcription factor that we identified in the AWM analysis with SVs and TRs. According to the Human Protein Atlas, ETV1 expression is specific to salivary glands and the brain, specifically the cerebellum and thalamus (https://www.proteinatlas.org/ENSG00000006468-ETV1; accessed August 2022). ETV1 is associated with the differential expression of 23 genes between HFP and LFP, and ETV1 binding sites are enriched in proximity to these genes (Fig. 3). Although genome-wide significance was closely missed for these associations, we strongly believe that the AWM approach by Reverter and Fortes [37] led to the discovery of a high-confidence set of variants for this complex trait. Among these putative ETV1 targets are CSF2RB, which has been associated with major depression and schizophrenia [60], and MIS18BP1, a gene implicated in the autism spectrum [61]. Furthermore, an ETV1 antibody coimmunoprecipitated the GABAA receptor α6 (GABAARα6) promoter region in mice [57], and ETV1 is a regulator of gene expression in CD4 and CD8 T cells [62]. Hence, ETV1 may not only impact GABA receptor expression but might also participate in neurodevelopment. As demonstrated by Pasciuto et al., a lack of CD4 T cells in the brain of mice leads to excess immature neuronal synapses and behavioral abnormalities. Among the top DEGs identified by single-cell RNAseq were the transcription factors Klf2 and Klf4 [16]. We previously postulated that FP behavior is the result of disturbances in embryonic neurodevelopment and identified KLF14 as a major regulator in this regard [8]. In that study, we also identified a Smad4 domain-containing transcription factor, namely ENSGALG00000042129 (CHTOP), but we were not able to detect the enrichment of binding sites for that transcription factor in proximity to its associated DEGs. However, in light of the fact that ETV1 forms functional complexes with Smad4 [63], we propose a model in which the transcription factors ETV1, CHTOP, and KLF14 have an additive effect on the density of T cells in the developing brain of HFP. This might lead to structural abnormalities in the brains of HFP and may explain, at least in part, their abnormal behavior. Multiple studies point toward a central role of Krüppel-Like factors in neurodevelopment and behavior [64‐68]. However, conclusive results on the function of Krüpple-like factors in the neurodevelopment in chickens have not been reported yet, and research is hampered by erroneous annotation of KLF orthologues [69].
Fig. 2
Gene–gene interaction map of significant correlations that were identified from an association weight matrix on expression genome-wide association studies with the PCIT algorithm
Fig. 3
a Graphical representation of the binding motif frequency matrix of the ETV1 transcription factor. b Result of transcription factor binding site enrichment analysis for ETV1 with genes from expression genome-wide associations studies with p-values < 1 × 10-4
×
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Apart from DMD, we also discovered the genes PPP1R9A, INPP4A, and COA5 in both eQTL-AWM analyses, one of which was performed with SVs and TRs (Fig. 2) and one with SNPs and InDels [8]. In schizophrenia and bipolar disorder, the prefrontal cortical expression of PPP1R9A was altered [70] and its gene product Neurabin regulates anxiety-like behavior in adult mice [71]. INPP4A has been implicated in multiple neurological conditions, namely schizophrenia, autism, epilepsy, and intellectual disability [72‐74]. An additional link to schizophrenia is a frameshift variant in the GPATCH8 gene in exon 9 of transcript GPATCH8-201 (Table 3), an ortholog of ZNF804A, which has been shown to impact various mental illnesses via pre-mRNA processing [75]. Multiple TRs and one large deletion in RO60 showed genome-wide association with at least 30 DEGs between HFP and LFP (Table 3). With 53 genome-wide associations, an intergenic TR seemed to have an impact on more than half of the top DEGs between HFP and LFP whole brains. The variant is located 2.7 kb downstream of the E3 ubiquitin ligase ENSGALG00000055092 (DCAF11) and 7.5 kb upstream of IQGAP3, which is required for proper cell cycle progression [76] and which influences immune cell infiltration and immune modulators [77]. A 294-bp intron deletion in RO60, a gene involved in the regulation of inflammatory gene expression [78], was associated with 30 DEGs, which might be responsible for the observation that 48.1% of DEGs between HFP and LFP belong to the PANTHER protein class defense/immunity (PC00090) [1]. A TR within an S100A16 intron yielded 47 significantly associated DEGs, which is also a gene related to the immune system [79]. An intergenic TR with 39 genome-wide associated DEGs was located between the two lncRNAs ENSGALG00000035908 and ENSGALG00000047338. They probably belong to the class of psychiatric ncRNAs, a term scored by Gandal et al. [80].
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We discovered multiple links between FP and human psychiatric disorders in our previous studies [1, 2, 14]. The fact that methylation of KLF14 correlates with psychosis severity in schizophrenia patients [81] strengthens our argument for the applicability of HFP chickens in the research of the basic mechanisms involved in human psychiatric disorders. Furthermore, by conducting multiple GWAS on two experimental populations of FP chickens with different variant classes with whole-genome marker density, we identified numerous genes that have previously been linked to psychiatric disorders or other neurological conditions or phenotypes (Table 4). These genes coherently fit the mechanistic hypotheses raised so far and, at the same time, underpin the complex nature of this trait. Among these are anxiety, depression, intellectual disability, autism, compulsive behavior, drug addiction, bipolar disorder, and schizophrenia. This raises the question of what common mechanisms these conditions share, which is a current matter of debate. Blokhin et al. proposed that future treatment of human psychiatric disorders should be tailored under the use of a genome-wide “targetome” [82], and Johnsson et al. discovered multiple genetic overlaps between anxiety behavior in chickens and numerous human psychiatric disorders [83]. The strong genetic burden of HFP chickens with mutations affecting neuropsychiatric genes makes this line of chickens a valid model system to study the effects of tailored drug treatment strategies.
Table 4
Summary of published data related to neurological disorders on the 20 highest associated genes from each genome wide association study on feather pecking behavior in laying hens
Associated with bipolar disorder [104] and involvement in the cell-surface expression of neuronal nicotinic receptors [105]
Acknowledgements
We acknowledge support from the Open Access Publication Funds of Göttingen University. We further thank the Gesellschaft für wissenschaftliche Datenverarbeitung mbH Göttingen (GWDG) for the access to their high-performance computing cluster.
Declarations
Ethics approval
The research protocol was approved by the German Ethical Commission of Animal Welfare of the Provincial Government of Baden-Wuerttemberg, Germany (code: HOH 35/15 PG, date of approval: April 25, 2017).
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Not applicable.
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Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
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