Tissue-specific and functional loci analysis of CASP14 gene in the sheep horn

Abstract

Under the current context of intensive farming, small-horned animals are more suitable for large-scale breeding. The CASP14 gene, closely associated with skin and keratin formation, may influence horn size due to its link with skin development. This study comprehensively analyzed the tissue-specific expression of CASP14 using RNA-Seq data, identified functional sites through whole-genome sequencing (WGS), and investigated allele-specific expression (ASE) validated by KASP assays. Results showed significantly higher CASP14 expression in the scurred group com-pared to the SHE group, with pronounced expression in the skin. Interbreed comparisons also revealed elevated CASP14 levels in the scurred group. Analysis of potential functional sites indicated structural similarities in the CASP14 protein among horned mammals. Five ASE events were discovered, and intersecting these with SNPs and high fixation index loci (Fst > 0.05) identified three potential functional sites: 7941628, 7941817, and 7941830. The SNP site 7944295 was selected for T-test analysis and further validated through KASP assays, corroborating the role of CASP14 in sheep horn phenotypes. Our findings suggest that CASP14 plays a significant role in horn development, offering insights into breeding strategies for small-horned animals.

Fig 1. Expression of CASP14 gene in scurred and SHE groups.

(A) Differential expression of CASP14 gene between scurred and SHE Groups. (B) Expression of CASP14 exons in scurred and SHE groups. The expression refers to the estimated values derived from the fitted expressions in the GLM regression, where E001 to E007 represent the number of exons. (C) Differential expression of CASP14 in horn and other tissues.

Fig 2. Expression differences of CASP14 gene.

(A) Differential expression of CASP14 gene in four species. (B) Sex differences in CASP14 gene expression across various sheep tissues. (C) Ex-pression of CASP14 gene in various sheep breeds.

Fig 3. Structural and evolutionary insights of the CASP14 gene.

(A) Evolutionary tree of the CASP14 gene. (B) Three-Dimensional (3D) structure of sheep CASP14 protein. (C) Amino acid sites of CASP14 gene unique to horned animals.

Fig 4. Allele count of 5 ASEs differing between scurred and SHE groups.

Fig 5. Genetic variation and population structure of the CASP14 gene.

(A) Three-dimensional (3D) principal component analysis (PCA) of the CASP14 gene. Each point represents a sheep breed, which is categorized into scurred and SHE types based on breed information. (B) F-statistics (Fst) of functional loci of the CASP14 gene.

Fig 6. Genomic insights into the CASP14 gene.

(A) Intersections of ASEs, SNPs and Fst. (B) LD heatmap of CASP14 gene. Darker colors represent higher LD values, and black triangles denote LD blocks, which are collections of SNPs with higher LD values.

Fig 7. Phenotypic and genotypic analysis of the CASP14 gene.

(A) Box plot of individual horn lengths for different genotypes. Calculating the p-value based on the dominant genetic model and t-test. (B) The KASP assay results for the CASP14 gene.