Gene function & annotation

Integrative analysis of splice-site variants and transcript isoforms expression underlying resistance to gastrointestinal nematode infections in sheep

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Abstract

Gastrointestinal nematode (GIN) infections impose substantial economic losses on global sheep production by compromising animal health, welfare, and productivity, emphasizing the need for sustainable genetic strategies to enhance resistance. Alternative splicing (AS) is a key post-transcriptional mechanism that generates multiple mRNA isoforms and modulates gene expression, thereby influencing host-pathogen interactions and immune responses. However, its role in GIN resistance remains poorly understood. This study aimed to characterize functional single-nucleotide polymorphisms (SNPs) affecting AS, particularly splice-site effects (SSEs), and to identify differentially expressed (DE) mRNA isoforms (DEIs) associated with resistance to GIN infections using RNA-Sequencing (RNA-Seq) data. Publicly available RNA-Seq data (GEO Accession ID: GSE63547) from abomasum lymph node tissues of resistant (R; n=10) and susceptible (S; n=10) Scottish Blackface lambs, pre-selected based on the fecal egg counts and experimentally challenged with Teladorsagia circumcincta L3 larvae, were analyzed. Quality control and sequence alignment to the Oar rambouillet v.3 reference genome were performed using CLC Genomics Workbench v24.0. SNPs located within canonical splice donor and acceptor regions were identified using RNA-Seq reads using the fixed ploidy variant detection tool. Variants uniquely fixed in the R and S groups were retained for downstream analysis. Their potential impact on splicing was predicted using Ensembl VEP, and pathway enrichment was assessed using Reactome. DEIs were identified using the large-gap read mapping tool in CLC Genomics Workbench v24.0, and functional enrichment analysis of DEIs was also performed using Reactome. In total, 450 and 442 SNPs with predicted SSEs were uniquely identified in R and S groups, respectively. Metabolic pathways linked to immune regulation, mitochondrial fatty acid β-oxidation, and transcriptional control were enriched in R sheep, whereas pathways associated with tissue remodelling, platelet activation, and stress-related immune responses predominated in S sheep (p< 0.05). Complementary transcriptome analysis comparing R and S groups at 7 and 14 days post-infection (dpi) identified 240 and 216 DEIs, respectively (FDR < 0.01; |FC| > 2). R sheep exhibited upregulation of immune and inflammatory pathways at 7 dpi, while S sheep showed enrichment of pathways linked to immune dysregulation, tissue remodeling, and stress responses at 14 dpi. Integration of mRNA isoforms and SNP data revealed overlaps between DEIs and splice-site SNPs, many of which were novel transcripts. Functionally relevant candidates included FERMT3 and NCOA7, which are involved in leukocyte adhesion and the oxidative stress response, respectively, alongside TEP1 (telomerase function and cellular maintenance), and SENP6 (protein desumoylation and chromatin organization). Collectively, these findings suggest that splice-site polymorphisms may influence immune-cell adhesion, transcriptional and post-transcriptional regulation, and stress-response pathways. These findings provide novel insights into the genetic basis of GIN resistance and highlight potential functional candidate markers for genomic selection.

Keywords: 2026

How to Cite:

Sinhalage, K., Karrow, N., Asselstine, V., Schenkel, F. & Cánovas, Á., (2026) “Integrative analysis of splice-site variants and transcript isoforms expression underlying resistance to gastrointestinal nematode infections in sheep”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2287338. doi: https://doi.org/10.31274/wcgalp.24269

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