219 Identification of Functional Single Nucleotide Polymorphisms Responsible for Alternative Splicing Affecting Gastrointestinal Nematodes Resistance in Grazing Sheep
Authors: Cunha Samla M F , Willoughby Olivia B , Schenkel Flavio S , Mallard Bonnie , Karrow Niel A , Cánovas Angela 
Published: November 2023

Abstract
Sheep infected with gastrointestinal nematodes (GIN) can show weight loss, anemia, diarrhea, decrease appetite, and hypoproteinemia, which can sometimes lead to death. These signs of infection decrease productivity and cause economic losses. Economic losses can also be associated with treatment without efficacy due to GIN resistance to most anthelmintic classes. Combined approaches need to be used to reduce the reliance on anthelmintic as the only control method to GIN. The aim of this study is the identification of potential functional single nucleotide polymorphisms (SNPs) responsible for alternate splicing in Rideau X Dorset crossbred sheep with different immune profiles using RNA-Sequencing (RNA-Seq). Animals were exposed to GIN while grazing, after that those with the greatest and least gastrointestinal tract parasite burdens were selected for liver RNA extracting. These animals can be separated into groups of high (H; n = 5) and medium (M; n = 6) innate immune responses. In addition, liver RNA was extracted for GIN-unexposed control lambs (U; n = 4). RNA-Seq analysis was performed using the CLC genomics workbench software, version 20.0.4 (CLC Bio, Aarhus, Denmark). Quality control analyses, including guanine-cytosine (GC) content, ambiguous base content, Phred score, base coverage, nucleotide contributions, and over-represented sequences parameters, were performed on fastq files. Sequence reads were aligned to the annotated Oar_rambouillet_v.1 (release 109) ovine reference genome. After filtering steps, mRNA isoforms with multiple (³ 2) expressed mRNA isoforms with at least one mRNA isoform differently expressed (DE) between H- and M-innate immune response and GIN-unexposed control groups (FDR &lt; 0.05 and |FC| &gt; 2) were keep. A total of 1, 75, and 58 DE mRNA isoforms were identified for M versus H, U versus M, and U versus H comparisons, respectively. Gene ontology (GO) analysis including the biological process, molecular function, and cellular component GO categories and metabolic pathways analysis were performed using STRING-db software. Enriched biological processes and metabolic pathways related to the immune system, as well as cholesterol and other sterol metabolism (e.g., sterol biosynthesis, steroid metabolism, cholesterol biosynthesis, cholesterol metabolism, ascorbate and aldarate metabolism, drug metabolism, and terpenoid biosynthesis) were identified (FDR &lt; 0.05). Next, the list of DE mRNA isoforms identified will be deeply studied by performing SNP discovery analysis to validate if specific functional SNPs are responsible for the alternative splicing affecting GIN resistance in grazing sheep. The complete results will help to better understand the biological mechanisms that may be regulating the immune response to GIN. Candidate functional SNPs have the potential to be included in a commercial SNP panel for selection of more resistant animals to improve GIN resistance in sheep flocks.