Genome-wide association study and subsequent functional analysis reveal regulatory mechanism underlying piglet diarrhea

Abstract

Objective: Piglet diarrhea poses a serious threat to piglet health and the livestock economy, and is one of the most pressing problems in animal husbandry. This study aims to investigate the genetic factors involved in piglet diarrhea and to identify key genes that regulate this condition.

Methods: We screened 600 diarrheal piglets based on unique diarrhea scores for resequencing and conducted a genome-wide association study (GWAS). Through this process, we identified 308 single nucleotide polymorphisms (SNPs) and annotated 151 candidate genes. Extensive functional validation and systematic analysis were performed on key candidate genes KSR1, SKAP1, SLC35F6, and OR12.

Results: The study found that the four key genes were involved in the regulation of piglet diarrhea through various mechanisms. OR12 affects the levels of ZO-1 and claudin-1. Changes in the expression levels of KSR1 could alter the expression of IL1-β, IL6, and TNF-α, as well as cell migration and proliferation. SKAP1 could affect the expression of CD3 and CD4, and influence the migration and proliferation ability of cells. SLC35F6 is involved in cell apoptosis through the Bcl2/BAX/caspase3 pathway and can also affect mitochondrial membrane potential.

Conclusion: The results of this study provide strong support for breeding programs aimed at disease resistance and offer potential solutions to the problem of piglet diarrhea.

Keywords: Diarrhea; Genome-Wide Association Study (GWAS); Immunity; Inflammatory; Pig.

Figure 1

Diarrhea grade and description in piglets.

Figure 2

GWAS for piglet diarrhea. (A) The Manhattan plot for the diarrhea piglets in GWAS. (B) The QQ plots of diarrhea piglets’ trait in GWAS. (C) GO enrichment analysis of candidate genes. (D) KEGG enrichment analysis of candidate genes. GO, gene ontology; GWAS, genome-wide association study; KEGG, Kyoto encyclopedia of genes and genomes; QQ, quantile-quantile.

Figure 3

Intestinal changes in piglets with diarrhea. (A) Hematoxylin and eosin (H&E) were demonstrated in six intestinal analyses of diarrhea piglets and normal piglets, including duodenum⊠ jejunum, ileum, colon, caecum, rectum. (B) KSR1, SKAP1, SLC35F6, OR12 mRNA expression in diarrhea piglets and normal piglets. NC, negative control. * p<0.05, ** p<0.01, *** p<0.001.

Figure 4

Effects of knockdown and overexpression of KSR1 on inflammatory, proliferation and apoptosis in IPEC-J2 cells. (A) The mRNA expression of KSR1 after LPS (10 μg/mL, 20 μg/mL) treatment of IPEC-J2. (B) The mRNA expression of KSR1-OEand KSR1-siRNA. (C) The mRNA expression of TNF-α, IL1-β and IL6 in IPEC-J2 cells by KSR1 overexpression and knockdown. (D) EdU results of KSR1-OE, KSR1-siRNA and NC at 24h in IPEC-J2 cells. EdU marks the proliferating cells, Hoechest represents the nucleus, and Merge represents an overlay of EdU and Hoechest. (E) Scratch assays and quantitative analysis were performed to detect the migration of IPEC-J2 cells after treatment with KSR1 knockdown and overexpression for 24 h. NC, negative control; LPS, lipopolysaccharide; IPEC, intestinal porcine epithelial cells; EdU, 5-ethynyl-2′-deoxyuridine; TNF, tumor necrosis factor; IL, interleukin. * p<0.05, ** p<0.01, *** p<0.001.

Figure 5

Effects of knockdown and overexpression of SKAP1 on immunization, proliferation and apoptosis in IPEC-J2 cells. (A) The mRNA expression of KSR1 after M1 macrophage supernatant for 24 hours in IPEC-J2 cells. (B) The mRNA expression of SKAP1-OE and SKAP1-siRNA. (C) The mRNA expression of SKAP1-OE and SKAP1-siRNA. (C) The mRNA expression of CD3 and CD4 in IPEC-J2 cells by SKAP1 overexpression and knockdown. (D) EdU results of SKAP1-OE, SKAP1-siRNA and NC at 24h in IPEC-J2 cells. EdU marks the proliferating cells, Hoechest represents the nucleus, and Merge represents an overlay of EdU and Hoechest. (E) Scratch assays and quantitative analysis were performed to detect the migration of IPEC-J2 cells after treatment with SKAP1 knockdown and overexpression for 24 h. NC, negative control; DAPI, 4′,6-diamidino-2-phenylindole; EdU, 5-ethynyl-2′-deoxyuridine; IPEC, intestinal porcine epithelial cells. * p<0.05, ** p<0.01, *** p<0.001.

Figure 6

Effects of knockdown and overexpression SLC35F6 on apoptosis in IPEC-J2 cells. (A) The mRNA expression of SLC35F6-OE and SLC35F6-siRNA. (B) The mRNA expression of Bcl2, BAX and caspase3 in IPEC-J2 cells by SLC35F6 overexpression and knockdown. (C) Apoptosis was verified through flow cytometry with SLC35F6-OE and SLC35F6-siRNA treatment. (D) MitoTracker Red with SLC35F6-OE and SLC35F6-siRNA treatments. The ROS level was measured using MitoTracker Red, a reduced mitochondrial dye that does not fluoresce until it is oxidized by ROS. (E) Detection of SLC35F6 knockdown or overexpression of ROS using the fluorescent probe DCFH-DA. NC, negative control; ROS, reactive oxygen species; IPEC, intestinal porcine epithelial cells; DCFH-DA, dichloro-dihydro-fluorescein diacetate. ** p<0.01, *** p<0.001.

Figure 7

Effects of knockdown and overexpression OR12 on tight junctions in IPEC-J2 cells. (A) The mRNA expression of SLC35F6-OE and SLC35F6-siRNA. (B) The mRNA expression of Claudin-1 and ZO-1 in IPEC-J2 cells by SLC35F6 overexpression and knockdown. (C) Differential Claudin-1 protein expression after KSR1 overexpression and knockdown in IPEC-J2 cells (100×). (D) Differential ZO-1 protein expression after KSR1 overexpression and knockdown in IPEC-J2 cells (200×). C, negative control; ROS, reactive oxygen species; DAPI, 4′,6-diamidino-2-phenylindole; IPEC, intestinal porcine epithelial cells. *** p<0.001.

Figure 8

Integrated regulation of piglet diarrhea.