Integrative Analysis Between Genome-Wide Association Study and Expression Quantitative Trait Loci Reveals Bovine Muscle Gene Expression Regulatory Polymorphisms Associated With Intramuscular Fat and Backfat Thickness

Abstract

Understanding the architecture of gene expression is fundamental to unravel the molecular mechanisms regulating complex traits in bovine, such as intramuscular fat content (IMF) and backfat thickness (BFT). These traits are economically important for the beef industry since they affect carcass and meat quality. Our main goal was to identify gene expression regulatory polymorphisms within genomic regions (QTL) associated with IMF and BFT in Nellore cattle. For that, we used RNA-Seq data from 193 Nellore steers to perform SNP calling analysis. Then, we combined the RNA-Seq SNP and a high-density SNP panel to obtain a new dataset for further genome-wide association analysis (GWAS), totaling 534,928 SNPs. GWAS was performed using the Bayes B model. Twenty-one relevant QTL were associated with our target traits. The expression quantitative trait loci (eQTL) analysis was performed using Matrix eQTL with the complete SNP dataset and 12,991 genes, revealing a total of 71,033 cis and 36,497 trans-eQTL (FDR < 0.05). Intersecting with QTL for IMF, we found 231 eQTL regulating the expression levels of 117 genes. Within those eQTL, three predicted deleterious SNPs were identified. We also identified 109 eQTL associated with BFT and affecting the expression of 54 genes. This study revealed genomic regions and regulatory SNPs associated with fat deposition in Nellore cattle. We highlight the transcription factors FOXP4, FOXO3, ZSCAN2, and EBF4, involved in lipid metabolism-related pathways. These results helped us to improve our knowledge about the genetic architecture behind important traits in cattle.

Keywords: Nellore cattle; RNA-Seq; SNP; backfat thickness; carcass and meat quality; expression quantitative trait loci; intramuscular fat content.

FIGURE 1

Manhattan plot of the posterior means of the percentage of genetic variance explained by each 1 Mb SNP window across the 29 autosomal chromosomes for intramuscular fat content (IMF) (A) and backfat thickness (BFT) (B). The X-axis represents the chromosomes, and the Y-axis, the percentage of genetic variance explained by each SNP window. Red dashed lines delimit the relevant QTL regions.

FIGURE 2

Scatter plot of the affected genes and eQTL (FDR < 0.05). The Y-axis represents gene order in relation to chromosome position in the Bos taurus genome, and X-axis represents the SNP order in relation to chromosome position in the Bos taurus genome. Points scattered diagonally indicates cis-eQTL. Points scattered vertically indicate trans-eQTL. The vertical blue lines denote individual autosomal chromosomes.

FIGURE 3

SNP-gene regulation networks representing the eQTL variants located within QTL windows associated with intramuscular fat content (IMF) and the genes regulated by them, focusing on the variants regulating transcription factors (TFs) and their direct connections. The colors are coded by QTL window: (A) BTA21_22, eQTL represented in orange; (B) BTA23_15, eQTL represented in lightgreen; (C) BTA7_2, eQTL represented in pink. All the formats are described in the legend. New variants are represented by chromosome: position. Gray lines represent a positive beta-value and red ones represent a negative beta-value.

FIGURE 4

SNP-gene regulation networks representing the eQTL variants located within QTL windows associated with backfat thickness (BFT) and the genes regulated by them, focusing on the variants regulating transcription factors (TFs) and their direct connections. The colors are coded by QTL window: (A) BTA21_23, eQTL represented in blue; (B) BTA13_52, eQTL represented in green. All the formats are described in the legend. Gray lines represent a positive beta-value and red ones represent a negative beta-value.

FIGURE 5

Top 10 pathway maps enriched for the genes regulated and containing the eQTL encompassing relevant QTL regions associated with intramuscular fat content (IMF) (A) and backfat thickness (BFT) (B) in a Nellore cattle population.