An Epigenome-Wide DNA Methylation Map of Testis in Pigs for Study of Complex Traits

Abstract

Epigenetic changes are important for understanding complex trait variation and inheritance in pigs that are also a valuable biomedical model for human health research. Testis is the main organ for reproduction and boar taint in pigs; however, there have been no studies to-date on adult pig testis epigenome. The main objective of this study was to establish a genome-wide DNA methylation map of pig testis that would help identify candidate epigenetic biomarkers and methylated genes for complex traits such as male reproduction, fertility or boar taint. Reduced Representation Bisulfite Sequencing (RRBS) was used to study methylation levels of cytosine in nine pig testis samples. The results showed that genome-wide methylation status of nine samples overlapped greatly and their variation among pigs were low. The methylation levels of promoter, exon, intron, cytosine and guanine dinucleotide (CpG) islands and CpG island shores regions were 0.15, 0.47, 0.55, 0.39, and 0.53, respectively. Cytosines binding to CpG islands showed different methylation levels between exon and intron regions. All methylation levels of CpG islands were lower than CpG island shores in different genic features. The distribution of 12,738 differentially methylated cytosines (DMCs) within CpG islands, CpG island shores and other regions was 36.86, 21.65, and 41.49%, respectively, and was 0.33, 1.71, 5.95, and 92.01% in promoter, exon, intron and intergenic regions, respectively. Methylation levels of DMCs in promoter, exon and intron regions were significantly different between CpG islands and CpG island shores ( P < 0.05). A total of 898 genes with 2089 DMCs were enriched in 112 Gene Ontology (GO) terms. Fifteen methylated genes from our study were associated with fertility or boar taint traits. Our analysis revealed the methylation patterns in different genic features and CpG island regions of testis in pigs, and summarized several candidate genes associated with DMCs and the involved GO terms. These findings are helpful to understand the relationship between DNA methylation and genic CpG islands, to provide candidate epigenetic regions or biomarkers for pig production and welfare and for translational epigenomic studies that use pigs as an animal model for human research.

Characterization of Global DNA Methylation in Different Gene Regions Reveals Candidate Biomarkers in Pigs with High and Low Levels of Boar Taint

“…Only cytosines in the cytosine and guanine dinucleotide (CpG) sites were used in our study. Quality control (i.e., removal of adapter and short reads) and alignment of RRBS reads were performed using Trimmomatic software [ 30 ] and Bismark software [ 31 ], respectively, based on the same parameters used in our previous study [ 28 , 29 ]. Furthermore, the unqualified read coverages of cytosines (i.e., ≤10 counts and ≥99.9th percentile) were also trimmed following previous studies [ 28 , 29 , 32 , 33 ].…”

“…Quality control (i.e., removal of adapter and short reads) and alignment of RRBS reads were performed using Trimmomatic software [ 30 ] and Bismark software [ 31 ], respectively, based on the same parameters used in our previous study [ 28 , 29 ]. Furthermore, the unqualified read coverages of cytosines (i.e., ≤10 counts and ≥99.9th percentile) were also trimmed following previous studies [ 28 , 29 , 32 , 33 ]. Based on the publicly available information of experimental animals published in our previous study [ 28 , 34 ], nine testis tissue samples from three BT groups (i.e., low, medium and high BT groups) of pigs were used in this study, so each BT group had three pigs as replicates.…”

“…We have previously published descriptions of the epigenetic characteristics of the porcine genome and its relationship with boar taint level [ 28 , 29 ]. However, a detailed DNA-methylation characterization of different gene regions was not reported in this previous study, despite the important relation of different levels of differentially methylated regions (DMRs) in gene components (e.g., promoters, exons and introns) to BT level.…”

GeneDMRs: An R Package for Gene-Based Differentially Methylated Regions Analysis

“…When a polymerase chain reaction experiment suffers from duplication bias, some clonal reads will impair accurate determination of methylation (Akalin et al, 2012). In addition, lower read coverages (e.g., lower than 10) will cause the biases for methylation percentage calculation (Wang and Kadarmideen, 2019b). Therefore, cytosines with a percentile of read coverage higher than the 99.9th and read coverages lower than 10 were discarded for the qualified reads by Methfile_QC(high_quantile = 99.9, low_ coveragenum = 10).…”

A look into the use of Raman spectroscopy for brain and breast cancer diagnostics: linear and non-linear optics in cancer research as a gateway to tumor cell identity

“…Now is just the beginning of our understanding of epigenetic factors involved in the development of cancer diseases, but it has become evident that cancer cells have many other epigenetic differences including DNA methylation. DNA methylation is constantly the best-studied epigenetic mechanism [96,97]. Abnormal methylation, that provides growth advantages, is directly connected to cancer development.…”