DNA methylation patterns and gene expression associated with litter size in Berkshire pig placenta

<div><p>Increasing litter size is of great interest to the pig industry. DNA methylation is an important epigenetic modification that regulates gene expression, resulting in livestock phenotypes such as disease resistance, milk production, and reproduction. We classified Berkshire pigs into two groups according to litter size and estimated breeding value: smaller (SLG) and larger (LLG) litter size groups. Genome-wide DNA methylation and gene expression were analyzed using placenta genomic DNA and RNA to identify differentially methylated regions (DMRs) and differentially expressed genes (DEGs) associated with litter size. The methylation levels of CpG dinucleotides in different genomic regions were noticeably different between the groups, while global methylation pattern was similar, and excluding intergenic regions they were found the most frequently in gene body regions. Next, we analyzed RNA-Seq data to identify DEGs between the SLG and LLG groups. A total of 1591 DEGs were identified: 567 were downregulated and 1024 were upregulated in LLG compared to SLG. To identify genes that simultaneously exhibited changes in DNA methylation and mRNA expression, we integrated and analyzed the data from bisulfite-Seq and RNA-Seq. Nine DEGs positioned in DMRs were found. The expression of only three of these genes (<i>PRKG2</i>, <i>CLCA4</i>, and <i>PCK1</i>) was verified by RT-qPCR. Furthermore, we observed the same methylation patterns in blood samples as in the placental tissues by PCR-based methylation analysis. Together, these results provide useful data regarding potential epigenetic markers for selecting hyperprolific sows.</p></div

Distribution of CpG methylation levels in CpG island, HCP, ICP, and LCP of each group.

<p>The percent methylation for each CpG site is shown in the x-axis, and the y-axis shows the number of CpG sites (x10²).</p

Genome-wide methylation levels in the two groups.

<p>Genome-wide methylation levels in the two groups.</p

GO enrichment analysis.

<p>GO enrichment analysis was performed for the upregulated (A) and downregulated (B) DEGs. The <i>x</i> axis indicates the significance of DEGs as −log (<i>p</i> value). The number of genes in each category is shown on the <i>y</i> axis, as indicated in parentheses.</p

Distribution of DMRs for different genomic regions in LLG compared to SLG.

<p>The different genomic regions are shown in the x-axis, and the y-axis shows the number of DMRs.</p

RT-qPCR analysis of <i>EGR2</i>, <i>PHEROC</i> and <i>LIPG</i> in placental tissue.

<p>Three different placentas from LLG and SLG were prepared immediately following delivery and subjected to RT-qPCR analysis. The fold change of validation (upper panel) and RNA-Seq (lower panel) for the (A) <i>EGR2</i>, (B) <i>PHEROC</i> and (C) <i>LIPG</i> genes are presented. The RT-qPCR data were analyzed by relative quantification using 2^-ΔΔCt. Data are expressed as means ± standard deviation (S.D.). Each experiment was performed in triplicate. *, <i>p</i> < 0.05 versus SLG.</p

RT-qPCR analysis to assess <i>TCF12</i>, <i>CTNNB1</i>, <i>WNT11</i>, <i>WNT9B</i> and <i>IL-6</i> mRNA expression.

<p>RNAs from three different individual placentas each were prepared from LLG and SLG. RT-qPCR was performed using gene-specific primers. <i>PPIA</i> was used as a universal control. The RT-qPCR data were analyzed by relative quantification using 2^-ΔΔCt. Data are expressed as means ± standard deviation (S.D.). Each experiment was performed in triplicate. *, <i>p</i> < 0.05 versus SLG.</p

Primer sequences used in RT-qPCR.

<p>Primer sequences used in RT-qPCR.</p

List of DEGs positioned in DMRs by the litter size.

<p>List of DEGs positioned in DMRs by the litter size.</p

Distribution of DEGs.

<p>(A) The dot graph shows each DEG between the LLG and SLG. log₂-fold change between fragments per kilobase of exon per million reads mapped (FPKM) in the SLG and LLG according to the Cufflinks package are presented along the <i>x</i> axis. Red dots represent significantly altered DEGs between the LLG and SLG (<i>p</i> < 0.05, fold change > 1.5, the decimal system). The <i>y</i> axis indicates significance as -log (<i>p</i> value). (B) The number of downregulated and upregulated DEGs in the LLG compared with the SLG.</p