Genome-Wide DNA Methylation Analysis and Functional Validation of Litter Size Traits in Jining Grey Goats

Abstract

DNA methylation (DNAm) is associated with the reproductive system. However, the genetic mechanism through which DNAm regulates gene expression and thus affects litter size in goats is unclear. Therefore, in the present work, genome-wide DNAm profiles of HP and LP Jining Grey goat ovary tissues were comprehensively analyzed via WGBS, and RNA-Seq data were combined to identify candidate genes associated with litter size traits in the Jining Grey goat. Finally, BSP and RT-qPCR were used to verify the sequencing results of the key genes. Notably, the DNMT genes were downregulated at the expression level in the HP group. Both groups exhibited comparable levels of methylation. A total of 976 differentially methylated regions (DMRs) (973 DMRs for CG and 3 DMRs for CHG) and 310 differentially methylated genes (DMGs) were identified in this study. Through integration of WGBS and RNA-Seq data, we identified 59 differentially methylated and differentially expressed genes (DEGs) and ultimately screened 8 key DMGs (9 DMRS) associated with litter size traits in Jining Grey goats (SERPINB2: chr24_62258801_62259000, NDRG4: chr18_27599201_27599400, CFAP43: chr26_27046601_27046800, LRP1B. chr2_79720201_79720400, EPHA6: chr1_40088601_40088800, TTC29: chr17_59385801_59386000, PDE11A: chr2_117418601_117418800 and PGF: chr10_ 16913801_16914000 and chr10_16916401_16916600). In summary, our research comprehensively analyzed the genome-wide DNAm profiles of HP and LP Jining Grey goat ovary tissues. The data findings suggest that DNAm in goat ovaries may play an important role in determining litter size.

Keywords: DNA methylation; gene expression; goat ovary; prolificacy.

Figure 1

Genome-wide DNAm profiles of HP and LP Jining Grey goat ovaries. (A) Experimental design. (B) Mean proportion of diverse methylation contexts in the ovaries of Jining Grey goats. Blue, yellow and green represent methylated mCG, mCHH and mCHG, respectively. (C) Methylation levels in different genomic elements. Horizontal coordinates represent genomic elements, and vertical coordinates represent methylation levels; the value is the average methylation level within a gene element, with different colors representing different groups. (D) Distribution of DNAm level at 3K upstream/downstream. Horizontal coordinates show diverse regions, vertical coordinates represent methylation levels, with different colors representing different samples. Genebody: the active coding region of a gene from the beginning to the end of active coding (includes the exon and intron of the gene); UP3K: refers to 3000 bp before starting from the transcription start site (TSS); DOWN3k:refers to the transcription termination site (TTS) after 3000 BP.

Figure 2

Identification of DMRs and DMGs. (A) Number of DMRs distributed in mCG, and mCHG, and number of hypo and hyper DMRs in CG. (B) Number of DMG distributions in mCG, and mCHG, and number of hypo and hyper DMG in CG. (C) Distribution of DMR methylation levels in CG. The horizontal axis indicates HP and LP groups, and the vertical coordinate indicates methylation level values. (D) DMR anchor region in CG. On the x-axis, each region’s type is indicated, while the number of hyper/hypo DMRs in each region is represented on the y-axis.

Figure 3

DMG functional enrichment analysis. (A) GO. horizontal coordinate represents −lg (p value), and vertical coordinate represents the corresponding GO terms. (B) KEGG: vertical and horizontal coordinate indicate enriched pathway and the corresponding pathway, respectively. The size of the dots indicates the amount of DMG contained in each pathway, and the color of the dots reflects the −log10 (p-value) of each pathway.

Figure 4

Combined DNAm and transcriptome analysis of HP and LP Jining Grey goat ovaries. (A) Venn diagram represents the intersection of DMG and DEG in WGBS and RNA-Seq; red represents DMG; and blue represents DEG. (B) DMG-DEG divided into groups according to hyper and hypo; vertical coordinate indicates the number of DMG-DEG in each group.

Figure 5

Key candidate gene validation. Methylation levels of (A) NDRG4, (B) SERPINB2, (C) EPHA6, and (D) LRP1B genes were validated by bisulfite sequencing PCR (BSP). Each circle represents a CpG dinucleotide, and black and white denote methylated and unmethylated sites, respectively. RT-qPCR was utilized to examine the mRNA expression of the (E) DNMTs in the ovaries and the (F) eight relative mRNA expressions of key candidate genes; mean ± SEM of 3 biological replicates of 2^−ΔΔCt value.