Genome-wide identification of methylation markers associated with Streptococcus agalactiae resistance in cfDNA of GIFT tilapia.

Traditional evaluation of DNA methylation in fish using invasive tissue biopsy limits its application for in vivo monitoring and genetic selection for resistant individuals. As a non-invasive approach, plasma circulating cell-free DNA (cfDNA) testing has become a prospective strategy for studying individual epigenetic states and immune responses recently. In this study, the genome-wide methylation profiles in cfDNA by whole-genome bisulfite sequencing (WGBS) were evaluated for the GIFT strain of Nile tilapia (Oreochromis niloticus) following Streptococcus agalactiae infection. The cfDNA concentration in the susceptible group (2.72 ± 1.46 ng/μL) was significantly higher than that in the control (1.12 ± 0.38 ng/μL) and resistant group (1.23 ± 0.72 ng/μL). A total of 3076 differentially methylated regions (DMRs) were identified between resistant and susceptible groups, and the DMR-associated genes were significantly enriched in pathways related to transcriptional regulation, immune response, and energy metabolism. By integrating published spleen methylation data and conducting RNA-seq in spleen, we further explored the potential tissue origin of cfDNA methylation markers. Approximately 7.6% of cfDNA DMRs (234) overlapped with the spleen DMRs, and 111 overlapping DMRs with consistent methylation direction showed a strong correlation between the cfDNA and spleen datasets (R = 0.928, p < 1.31e - 48). Promoter methylation levels in cfDNA were negatively correlated with gene expression in spleen transcriptome data. 100 differentially expressed genes (DEGs) that were detected in spleen data were also observed in the cfDNA gene dataset with differentially methylated promoters (DMPs). BS-PCR and qRT-PCR analyses confirmed significant differences in methylation and expression of candidate genes b4galt1l, pigr and adsl across four immune-related tissues (spleen, head kidney, liver, and hindgut) between the control and infected groups. In summary, this study provides a valuable epigenetic dataset for developing epigenetic biomarkers in GIFT tilapia breeding for resistance to S. agalactiae and indicates that spleen could be one of the potential tissue sources of cfDNA methylation marks.