Differential methylation reveals pathways associated with sex differentiation in Chinese tongue sole (Cynoglossus semilaevis)

Sex reversal is a complex biological phenomenon that occurs in Cynoglossus semilaevis . Some genetic females may irreversibly convert to pseudomales, thus increasing aquaculture costs because males grow much more slowly than females. In recent years, our studies have shown that genetic background is the primary factor contributing to sex reversal in tongue sole, and two linked loci on the Z chromosome were found to be associated with sex reversal. However, the role of epigenetics in sex reversal and temperature-independent sex determination has yet to be determined. In this study, we used reduced representation bisulfite sequencing (RRBS) to investigate the differences in methylation among female, male and pseudomale tongue sole and explored the possible pattern of DNA methylation associated with sex differentiation and sex reversal. A total of 32200, 341115 and 2691 DMRs with average lengths of 163.45, 170.15 and 128.77 bp, respectively, were identified between pseudomales and females, males and females, and pseudomales and males. Notably, the sex chromosome (Chr. Z) contained the greatest number of DMRs (303) between pseudomales and males. All the DMGs in each pairwise comparison were enriched in ubiquitin-mediated proteolysis, biosynthesis of secondary metabolites, thermogenesis and metabolic pathways, etc. In addition, differentially methylated genes possibly related to sex differentiation, such as dmrt1 , nobox , foxl2 , pdgfrb , rspo1 , lhx9 , emx2 , arx , and piwil2, were screened. Methylation differential genes and metabolic pathways associated with sex differentiation have been identified, suggesting the vital role of methylation in gender reversal and sex determination. This study provides valuable insight into epigenetic mechanisms involved in sex differentiation and sex reversal in C. semilaevis .