A Cautionary Tale of Sexing by Methylation: Hybrid Bisulfite-Conversion Sequencing of Immunoprecipitated Methylated DNA in Chrysemys picta Turtles with Temperature-Dependent Sex Determination Reveals Contrasting Patterns of Somatic and Gonadal Methylation, but No Unobtrusive Sex Diagnostic

SIMPLE SUMMARY: Identifying the sex of turtle hatchlings is important to assess the sex ratio of populations, which is important to study their ecology and evolution and for conservation programs. However, turtle hatchlings rarely display morphological differences detectable to the naked eye, and existing sexing techniques are either harmful, lethal, or non-viable for turtles with temperature-dependent sex determination. We investigated two methodologies that rely on differences in DNA methylation, a modification that occurs naturally in the DNA without changing its sequence, but that affects the expression of genes. As DNA methylation is known to differ in the gonads of male and female painted turtle hatchlings, we investigated whether the same is true in their tails We found that the painted turtle displays differential DNA methylation in the gonads, but not in the tails. We conclude that DNA methylation is tissue-specific in the painted turtle and that this epigenetic modification plays an important role in sexual development in this species but not in the somatic tissue of the tails. ABSTRACT: Background: The gonads of Chrysemys picta, a turtle with temperature-dependent sex determination (TSD), exhibit differential DNA methylation between males and females, but whether the same is true in somatic tissues remains unknown. Such differential DNA methylation in the soma would provide a non-lethal sex diagnostic for TSD turtle hatchings who lack visually detectable sexual dimorphism when young. Methods: Here, we tested multiple approaches to study DNA methylation in tail clips of Chrysemys picta hatchlings, to identify differentially methylated candidate regions/sites that could serve as molecular sex markers To detect global differential methylation in the tails we used methylation-sensitive ELISA, and to test for differential local methylation we developed a novel hybrid method by sequencing immunoprecipitated and bisulfite converted DNA (MeDIP-BS-seq) followed by PCR validation of candidate regions/sites after digestion with a methylation-sensitive restriction enzyme. Results: We detected no global differences in methylation between males and females via ELISA. While we detected inter-individual variation in DNA methylation in the tails, this variation was not sexually dimorphic, in contrast with hatchling gonads. Conclusions: Results highlight that differential DNA methylation is tissue-specific and plays a key role in gonadal formation (primary sexual development) and maintenance post-hatching, but not in the somatic tail tissue.