Supplementary Material for: Characterization of DMRT1 variants for testis determination and differentiation in emu

Introduction: DMRT1 on the Z chromosome is a conserved male sex-determining gene in birds. In chickens, a representative model species of Neognathae, the function of DMRT1 has been well characterized. In contrast, Palaeognathae species such as the emu possess less differentiated sex chromosomes and thus provide a valuable system for investigating avian sex determination, yet molecular studies remain limited. We investigated the timing of sex determination and the expression of key genes involved in gonadal differentiation in emu, and further characterized DMRT1 variants. Methods: Sex determination stage was identified by anatomical comparison of male and female embryonic gonads. Expression of seven genes (DMRT1, AMH, SOX9, NR5A1, FOXL2, CYP19A1, and RSPO1) was examined by mRNA-seq and RT-PCR. DMRT1 splicing variants were predicted by in silico analysis and 3’ RACE was used to identify alternative polyadenylation (APA) variants. Results: The gonadal differentiation occurred at HH25–28 based on gonadal morphology. Gene expression analysis revealed emu-specific patterns not observed in chickens. Notably, RSPO1 was highly expressed in females at HH24–25, preceding DMRT1 expression in males at HH28–29, suggesting ovarian differentiation begins earlier. We identified three splicing variants and four APA variants of DMRT1, with variant 1 predominant during gonadal development. Conclusion: These findings suggest that while molecular sex differentiation mechanisms are largely conserved between Palaeognathae and Neognathae, they differ in parts. In particular, early RSPO1 expression may initiate ovarian differentiation prior to testis determination by DMRT1. The presence of emu-specific DMRT1 variants further indicates possible species-specific mechanisms in testis development.