Identification of sex-linked molecular markers in the Asiatic toad (Bufo gargarizans Cantor, 1842) reveals an XX/XY sex determination system

Background Previous karyotypic analyses of Bufo gargarizans reported 11 pairs of homomorphic chromosomes (2n = 22) and no cytologically distinguishable sex chromosomes. A ZW-type sex determination system had been hypothesized based on differential replication patterns in chromosome pair 10, but this remained speculative in the absence of molecular evidence. Result Here, we identified male-specific genetic markers, providing the first definitive molecular evidence for an XY-type sex determination system in this species. Chromosomal localization of these markers confirmed chromosome 1 as the sex chromosome. By calculating Fst between males and females, we delineated the non-recombining region (NRR) on chromosome 1, which spans approximately 212 Mb (246.69 Mb to 458.35 Mb), revealing a remarkably large sex-differentiating region with low recombination rates. We further detected non-homologous sex chromosome transitions between B. gargarizans and B. bufo , highlighting the dynamic evolution of sex chromosomes within the genus Bufo . Conclusion These findings revise the current understanding of sex determination in B. gargarizans , define the extent of the NRR, and establish this species as a valuable model for studying the origin and differentiation of sex chromosomes, thereby contributing important insights into the diversity of sex-determining mechanisms in amphibians and vertebrates.