Identification of the sex determination system and development of sex-specific molecular marker in Strauchbufo raddei based on whole-genome resequencing

Amphibians exhibit remarkable diversity in sex determination systems, whereas their sex chromosomes are often morphologically undifferentiated. Strauchbufo raddei possesses homomorphic sex chromosome, and its sex-determining region (SDR) and sex determination system remain unclear. In this study, whole‑genome resequencing was performed to identify sex-associated genetic differences between male and female populations of S. raddei, thereby localizing the SDR and elucidating its sex determination system. Genome‑wide association and genetic differentiation analyses identified chromosome 4 as the sex chromosome of this species, supporting a female-heterogametic (ZW/ZZ) sex determination system. According to read‑depth distribution, the 0–3.8 Mb interval of chromosome 4 corresponds to the SDR of the W chromosome, and the 3.9–4.8 Mb interval corresponds to that of the Z chromosome. Furthermore, based on the female‑specific insertion fragment within the W‑chromosomal SDR, we developed the first molecular marker for genetic sex identification in S. raddei. These results provide genomic evidence supporting a ZW‑type sex determination system in S. raddei and provide a foundation for future studies on amphibian sex‑chromosome evolution and the genetic mechanisms underlying sex determination.