Identification of Male-Specific Markers by Genotyping-by-Sequencing in the Giant Spiny Frog, Quasipaa spinosa

Background/Objectives: Quasipaa spinosa, a large-sized spiny frog, has high commercial value in the food trade. Although the sexual dimorphism in body size between males and females has been investigated, the sex-determining mechanism in this frog remains unknown. Methods: This study employed a genotyping-by-sequencing (GBS) method to identify sex-associated genomic markers and elucidate the sex determination mechanism in the species. Results: We obtained 853 candidate sex-specific GBS tags, with 811 tags (95.07%) demonstrating a male heterozygous system (XX/XY). The diagnostic specificity of the nine markers was further demonstrated by PCR analysis across multiple adult individuals from seven distinct geographic populations of the frog. Four sex-specific markers were aligned with the DMRT1 gene, representing a master regulator of sex determination and gonadal differentiation across the animal kingdom. Conclusions: Our results deciphered the genetic mechanisms governing sex determination in Q. spinosa and presented effective strategies for mono-sex breeding.