Identification of a Candidate Sex Determination Region and Sex-specific Molecular Markers of Spinibarbus caldwelli

Spinibarbus caldwelli is a commercially significant aquaculture species in China that exhibits pronounced sexual dimorphism in growth-related traits. However, identifying sex based on morphological characteristics remains challenging before sexual maturation, necessitating the development of sex-specific molecular markers for sex-controlled breeding. In this study, whole-genome resequencing was performed on 50 individuals (25 females and 25 males) with an average sequencing depth of 10.05×. A genome-wide association study (GWAS) identified 140 loci significantly associated with sex, all located within a narrow 7-kb region (26.033–26.040 Mb) on chr 46. Analysis of fixation index (FST) revealed a prominent peak within the same interval, corroborating the GWAS results and confirming a strong association between this locus and sex determination. Utilizing seven InDel polymorphisms within this region (comprising a 20 bp total deletion), we developed the primer pair P11-3. Electrophoretic genotyping using this marker demonstrated 100% accuracy, showing complete concordance between genotypes and phenotypic sex. This study fine-mapped a candidate sex-associated region in S. caldwelli, providing robust evidence for a male heterogametic (XX/XY) sex determination system. Furthermore, we established a simple and highly accurate molecular method for sex identification. These findings provide reliable technical support for broodstock selection, early sexing of juveniles, and germplasm improvement in S. caldwelli, while offering valuable insights into the mechanisms of sex determination in cyprinid fishes.