Cytogenetic Evidence for a Male-Heterogametic (XY) Sex Determination System in Clarias gariepinus

Abstract Clarias gariepinus is quite widespread in Africa and Asia due to its high adaptability and is an important species for aquaculture. A detailed cytogenetic analysis was conducted in a Sakarya population of C. gariepinus to elucidate karyotype organization, heterochromatin distribution, nucleolar organizer region (NOR) patterning, and sex chromosome differentiation. Mitotic chromosomes from morphologically and gonadally sexed males and females were examined using Giemsa staining, C-banding, Ag-NOR staining, CMA3/DAPI staining, and telomeric FISH. Both sexes exhibited a conserved diploid chromosome number of 2n = 56; however, comparative karyotype analysis revealed a heteromorphic chromosome pair in males and a corresponding homomorphic pair in females, providing clear cytogenetic evidence for a male-heterogametic XX/XY sex determination system. The male karyotype formula was 20m + 16sm + 12st + 6a + XY, with a fundamental number (NF) of 92 and an autosomal arm number (NFa) of 90. C-banding demonstrated prominent pericentromeric heterochromatin blocks on all chromosomes, with additional heterochromatic segments associated with NOR-bearing regions. Ag-NOR and CMA3 staining consistently identified a single GC-rich, transcriptionally active NOR located on a subtelocentric chromosome pair, while a terminal CMA3-positive but Ag-NOR-negative site on an acrocentric chromosome pair indicated the presence of an inactive NOR. Telomeric signals were exclusively restricted to terminals of chromosomes, with no evidence of interstitial telomeric sequences. These results reveal pronounced population-specific variation in karyotype structure and provide robust cytogenetic support for an XY sex chromosome system in C. gariepinus, underscoring the dynamic and independent evolution of sex chromosomes and repetitive DNA elements within the genus Clarias.