Identification of sex markers by cDNA-AFLP in Takifugu rubripes

We searched for sex markers in mature male and female Takifugu rubripes gonads and tailfins by cDNA-amplified fragment length polymorphism (AFLP) performed on pooled cDNA samples. Five sex markers (TDF1-5) were identified, cloned, characterized and confirmed on individual samples of cDNA and genomic DNA, showing good agreement with phenotypic sex. Among them, four markers (TDF1-4) derived from gonads were detected in immature T. rubripes (at months 6, 12, 18 and 24) testis or ovary cDNA, while the TDF5 from tailfins was detected in immature T. rubripes (at months 6, 12, 18 and 24) tailfin cDNA and T. rubripes tailfin genomic DNA. Therefore, the sex specific primer or probe of TDF5 could be used in quick detection of phenotypic sex in juvenile and even larval stages without the need to wait for the offspring to become sexually mature or dissect the fish. When tested on one closely related species from the Takifugu genus, TDF5 could amplify a specific fragment from Takifugu pseudommus tailfin genomic DNA, indicating that homologues of sex markers might exist in other species of the Takifugu genus.