Polymorphism in a sex-linked DNA marker located on LG23 in Hainan strain of Nile tilapia (Oreochromis niloticus)

Marker-5, which is linked to the major sex-determination gene amhy on LG23, has been used for sex genotyping of various strains of Nile tilapia. Marker-5 specifically amplifies fragments of approximately 1,422 and 982 bp of the X and Y chromosomes, respectively. Herein, we evaluated Marker-5 polymorphism in Nile tilapia of Hainan strain, China. Agarose gel electrophoresis showed that in 13 out of 85 female fish, Marker-5 amplified a larger band, distinct from the standard X-specific band. Sanger sequencing showed that this larger band was 1,688 bp in length and showed high similarity with both 1,422-bp X-specific and 982-bp Y-specific fragments. The larger band was proved to be a female sex-linked allele of Marker-5 and was labeled as X-L. When the polymerase chain reaction (PCR) products of XLY fish, amplified by Marker-5, were tested by agarose gel electrophoresis, an unusual band, smaller than X-L but larger than that of Y fragment, was observed. The unusual band could be a heteroduplex of the X-L and Y fragments. In very few XXL male, amhy was found to be linked with 1,422-bp fragment amplified by Marker-5. Understanding the Marker-5 is useful for its utilization in sex-controlled breeding in farmed Nile tilapia.