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Variation in the TGF-β signaling pathway is emerging as an important mechanism by which gonadal sex determination is controlled in teleosts. Here we show that  amhy , a Y-specific duplicate of the  anti-Müllerian hormone  ( amh ) gene, induces male sex determination in Nile tilapia.  amhy  is a tandem duplicate located immediately downstream of  amhΔ-y  on the Y chromosome. The coding sequence of  amhy  was identical to the X-linked  amh  ( amh ) except a missense SNP (C/T) which changes an amino acid (Ser/Leu92) in the N-terminal region.  amhy  lacks 5608 bp of promoter sequence that is found in the X-linked  amh  homolog. The  amhΔ-y  contains several insertions and deletions in the promoter region, and even a 5 bp insertion in exonVI that results in a premature stop codon and thus a truncated protein product lacking the TGF-β binding domain. Both  amhy  and  amhΔ-y  expression is restricted to XY gonads from 5 days after hatching (dah) onwards. CRISPR/Cas9 knockout of  amhy  in XY fish resulted in male to female sex reversal, while mutation of  amhΔ-y  alone could not. In contrast, overexpression of Amhy in XX fish, using a fosmid transgene that carries the  amhy/amhΔ-y  haplotype or a vector containing  amhy  ORF under the control of CMV promoter, resulted in female to male sex reversal, while overexpression of AmhΔ-y alone in XX fish could not. Knockout of the  anti-Müllerian hormone receptor type II  ( amhrII ) in XY fish also resulted in 100% complete male to female sex reversal. Taken together, these results strongly suggest that the duplicated  amhy  with a missense SNP is the candidate sex determining gene and  amhy/amhrII  signal is essential for male sex determination in Nile tilapia. These findings highlight the conserved roles of TGF-β signaling pathway in fish sex determination.

plos genetics

A Tandem Duplicate of Anti-Müllerian Hormone with a Missense SNP on the Y Chromosome Is Essential for Male Sex Determination in Nile Tilapia, Oreochromis niloticus