A rapid method to generate an all-female population in common carp (Cyprinus carpio) using the neomale carp of F0 population with targeted aromatase mutations

Teleosts are excellent models for elucidating the regulatory mechanisms of gonadal differentiation. Although the essential role of ovarian aromatase (encoded by cyp19a1a) in ovarian differentiation has been documented in several species of fish, this remains poorly understood in common carp (Cyprinus carpio) due to its long period of sexual maturity and the complexity of the multiple loci in its allotetraploid genome. Here, using the CRISPR/Cas9 strategy, some neomale carp with chimeric mutation (CM) of cyp19a1aA, cyp19a1aB or cyp19a1aA;cyp19a1aB were found in the F0 population at 1 year post-hatching (yph). The mutation ratios of cyp19a1aA, cyp19a1aB, or cyp19a1aA;cyp19a1aB were positively correlated with the ratio of sex reversal from female to male. Concentrations of estradiol were significantly decreased in female and neomale of chimaeras carp compared to wild-type (WT) control female carp. The progeny from artificial fertilization between WT female carp and these neomale carp were confirmed as all-female (AF) carp, as evidenced by genetic identification of male-specific DNA marker and histological analysis of progeny gonads. Finally, in comparison to the control group, which comprised both male and female carp reared in the same pond, AF carp at 4 months post-hatching (mph) demonstrated enhanced 12.06 % growth performance. In conclusion, we have shown that both Cyp19a1aA and Cyp19a1aB are required for estrogen synthesis, which is essential for ovarian differentiation in common carp. Therefore, a rapid method to generate AF populations using the F0 CM populations has been established by genetic manipulation targeting the loci of the cyp19a1a genes. The present study has revealed the non-redundant roles of cyp19a1aA and cyp19a1aB in ovarian differentiation and also provides an efficient and rapid method of controlling gonadal differentiation in fish without knowledge of the specific sex-determining gene (SDG) of the particular fish.