| Fang Bo "Xianfeng No.2" is a new subfamily variety formed from the selected M.amblycephala as female parent and A.nigrocauda as male parent.This new variety,which has the advantages of fast growth,strong resistance to disease and adversity,low breeding cost,easy fishing and so on,is an important economic fish in China.Through long-term breeding,it is found that the female grows faster than male,and breeding all-female system can bring higher market economic value.The development of sex molecular markers is very important for large-scale monosexual breeding and directional breeding,but there is no related research on the development of sex molecular markers of this hybrid.In this study,nine candidate male specific molecular markers were mined by2b-RAD technique;Then,the candidate male specific molecular markers were verified in hybrid and A.nigrocauda populations.At the same time,two male specific molecular markers(including one gene encoding region)were obtained by analyzing the transcriptomic data of male and female gonads of hybrid populations.The male specific molecular markers of the hybrid were developed from the level of genome and transcriptome expression profile;It was applied to the sex identification of hybrid gynogenetic female population F1 and pseudomale fish induced by different concentrations of androgen,and the optimal androgen treatment concentration was determined by the success rate of induction,which laid a foundation for the large-scale breeding of all-female population of the hybrid.The results mainly include the following aspects:(1)Screening of gender specific molecular markers of Fang Bo "Xianfeng No.2"20 individuals(10 females and 10 males)of Fang Bo "Xianfeng No.2" were selected for 2b-RAD simplified genome sequencing analysis.Nine candidate male specific molecular markers(ref-57033-1,ref-57660-1,ref-85149-1,ref-106035-1,ref-119563-1,ref-206431-1,ref-231141-1,ref-305044-1,ref-330456-1)were excavated from the DNA genome level;The candidate male specific molecular markers were verified by PCR in 20 individuals of 2b-RAD sequencing population;The results showed that ref57033-1 and ref57660-1 existed only in male individuals,but not in female individuals.After verification,two effective male specific molecular markers were preliminarily obtained;Then,the samples were expanded and the two molecular markers were verified by PCR in 30 individuals of Fang Bo "Xianfeng No.2" and 30 individuals of A.nigrocauda.The results showed that there was no band amplification in all female individuals,but a single bright band in male individuals;we can accurately distinguish male and female,and further verified the universality of the two molecular markers.(2)Comparative analysis of male and female gonad transcriptome of Fang Bo "Xianfeng No.2" and expression verification of gender specific markersThe mature gonads of Fang Bo "Xianfeng No.2" were analyzed by male and female comparative transcriptome analysis.A total of 21,324 differentially expressed genes(DEGs)were identified in the male groups.Compared with the female group,15,299 DEGs were up-regulated and 6,025 DEGs were down regulated in the male group.Among these DEGs,3,122 DEGs were expressed only in males but not in females.By combined analysis of 2b-RAD simplified genome sequencing and transcriptomic sequencing,two fragments of male specific molecular markers were blasted with DEGs.Ref-57660-1(326 bp)was successfully matched with Trinity DN17103(770 bp)and only expressed in males.The accuracy of the results was further confirmed by semi-quantitative experiments.Then,the differential genes Trinity DN17103 was blasted in the NCBI database,which shared 80% identity with a paternal microsatellite locus(EHWB15)of A.nigrocauda.It further verified the reliability of the molecular marker.(3)The application of male specific molecular markers in preliminary study on sex reversal of gynogenetic population of Fang Bo "Xianfeng No.2"Inactivated sperm of Cyprinus carpio var.Singuonensis was used to stimulate the eggs of Fang Bo "Xianfeng No.2",and the gynogenetic population was obtained by inhibiting the release of the second polar body through cold shock.The gonadal development was detected to be female;The microsatellite markers of Cyprinus carpio var.Singuonensis were used to genetic verification.It was found that the gynogenetic population had no penetration of paternal genetic material,so it was speculated that the sex determination mechanism of Fang Bo "Xianfeng No.2" was XX-XY type.After that,the sexual reversal study was carried out based on the gynogenetic population.The fish fries were fed with different concentrations of methyltestosterone androgen(0 mg/kg,100mg/kg,200 mg/kg)from 30 to 150 days after hatching.Combined with the male specific molecular marker of the hybrid and gonadal tissue sections,the sex of gynogenetic individuals treated with different concentrations of gradient methyltestosterone was identified.The results showed that the male induction rate of 100 mg/kg androgen treatment group was 30%;The male induction rate of 200 mg/kg androgen treatment group was 45%.The pseudomale fish was accurately identified from the perspectives of physiology and genetics,which laid a foundation for the cultivation of its all-female population. |
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