Screening And Preliminary Functional Studies Of Genes Associated With Sex Determination And Differentiation In Red-tail Catfish(Hemibagrus Wyckioides)

Red-tail catfish(Hemibagrus wyckioides),a freshwater species of the family Hemibagrus,Bagridae,Siluriformes,is mainly distributed in multiple Southeast Asian countries and the Lancang River of the Yunnan Province,China.The red-tail catfish has shown a sustained increase in its culture and has become an important economic aquaculture species in Southern China in recent years.Like many catfish species,the red-tail catfish shows apparent sexual dimorphism in growth,with males growing faster than females,which suggests that the production and farming of the all-male population of red-tail catfish will be of greater economic benefit.The investigation of sex determination and differentiation mechanisms is necessary for the breeding of all-male population,however,little research has been performed on the sex determination and differentiation of red-tail catfish,and there is still a long way to go to achieve the production of all-male population and large-scale farming.In this study,we performed the first transcriptome sequencing analysis of testis and ovary tissues at four stages of early gonadal development(10,18,30 and 48 days after hatching)in red-tail catfish,based on the determination of the timing of sexual differentiation in red-tail catfish.The results showed that a total of 23 588 genes were identified in 24 sequenced samples,and 28,213,636 and 1 381 differentially expressed genes were screened at four developmental stages.Seven shared genes were further identified by taking intersections of differentially expressed genes at four developmental stages.Further validation by quantitative real-time PCR showed that amh,gdf6 a,tsga10 and cyp17 a were highly expressed mainly in males,and cyp19a1 b,foxl2 and hsd17b1 were highly expressed in females.The KEGG pathway enrichment showed that these identified genes were mainly involved in steroid hormone biosynthesis pathway and TGF-β signaling pathways.Based on these results,discussion and analysis,amh and gdf6 a may be the male biased expression genes,and foxl2,cyp19a1 b and hsd17b1 may be the female biased expression genes of red-tail catfish.These five genes may be involved in gonadal differentiation and development and may be candidates for genes related to sex determination and differentiation in red-tail catfish.This study then carried out preliminary functional validation of candidate sex determination and differentiation genes.Based on our previous research on the identification of sex-specific markers and the construction of the genetic linkage map,we have identified a gene,cfap58,that may be involved in sex determination in red-tail catfish.In this study,the cfap58 knockout F0 generation was constructed by applying CRISPR/Cas9 technology to knock out this screened candidate sex-determining gene cfap58.No significant differences were observed in their gonadal developmental morphology and expression of key genes compared to the wild type.The function of this gene still needs to be verified by further experiments and analysis.The results of this study provide an important theoretical basis and data support for the subsequent screening of sex-determining genes,revealing of gene function and breeding of all-male population in red-tail catfish.