Study On The Characteristics Of Gonadal Differentiation Of Hybrid F₁ Of Grass Carp（♀）×Barbel Chub（♂）

With the rapid development of fish hybrid breeding,various hybrid varieties came into being.To improve the anti-GCRV ability of Ctenopharyngodon idella,our team had carried out the hybridization experiment of C.idella and Squaliobarbus curriculus since 2010,and successfully obtained the 1-year-old to 5-year-old hybrid F₁ between C.idella（♂）×S.curriculus（♀）.The hybrid F₁ had heterosis in meat quality and disease resistance,but the latest research results showed that the hybrid F₁ had abnormal sex differentiation.Only part of hybrid F₁ females was fertile,and fertility varied widely between individuals,hybrid F₁males could produce sperm cells,but mature sperm and normal spermatozoa expulsion had not been observed.In this study,the morphology of early gonadal tissue of C.idella,S.curriculus and hybrid F₁ was determined.The transcriptome sequencing combined with the whole genome information of C.idella and S.curriculus was used to carry out the multi-omics joint analysis.The genes related to gonad development of C.idella,S.curriculus and hybrid F₁ were screened and their expression levels were detected.At the same time,the serum levels of neutral steroid hormones in male F1 hybrids at different ages were measured to analyze the expression characteristics of key factors of gonadal development in male testis at different developmental stages.The aim was to provide a reference for the study of gonadal differentiation mechanism of C.idella,S.curriculus and hybrid F₁.The main results are as follows:（1）There were a few primary oocytes in phase I in the gonadal tissue of S.curriculus at120 days,that is,the gonadal differentiation was completed before 120 days.The oocytes of C.idella were in division stage at 120 d,and gonadal differentiation was completed at 150 d.At 120 d,the hybrid F₁ showed transparent thread-like gonads,and the gonadal differentiation was between S.curriculus and C.idella.（2）By conducting full-length transcriptome and second-generation transcriptome sequencing of the pituitary,hypothalamus and gonad tissues related to the regulation of gonadal development of C.idella,S.curriculus and their hybrid F₁,transcriptome sequencing of 27 samples was completed,and a total of 200.23 G of clean data were obtained,with Q30reaching 85%.By structural analysis of the redundant transcripts of each sample,4016,5331,6182 alternative splicing sequences were obtained for each sample.A total of296,632 SSRs and 142,391 complete CDS regions were predicted by structural analysis of the combined and redundant transcripts.A total of 24,505 lnc RNAs were obtained by predicting the combined and redundant transcripts.A total of 154,293 transcripts were annotated by functional annotation of the redundant transcripts after merging.KEGG pathway analysis showed that the enrichment pathways with high significant difference in the hypothalamic transcriptome data of C.idellus and S.curriculus included Oxytocin signaling pathway,Gn RH signaling pathway,SNARE interactions in vesicular transport SNARE,Renin secretion and Oocyte meiosis.（3）HSD3B7,HSD17B1,HSD17B3,HSD20B2,CYP17A2,CYP1B1,CYP2AA12,UGT2A1,UGT1A1,FSHR,a total of 10 key molecules regulating gonad development by transcriptome screening,such molecules have significant differences in the hypothalamus,pituitary and gonad of the three species of fish.q PCR was used to detect the expression differences of these molecules in hypothalamus,pituitary and gonad tissues of C.idella,S.curriculus and their hybrid F₁.The results showed that the expression level of UGT1A1 in the gonads of hybrid F₁ was significantly higher than that of C.idella and S.curriculus.The expression levels of HSD3B7 and CYP17A2 in pituitary gland and gonad of S.curriculus were significantly higher than those of C.idella and hybrid F₁.The expression of CYP2AA12 in hypothalamus of S.curriculus was significantly higher than that of C.idella and hybrid F₁,but in pituitary,the expression of CYP2AA12 was the most significant in hybrid F₁.Except for UGT1A1,the expression levels of the other 9 molecules in pituitary tissues of the three fish were significantly different,and the expression levels of HSD17B1,HSD17B3,HSD20B2,CYP1B1,UGT2A1 and FSHR in pituitary tissues of C.idella were significantly higher than those of S.curriculus and hybrid F₁.（4）The serum concentrations of luteinizing hormone（LH）,follicle-stimulating hormone（FSH）,testosterone（T）and 11 ketone testosterone（11-KT）in 2-6 age hybrid male F₁ individuals were determined.The general trend of FSH was that the hormone concentration increased gradually with the increase of age.The concentrations of FSH and LH reached the maximum at the 4th instar（5.12m IU/m L,9.61 m IU/m L,respectively）.There was no significant change in T and 11-KT concentrations,but the concentrations of T and11-KT were the highest in the third age（11.81nmol/L and 2305ng/L,respectively）.The expression levels of dmrt1 and sox9 genes,which are closely related to testis development,were detected by q PCR.The results showed that dmrt1 expression was the highest in the testis of the second instar,and then decreased gradually.The highest expression of sox9 was found in the testis of the 4th instar.To sum up,it was found that the early gonadal differentiation traits of hybrid F₁ were between S.curriculus and C.idella.The transcriptome sequencing combined with the whole genome information of C.idella and S.curriculus was used to carry out multi-omics joint analysis,and the expression differences of some gonadal-related molecules in the hypothalamus,pituitary gland and gonad of C.idella,S.curriculus and their hybrid F₁ were identified,which is preliminarily concluded that the gonadal differentiation of red eye trout and hybrid F₁ was completed before 150 days of age,while that of grass carp was completed between 150 and 180 days of age.At the same time,the gonadal development and expression characteristics of hybrid F₁ males of different ages were investigated.It was found that the expression of T and 11-KT affected the spermatogenesis and sperm maturation of hybrid F₁,the abnormal expression of dmrt1 gene might be related to the abortion of hybrid F₁ males.The results will lay a scientific foundation for future studies on the gonadal differentiation mechanism of C.idella,S.curriculus and hybrid F₁.