Induction Of Neo-male And Screening Analysis Of Sex-related Genes In Yellow Drum

As an important mariculture fish in China,the yellow drum(Nibea albiflora)has the advantages of fast growth,wide adaptability and strong stress resistance.The females of yellow drum grow 30% faster than males at 15 month old.Therefore,it is of great significance to perform the sex control and study sex determination of yellow drums for the development of large-scale production of all-female fish.This study optimized the induction method of neo-males,and analysis the sex-related genes using transcriptome sequencing technology.The results are as follows:1.Induction of sex reversal in gynogenetic yellow drum(25 dph)by oral administering letrozole(10/100 mg/kg)and MT(10 mg/kg)at different doses,which set up normal yellow drum and gynogenetic yellow drum as the control groups,to study the effects on growth performance,sex ratio,and histological structure of gonads,and then to optimize suitable methods to induce neo-male.The results showed that letrozole(10/100 mg/kg)can successfully induce all the gynogenetic yellow drum to be reversed to phenotypic male,and promote the spermatogenesis.However,there was a slight inhibitory effect on the growth of gynogenetic yellow drum during treatment.Observation of tissue sections revealed that the gonads of neo-males induced by letrozole had normal testicular structures with good development,and maintained their normal testicular structures after the letrozole treatment withdrawal.there was a certain proportion of abnormalities in the MT group,including toxic physicochemical phenomena such as vacuolization.In addition,the gonadosomatic index(GSI)at 150 dph and 210 dph also showed no significant difference between the treatment group and the control group.2.We collected a total of 26 brain and gonadal tissue samples from neo-male,gynogenetic yellow drum and normal female and male yellow drum for transcriptome sequencing.Each sample produced 6.41 Gb of data after quality control on average,.The average rate of the samples was blasted with the genome was 86.94%.A total of 58,282 genes were detected,of which 56,964 were known genes and 1,318 new genes were predicted.Analysis of differentially expressed genes showed that the gonad of normal female and neo-male had the largest number of differential genes,up to 8958 genes;the normal male and female brains had the least differential genes,with only 30 genes.We analyzed the transcription maps of the brains and gonads of the gynogenetic yellow drum,neo-male and normal male and female yellow drum,and found that the gene expression level in the ovary of the gynogenetic yellow drum and normal female was consistent,and the neo-male was consistent with the normal male testis.The transcription profiles of the brains of the samples were consistent.Using GO functional annotation,the brain is generally enriched in the regulation of biological processes and catalytic activity,and the gonads are enriched in many reproductive related items.The gonads are also enriched in p53 and PPAR signaling pathways through KEGG annotation.3.The qRT-PCR analysis was performed on four female-related genes(cyp19a,zp3,zp4,foxl2),four male-related genes(dmrt1,gsdf,amh,ar)and two germ cell genes(dnd and vasa)to verify the reliability of transcriptomic results.The specific expression of cyp19 a,dmrt1,foxl2 and vasa in the tissues of normal yellow drum was detected.It was found that cyp19 a was only expressed in the ovary,while dmrt1 was only expressed in the testis.Foxl2 is expressed in both the gill and gonad,while vasa only expressed in the gonad as a germ cell gene.