Analysis Of Gonadal Transcriptome And Identification Of Candidate Female Sex Determining Gene In Blue Tilapia (Oreochromis Aureus)

Blue tilapia(Oreochromis aureus),native to Egypt,Israel and Saudi Arabia,is a representative species of Cichlidae and Oreochromis.It has the advantages of wide feeding habits,fast growth rate and strong disease resistance.It is one of the widely cultivated tilapia species in China.The blue tilapia possesses a ZW(female heterogametic)sex determination system with a female sex determining locus on LG3.At present,the identified sex determining genes are all male determiners in teleost,thus it is of great significance to isolate the female sex determining gene of blue tilapia.The divergence time of blue tilapia and Nile tilapia is less than 4 million years.However,they have completely different sex determination systems(ZW and XY)and sex chromosomes(LG3 and LG23),which makes them good models for studying the transition and rapid evolution of sex chromosomes and sex determination systems of cichlids.In addition,the growth rate of males is much faster than that of females in tilapias,so the monosex(allmale)culture has broad application prospects.Therefore,the isolation of female sex determining gene of blue tilapia would be helpful for promoting the research on the transition and rapid evolution of sex chromosome and sex determining system of tilapias,as well as the development of sex control breeding technology of this species.The highquality reference genomes of ZZ and WW blue tilapia were obtained,and the sex determining regions on Z and W chromosomes(Z-SDR and W-SDR)were successfully identified in our previous studies.In this study,we sequenced the transcriptome of male and female gonads of blue tilapia at 5,10,20 and 30 dah(days after hatching)and analyzed the dynamic changes of genes in the process of sex determination and differentiation.Through genome and transcriptome analysis,the candidate female sex determining gene,banf-W,of blue tilapia was screened out.Then,the gene was preliminarily verified,including phylogenetic analysis,gene knockout and so on.The main results are as follows:1 Sequencing and analysis of gonadal transcriptome of blue tilapia1.1 Gonadal transcriptome sequencing of blue tilapia at key stages of sex determination and differentiation,and clean reads mapped to WW genomeIn this study,we sequenced the gonadal transcriptome of male and female blue tilapia at 5,10,20 and 30 dah(3 repeats at each stage,24 samples in total)by Illumina technology,and the clean reads were mapped to WW reference genome.The statistics of the sequencing data showed that the sequencing quality of each sample was good.In terms of the amount of generated data,the average numbers of raw reads and clean reads of 24 samples were 62157129 and 61042644,respectively.The average error rate and GC content of 24 samples were 0.03% and 48.58%,respectively.The mapping rate of each sample was also good.The concordant pair alignment rates of all samples were more than81.80%,with an average value of 83.76%;the maximum and average values of multiple pair alignment rates were 8.90% and 6.25%,respectively.Besides,the discordant pair alignment rates of all samples were all less than 1.80%,with an average value of 1.28%.1.2 Analysis of differentially expressed genes(DEGs)In this study,Edge R software was used to screen DEGs of male and female gonads at different stages.In general,the number of DEGs increased accompanying with the gonad development,and the number of ZW female-differentially expressed genes(ZWDEGs)was higher than that of ZZ male-differentially expressed genes(ZZ-DEGs).At four development stages,the number of ZW-DEGs was 731,2657,4347 and 7375respectively;while the number of ZZ-DEGs was 628,919,1925 and 1184,respectively.The number of ZW-DEGs shared by all four stages was 156,76 of which were distributed on the sex chromosome LG3;while the number of ZZ-DEGs shared by all four stages was 39,with random chromosome distribution.The GO terms of ZW-DEGs and ZZDEGs shared by all four stages showed that these genes were enriched in the “biological process(BP)”,including GO terms involved in sex determination and gonadal development.1.3 The expression patterns of genes involved in steroids synthesis pathway,estrogen and androgen receptor genesThe total expression level of genes involved in steroids synthesis pathway was gradually increasing accompanying with the gonad development.In female gonads,the expression of cyp19a1 a was higher than that in the male gonads,while the expression of cyp11b2 was always at a low level.In male gonads,the expression of cyp19a1 a was relatively high at 5 dah,but decreased rapidly with the increase of the expression of cyp11b2.The total expression level of estrogen receptor genes remained almost unchanged during the four stages,among which esr2 a was the highest and esr2 b was the lowest.The total expression level of androgen receptor genes(ar1 and ar2)increased accompanying with the gonad development,but there was no significant difference between males and females.2 Screening and identification of candidate female sex determining gene in blue tilapia2.1 Screening of candidate female sex determining gene in blue tilapia using genomic and transcriptome dataIn this study,we used genome,re-sequencing and transcriptome data to screen candidate female sex determining gene in blue tilapia through a series of bioinformatics analysis.The number of ZW-DEGs,which were shared by all four stages and located in W-SDR,was 22.Detailly,all of them were located in a 6 Mb region of W-SDR,while 11 of them were concentrated in 0.4 Mb(47.0-47.4 Mb).Intriguingly,the 0.4 Mb region was consistent with the 0.6 Mb region of Z-SDR full of female-specific SNPs,and contained three genes(one banf and two paics genes)with more copies on the W chromosome than the Z chromosome.Genomic and transcriptome analysis showed that these three genes were all W-specific copies,but the expression of two paics genes at the key stage of sex determination was too low to be the candidate sex determining genes.Therefore,the Wspecific banf gene,which was named banf-W,was a candidate female sex determining gene of blue tilapia.2.2 The identification and preliminary functional analysis of banf-WIn this study,banf-W was identified by sequence alignment,amplified verification of different strains,phylogenetic analysis and gene knock-out.1)W-specificity: banf-W was W-specific based on PCR validation in 108 fish from lab,Wuxi and Guangzhou strains.2)The evolutionary history of banf-W and banf-LG3: banf-W was a speciesspecific duplication,originated from banf-LG3;while banf-LG3 was a tilapiine-specific duplication,probably originated from banf2.3)The knockout of banf-W/banf-LG3: F0 mutant fish with deletion of the third exon were successfully obtained by CRISPR/Cas9 technology.Compared with wild type,the gonads of F0 mutant fish(6 months old)were atrophied without large phase oocytes.The H.E.staining and fluorescence immunohistochemistry showed that: the gonads of F0 mutant fish had a lot of cavities;the number of oocytes decreased significantly;and a large number of granular substances,which were easy to be stained,occurred.RT-PCR showed that the expression of cyp19a1 a and foxl2 decreased,while the expression of cyp11b2,dmrt1 and amhr2 increased in F0 mutant fish.These indicated the F0 mutant fish might have masculinized.In summary,we screened out the DEGs of each stage by sequencing and analyzing the transcriptome of male and female gonads at the key stages of sex determination,and the ZW-DEGs shared by all four stages were mainly located on the sex chromosome LG3.Through the analysis of expression patterns of genes involved in steroids synthesis pathway,we found that estrogen and androgen were essential for the sex determination and differentiation of this species.Based on genomic and transcriptional data,the candidate female sex determining gene of blue tilapia,banf-W,was successfully isolated through a series of bioinformatics analysis.Using CRISPR/cas9 technology to delete the third exon of banf-W/banf-LG3,the F0 mutant fish with high positive rate masculinized,which indicated that banf-W might be the female sex determining gene of blue tilapia.This study can not only theoretically improve the understanding of female sex determining genes in teleost and promote the research on the transition and rapid evolution of sex chromosome and sex determining system of cichlids,but also lay a foundation for the sex control breeding of this species.