Identification Of Male Sex Determining Region And Candidate Sex Determining Gene On LG1 Of Tilapia Based On Resequencing And Transcriptomic Data

Tilapias are representative species of the cichlids which are used as classic model of adaptive radiation.They are also worldwide farmed fish.They have the characteristics of good meat quality,strong adaptation and strong disease resistance.In addition,tilapias also show striking sexual dimorphism,with the growth rate of male fish 40-50% faster than that of female fish.Thus,the all-male tilapia can not only avoid excessive breeding,but also improve farming efficiency.Therefore,sex control is of great significance in tilapia farming.Previous study from our group has showed that LG23 is the sex chromosome and amhy is sex determining gene of most strains of Nile tilapia even though there are reports showing that LG1 is the sex chromosome with a sex determining region of 9 M.On the other hand,there are also reports showing LG1 is the sex chromosome of the red tilapia strain in our laboratory and hybrids(ZX)between Nile tilapia and blue tilapia.However,the male sex determination region and sex determination genes on LG1 are still unclear.In this study,we performed whole-genome resequencing on laboratory strains of red tilapia(WL-red)and Guangzhou strains of red tilapia(GZ-red),and gonad transcriptome sequencing of hybrid fish ZX and female Nile tilapia XX at the critical stage of early sex determination and differentiation.The present study was intended to identify male sex determining region and candidate sex determining gene on LG1.Our laboratory has sequenced and assembled the whole genome sequences of nile tilapia named as WL-O.n-XX,WL-O.n-YY and blue tilapia named as WL-O.a-WW,WL-O.a-ZZ,and red tilapia of laboratory strain named as WL-red-XX,WL-red-XY,and red tilapia of Guangzhou strain named as GZ-red-XX,GZ-red-XY using the third generation sequencing.The main contents and results are as follows:1.Identification of sex chromosome of GZ-red.The 11 sex-linked markers suitable for ZX and WL-red fish,obtained in the previous study,were used to test GZ-red.The results showed that one of the sex-linked markers(Tmarker18)could distinguish the genotypes of male and female fish,indicating that LG1 is also the sex chromosome of GZ-red fish.2.Identification of the sex determining region of two strains of red tilapia by whole-genome resequencing.The whole-genome resequencing of 5 male and 5 female individuals of WL-red and GZ-red was performed on Illumina platforms.After quality control and filtering of raw data,an average of 24.08 G of clean data per sample was obtained.The clean data were mapped to the Nile tilapia reference genome assembly with an average mapping rate of 99.3%.Genome-wide association analysis of WL-red and GZ-red resequencing data showed that sex-linked SNPs are located on LG1 with high frequency and density.A total of 2021 sex-linked SNPs were identified between24.5-26.4 M on LG1,with the possible sex determining region spanning the 2 M region,much less than 9 M reported previously.3.Screening of sex-linked markers.The genomic sequences of the 2 M sex determining region were obtained from genome assembly of WL-O.n-XX,WL-O.n-YY,WL-O.a-ZZ,WL-O.a-WW,WL-red-XX,WL-red-XY,GZ-red-XX and GZ-red-XY.These genomic sequences were aligned using Bio Edit and primers were designed around structure variation(insertions or deletions)and named as marker1-marker7.These markers were used to discriminate ZX,WL-red,GZ-red female and male fish by PCR.Finally two sex-linked markers(Marker6 and 7)were obtained.These two sex-linked markers could distinguish genotypes of different tilapia strains with sex chromosome identified as LG1.4.Gonad transcriptome sequencing.The gonad transcriptome sequencing of ZX and XX fish were performed at 5,10,20,30 dah(days after hatching),the critical stages of early sex determination and differentiation.Differentially expressed genes between the ZX and XX fish were identified at 5,10,20,30 dah,with the number of1164,1494,4054,8154 genes at each stage respectively.Among them,there were 3male-biased genes(chs1,pbld,cgref1)within the 2 M sex determining region.The promoter region of chs1 showed obvious structural variations between the Z/Y and X allele of ZX,WL-red and GZ-red fish.Based on the above results,we speculate that chs1 might be a candidate sex determining gene for tilapias with LG1 as the sex chromosome.In summary,this study identified LG1 as the sex chromosome and narrowed the sex determining region from 9 M to 2 M by re-sequencing of two strains of red tilapia.Two sex-linked markers were screened out and widely applicable to different strains of tilapia with LG1 as sex chromosome.chs1 was identified as the candidate sex determining gene by bioinformatic and transcriptomic analyses.This study provided important theoretical foundation for the all-male breeding and molecular marker-assisted breeding of tilapia.It is also of great significance for analyzing the molecular mechanism of sex determination,the evolution of sex chromosomes in tilapia and the adaptive radiation of cichlids.