| Blue tilapia(Oreochromis aureus),belongs to Perciformes,Cichlidae and Oreochromis,was originated from west Africa,downstream Nile river and Israel.The blue tilapia are with strong adaptability,salt tolerance,hypoxia tolerance and disease resistance.It is one of the world’s important aquaculture fish and also an important freshwater aquaculture species in China.The growth of tilapia displays obvious sexual dimorphism,and males grows 40～50% faster than females.Therefore,the development of all-male stock would be of significant benefit for aquaculture.Currently,hormonal treatment is used to produce all male or nearly all male tilapia populations,which is not environmentally friendly.Sex controlled breeding of blue tilapia through genetic breeding can not only improve yield but also avoid food safety issues cause by hormonal treatment.Thus,it is urgent to develop sex-linked molecular markers and identify sex-determining gene in blue tilapia.Although there were some reports of sex-linked molecular markers in blue tilapia,they were only developed by family population and could not be applied to other populations which limits their wide application.Thus,it is of great significance to develop the universal sex-linked molecular markers for wide application of marker assisted selection in production of genetically male blue tilapia.In addition,tilapia is an important species of Cichlidae,a representative group of rapid radiation,and diversity of its sex determination mechanisms.There are male heterogametic(XX/XY)species,including Nile tilapia and female heterogametic(ZZ/ZW)species,including blue tilapia.Identification of sex-liked molecular marker and sex-determining gene of blue tilapia is helpful to analyze the evolution of sex-determining mechanism and improve our understanding of the molecular mechanism of rapid adaptive speciation.With the development of next generation sequencing(NGS)and the reduction of sequencing cost,development of molecular markers and mapping technology have been developed rapidly.Identification of the sex-determining region(SDR)and development of sex-liked molecular markers by NGS technology have been successfully applied to many species,which establish a solid foundation for SDR identification and sex-linked markers development in blue tilapia.In this study,whole genome re-sequencing data of five different populations,and high quality genomes of male(ZZ)and super-female(WW)of blue tilapia assembled in our lab were used to screen out sex-linked molecular markers and identify the SDR via whole genome association analysis.The main research results are as follows:1.Identification of SDR based on re-sequencing data of five different populations of blue tilapia.Totally,a male pool(50 male individuals)and 5 female individuals from laboratory family(WL),5 females from Wuxi Freshwater Fisheries Research Center(Wuxi)and 5 females from Guangdong Tilapia Breeding(GZ)were used for resequencing.In total,298.99 Gb clean reads were obtained.These data,incorporated with re-sequencing data of a female pool(22 female individuals)and a male pool(22 male individuals)and a female individual and a male individual of blue tilapia from NCBI platform were mapped to the WW reference genome.GWAS analysis was used to call sex-linked SNPs.In total,41 sex-linked SNPs have been identified,which were located on the region of 71,859,554-78,987,068 bp of LG3.These is the SDR of blue tilapia.Our analysis based on resequencing data from 5 populations has narrowed the SDR(～50 Mb)identify by the Kocher reaserch team based on pooled sequecing data form family population.Moreover,SDR identify by Fan Zheng based on SLAF sequencing data form family population,which is 3.85 Mb apart compared with the SDR identified in present study.2.Validation of sex-linked SNPs by PCR amplification Sanger sequencing.Of the 41 sex-linked SNPs,10 were randomly selected to extract the flanking 1000 bp sequencing on WW genome.Subsequently,primers were designed and used to amplify the genome of 7 males pools(35 male individuals)and 3 superfemales pools(15 male individuals)and 15 female individuals from WL,Wuxi and GZ population to identify the genotype.The Sanger sequencing results showed that 5 SNPs were consistent with the results of resequencing and complete linked to sex,which confirms the reliability of the SDR in blue tilapia.3.Screening of the sex-linked molecular markers in blue tilapia.In the sex determining region,genome sequences of ZZ and WW were aligned to identify the structure variation.Primers were designed to amplify the W-or Z-specific sequences.Four effective molecular markers consistent with phenotype sex were identified from200 pairs of primers,named Maker-1,2,3 and 4,respectively.The Marker-1,2 and 3were W-specific markers and Maker-4 was Z-specific marker.Totally,292 individuals,including 196 individuals form WL population,78 individuals from Wuxi and 18 individuals from GZ,were used to verify the specificity of these four markers.The results showed that Marker-1,2 and 3 can accurately identify the genetic sex of all individuals from three different populations,but could not distinguish between female(ZW)and super female(WW);Maker-4 can amplify Z specific bands in ZZ and ZW.Combining any one of Marker-1,2,3 with Marker-4 can identify the genotypes of ZZ,ZW and WW.The phenotypes of the blue tilapia identified by the four sex-linked molecular markers can be 100% concordance with the genotypes,which confirms the reliability of the SDR identified on LG3 and lays a foundation for sex control breeding and sex-determining gene identification in blue tilapia.In summary,the present study identified the sex determining region of blue tilapia,and four universal sex-linked molecular markers,which laid a foundation for sex control breeding,identification of sex-determining gene,the study of the molecular mechanism of sex determination of blue tilapia and understanding of the molecular mechanism of rapid radiation speciation in cichlids. |
