| Tilapia is one of the most productive and internationally traded freshwater food fish in aquaculture.Tilapia has been popular with consumers because of its delicious meat quality and fewer intermuscular bones.China is by far the largest producer and exporter of tilapia products and is currently producing one half of the global supply.Because the males grow faster and reach a larger size than females,mono-sex culturing of tilapia has important applications in aquaculture.The traditional way to produce monosex progeny is hormone treatment.Hormone treatment has raised concerns because it may affect consumer acceptance of the fish,and hormone residues may damage water quality and biodiversity.Another way to obtain all male tilapia is using the hybrid(cross of ♀ O.niloticus and ♂ O.aureus).Test crosses were needed to identify parental genotypes,due to the lack of accurate sex-linked molecular markers,which are time-consuming.There are molecular markers to distinguish XX,XY,and YY individuals in Nile tilapia.Recently,amhy has been identified to be the master sex determination gene in Nile tilapia.However,there is no practical molecular marker to accurately identify the sex and different genotypes of blue tilapia.Therefore,it is of great significance to develop the sex-linked molecular markers for blue tilapia.On the other hand,the blue tilapia and Nile tilapia are closely related species,with the genetic similarity higher than 95%,but with completely different sex chromosomes and sex determining systems.Nile tilapia have a male heterogametic(XX/XY)system,and the sex-determining locus is on either linkage group(LG)1 or 23,depending on the strain.In blue tilapia,the sex-determining loci include both a Y locus on LG1 and an epistatically dominant W locus on LG3.Thus,identification of sex-linked molecular markers and sex determining genes in tilapia will also improve our understanding of the rapid and adaptive radiation of cichlids in evolutionary biology.In this study,we first established a strain of blue tilapia,which provided sufficient experimental materials for subsequent studies.Using SLAF sequencing confirmed blue tilapia sex chromosomes in LG3,and find the sex determination region.The primers were designed to identify the sex-linked molecular markers in places where the ZZ and WW individual genomes were different within the sex determination region by using the resequencing data.A total of 236 individuals in the F2 generation were used to identify markers for narrow the region of sex determination.A candidate gene for sex determination of blue tilapia was fund and a new scheme for GMT was proposed.The results are as follows:1.F2 Progeny production.The F1 generation was obtained by cross ZZ(67958)with ZW(67013).Three different crossing combinations were carried out to produce F2 progeny.Type 1: By crossing normal ZW(♀)and sex reversed ZW(♂),93 individuals with male and female ratio of 3:1 and genotype ratio of 1(WW): 2(ZW): 1(ZZ)were obtained for SLAF sequencing.Type 2: By crossing normal ZZ(♂)and ZW(♀),103 individuals with male(ZZ)and female(ZW)ratio of 1: 1 were obtained,which were mainly used to verify the molecular markers.Type 3: By crossing WW(♀)and sex reversal ZW(♂),40 individuals of all females offspring with the genotype radio of 1(ZW): 1(WW)were obtained to obtain a large number of WW.2.SLAF-seq was used to identify sex chromosome and sex determining region.Totally,the aformentioned 93 individuals and their parents were used for SLAF-seq.In total,499.59 M pair-end reads and 822,487 SLAF loci were obtained.The sex determining region was located on the LG3 b within 0-7,319,395.3.Identification of sex-linked markers.Primers were designed to identify sex-linked markers based on SLAF survey of the genomic sequence differences between ZZ and WW.Five effective molecular markers from 200 pairs of primers linked to sex were obtained,named Marker-1,2,3,4,and 5,respectively.Marker-1 and Marker-2 could accurately discriminate ZZ,ZW and WW genotype.Marker-3,Marker-4 and Marker-5 could only identified female and male,but failed to distinguish ZW from WW.Marker-1 and Marker-2 could identified the genotype of all individuals accurately based on the analysis of individual genotypes.However,Marker-3,Marker-4 and Marker-5 were inconsistent in one and two individual,respectively.4.Narrowed the sex determination regions to 3.6 Mb and found a sex determination candidate gene.A schematic diagram about pseudo-LG3 of blue tilapia was proposed and the markers was mapped on this chromosome.The sex determining region was in the region of 0-3,649,243 on LG3 b.5.WW superfemale has been established by Marker-1 and Marker-2.With previously established YY supermale in our lab,we can obtain genomic male tilapia of WY.A new scheme producing all male tilapia with genotype WY was proposed for MAS..First,using Fadrozole to treat the offspring of ZZ(♂)× ZW(♀),sex reveal male ZW(♂)was obtained by sex-linked marker.Secondly,the superfemale WW(♀)was obtained using sex-linked markers from ZW(♀)× ZW(♂).Next,the hybrid male tilapia with the genotype of WY was obtained by WW(♀)× YY(♂).In addition,a number of WW superfemale could be obtained by WW(♀)× ZW(♂).In conclusion,the progeny of blue tilapia was established and five sex-linked markers was isolated in this study.And completed the progress of genetic male tilapia by MAS.What’s more,narrowed the sex determination region to 3.6 Mb. |
