Screening Of Sex-related Microsatellite Markers In Tilapia And Their Application In Breeding Of All-Male Tilapia

Tilapia is an important freshwater fish in the world,of which the sexual maturity time is early,the spawning cycle is short after sexual maturity.So the male tilapia has more growth advantages than the female fish.Moreover,the mixed culture of males and females will result in too large tilapia breeding density and uneven harvest specifications,which will have a great impact on the commercial production of tilapia.Therefore,the commercial production of tilapia mainly depends on the monogamous breeding of male tilapia at present.Tilapia“Yue Min No.1”is a new all-male tilapia variety bred by our research team.This tilapia variety is a hereditary male fish,which does not need to be treated with hormones and has excellent growth performance.In the breeding process of tilapia"Yue Min No.1",the XY-pseudo females and YY₁ super males tilapia need to be identified by traditional cross-breeding experiments,but the screening process is complex and the cycle is long.In this study,the optimum feminization conditions of Oreochromis niloticus were explored to cultivate XY-transformed females and YY super males,and to identify XY-transformed females and YY super males by developing microsatellite markers related to the sex of tilapia.The purpose of this study includes two aspects:on the one hand,to shorten the breeding cycle of"Yue Min No.1"and reduce the cost,on the other hand,to provide reference for the identification of genetic sex of tilapia.The main achievements are as follows:1.Screening of sex-related microsatellite markers in Oreochromis niloticus and Oreochromis aureusA total of 24 pairs of microsatellite primers were used to screen sex-related microsatellite markers in two O.niloticus populations and two O.aureus populations using conventional PCR and polyacrylamide gel electrophoresis.And a total of 6microsatellite markers that are UNH931,GM128,GM201,GM258,GM597 and UNH898 were identified to tilapia sex.These six microsatellite markers were then amplified in the two O.niloticus populations,two O.aureus populations,and the ZY₁,WY₁,YY₁,YY₂ tilapia populations using touchdown PCR and capillary electrophoresis.The genetic diversity parameters of each population were counted,six microsatellite markers detected 95 alleles in 298 samples from the above populations,ranging in size from 97 to 302 bp.The number of alleles in each population ranged from 1 to 13,the average number of observed alleles was 2.167⁹.333,the average effective allele number was 1.624⁴.966,the average observed heterozygosity was 0.324⁰.983,the average expected heterozygosity was 0.329⁰.782,and the average polymorphic information content was 0.275⁰.753.The two O.niloticus populations,WY₁ and YY₂ populations reached a high polymorphism（PIC>0.5）,and the two O.aureus populations,ZY₁ and YY₁ populations were moderately polymorphic（0.25<PIC<0.5）.Cluster analysis showed that all groups were clustered into two branches.two O.aureus populations and ZY₁population were clustered together.two O.niloticus populations,WY₁,YY₁ and YY₂populations were clustered into another branch.Sex association analysis revealed that GM597 was significantly associated with the sex of O.niloticus population of Wuxi,O.niloticus population of Gaoyao and O.aureus population of Panyu（P<0.05）.UNH898was significantly correlated with the sex of the O.niloticus population of Gaoyao（P<0.01）.The combined analysis of GM597 and UNH898 can 100%identify the sex of two O.niloticus populations.GM597 can be used as a candidate marker for screening XY-pseudo females and YY₁-super males,because it is significantly correlated with the sex of both O.niloticus populations.2.Exploration on the conditions of tilapia feminization and the verification of microsatellite marker GM597 in genetic improvement of farmed tilapia（GIFT）33^#XX female of GIFT were crossed with 36^#YY super male to obtain S1 family,33^#XX female of GIFT were crossed with 31^#XY male of GIFT to obtain S2 family,30^#XX female of GIFT were crossed with 31 XY male of GIFT to obtain S3 family.The embryos of 1 day post-fertilization（dpf）of each family were divided into four groups for feminization treatment,which included hormone immersion and hormone feeding.The control group was not treated.1 dpf embryos were immersed in EB for 4 hours,and fed normally after opening the mouth in group 1.In group 2,embryos were not treated with EB immersion but were fed for 60 days after opening the mouth with fodder added in EB.In group 3,the embryos of 1 dpf were immersed in EB for 4 hours,and then fed with fodder added in EB for 60 days after opening the mouth.The results showed that the ratio of male to female in S1 family control group was close to the theoretical value of 0:1,the ratio of female to male in S2 and S3 family control group was close to the theoretical value of 1:1.The ratio of female to male in 3 families group 1 was close to 2:1,the average female rate was 66.76%.The ratio of female to male in 3 families group 2 was about 1:0,the average female rate was 94.34%,and the ratio of female to male in 3 families group3 was about 1:0,with an average female rate of 94.64%.The female rate of group 2 and3 was the highest and the difference was not significant.In view of the simplicity of the operation,the feminization mode of experimental group 2 could be used in subsequent experiments.GM597 was amplified in three full-sib families and their parents of GIFT,the 127 bp allele was found to be linked to X chromosome of three families,and 137 bp allele was linked to Y chromosome of three families.3.Breeding and genetic sex identification of XY-pseudo females and YY super malesThe mixed population of GIFT was divided into two groups,about 1000 tilapias in each group.EB mixture was fed for 60 days in the experimental group and normal feeding in the control group was carried out.The results showed that the female rate of the control group was 48.23%,and that of the experimental group was 95%.A total of 166 samples were selected from the experimental group and 75 XY-pseudo females were confirmed by GM597.Hybridization experiments were carried out using male GIFT and selected XY-pseudo female.7^#XY transformed female and 36^#YY super male were crossed to obtain S4 family,11^#XY transformed female and 31^#XY male were crossed to obtain S5 family,19^#XX female and 28^#XY male were crossed to obtain S6 family.The method of dry fertilization was used and the fertilized eggs were hatched in incubator.Feeding bait was started after opening the mouth of juvenile fish.150 samples were randomly selected from each family to amplify GM597.Among the 150 samples of S4 family,the genotype of 72 individuals were identified as XY and the genotype of 78 individuals were YY,the ratio of which was close to the theoretical value of 1:1.Among 150 samples from S5family,the genotype of 36 individuals were XX,the genotype of 75 individuals were XY and the genotype of 41 individuals were YY,the ratio of the three genotypes was close to the theoretical value of 1:2:1.Among 150 samples from S6 families,the genotype of 38individuals were XX,the genotype of 73 individuals were XY and the genotype of 39individuals were YY.The ratio of the three was close to the theoretical value of 1:2:1.YY super male tilapias were successfully screened by GM597.