| Siniperca chuatsi,commonly known as mandarin fish,is a valuable fish in freshwater aquaculture in China,and exhibits typical dimorphism in growth and reproduction between males and females at diferent times and seasons.In this study,the whole-genome resequencing library was constructed and sequenced,and the whole-gename sequencing data were used to screen the male and female loci of S.chuatsi for differences in the genomic DNA of the two sexes,to find the molecular markers associated with their sex,and to validate them in multiple geographical populations.The first round of hormone fee,ding induction experiments was designed,and the second round of refined hormone feeding induction experiments was designed based on histological and anatomical observations and mortality rates of S.chuatsi to determine the optimal induction concentration and provide a theoretical basis for the preparation of all-male Mandarin chuatsi and the molecular mechanism of sex determination in Mandarin.The main study contents and results are as follows:1.A total of 48 mandarin chuatsi from eight populations(three male and three female from each population)were selected for whole-genome resequencing library construction and sequencing.The resequencing data were compared with the reference genome of mandarin chuatsi to obtain differential male and female loci,primers were designed for PCR amplification and Sanger sequencing to validate,and eight populations were used to detect and screen out universal male-and femalespecific molecular markers to construct a sex marker platform for mandarin chuats.The results showed that 1186 male-to-female differantial SNP loci were obtained,including 277 Indel loci.There were six types of candidate loci obtained,among which the most number of SNP loci were obtained for the female-deficient male heterozygous type,with a total of 497 loci,followed by 397 for the femaleheterozygous male deficient type,129 for the female-pure male heterozygous type,72 for the female-deficient male pure type,64 for the female-heterozygous male pure type and the least number of 27 for the female-pure male deficient type:the results were verified by designing primers for PCR and Sanger sequencing in eight geographical groups(30 male and 30 female).Five SNP markers,numbered SNP1SNP5,showed specificity for both males and females in all eight geographical groups;SNP1-SNP5 were validated by expanding the design of primers for Sanger sequencing using 30 tails of each male and female from the eight geographical groups,with Identification accuracy of 100%.2.By setting five concentration gradients of estradiol 500 mg/kg,1500 mg/kg,4500 mg/kg,13500 mg/kg and 50500 mg/kg(referred to as E1-E5)to induce common Mandarin reversal,the appropriate induction concentration was determined to be 1500 mg/kg;then a second round of refined induction experiments were set up,setting 1000 mg/kg,The results showed that in the two rounds of hormone feeding experiments,Mandarin chuatsi were treated with different concentrations of estradiol from 50 to 60 days of age.The inhibition was more pronounced as the concentration of estradiol increased.At 5 months of age,there was no significant difference between the concentration groups and the control group,indicating that the effect of estradiol on the growth and development of Mandarin chuatsi was reversible.The histological and anatomical study of the gonads revealed that the gonads of female treated with estradiol were differentiated at 34 days of age in the low concentration group,earlier than the control group at 40 days of age,and in the gonads of male fish,the gonads of the high concentration group were differentiated at 34 days of age,later than the control males at 27 days of age.The proportion of parthenogenetic gonads was higher in the low estradiol-treated group,while the proportion of malformed gonads was higher in the high estradiol-treated group,and the male gonads developed towards the spermathecae in the first half of the treatment period,but some of them degenerated to form parthenogenetic gonads,which were expressed as spermathecae like ovaries or incomplete ovaries.In the male Mandarin chuatsi,the GSI was significantly lower than that of the control group in the E4 and E5 groups,and significantly higher than that of the control group in the rest of the experimental groups.The results showed that the optimum concentration of estradiol to induce sex reversal in S.chuatsi was 1000 mg/kg,and at this concentration,the mortality rate of S.chuatsi was 41.3%,the proportion of complete and parthenogenetic ovaries was 92%together,and the induction rate was 100%.. |
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