Development Of Sex-specific Markers And Sex Reversal In Largemouth Bass(Micropterus Salmoides)

Largemouth bass（Micropterus salmoides）belongs to Perciformes,cehtrachidae and Micropterus.It was native to North America and was introduced from Taiwan to the mainland in 1983.Because of the advantages such as fast growth,strong disease resistance,high capture rate,suitable for intensive culture,it has become an important special economic fish in China which had a production of 478000 tons in 2020.In the past decades,the two new species of“You Lu 1”and“You Lu 3”bred by our team promoted the rapid development of breeding industry of largemouth bass.However,the good varieties for largemouth bass are still lacking,which could not meet the demand for its industry development.It has been suggested that the female population of largemouth bass was suitable for one-year breeding system due to its faster growth rate than the male population.While the male population was suitable for two-year breeding system because of its less energy consumption for the development for the gonad and thus a more efficient food conversion.The breeding of the mono-sex population of largemouth bass will benefit for the development of its industry and increase the production.In this study,we first developed the sex-specific makers available for the genetic sex identification of largemouth bass through whole-genome re-sequencing.Then the early gonadal development and the expression patterns of the sex-linked genes was explored in largemouth bass.Finally,we attempted several sex reversal experiments with the different concentration addition of exogenously sex hormones.The main results are as followed1.Development of SNPs and In Dels for genetic sex identification of largemouth bassIn this study,5 females and 5 males were randomly selected and sequenced with whole-genome re-sequencing technology.The validation of the 35 SNPs located at Chr7 in three distinct population（n=74）indicated that 2 SNPs could distinguish the females and males with 100%accuracies,which were genotyped as CC and CG,AA and AT in the females and males,respectively.Further,8 In Dels with sizes more than 18bp were finally screened out on Chr7.2 male-specific In Dels（rs3394676 and rs3496814）located at Chr7 could effectively distinguish the above females and males with a PCR-based agarose gel electrophoresis,which performed as one band in the females and two bands in the males,respectively.This study provided a rapid method for the genetic sex identification of largemouth bass and contributed to the selection of the parents in the breeding of mono-sex population.2.Early gonadal development of largemouth bassIn this study,the histological observation,and sex-linked gene expression pattern of the gonadal development in juvenile of largemouth bass were conducted with a continuous 60 days after hatching.The results indicated that the gonadal differentiation system of largemouth bass was hermaphroditism.In addition,the gonads in female differentiated earlier than that in male.Primordial gonads were observed for the first time at 12 dph（days post hatch）.The female gonads differentiated into primary oocytes at 37 dph while the male gonads differentiated into spermatogonia at 51 dph.Gene expression showed that the expression of Foxl2 and Cyp19a1a in gonads of female was significantly increased at 20 dph,then decreased,and reached the second peak at 50 dph.In male largemouth bass,the expression of Dmrt1 reached the first peak at 30 dph,then decreased,and reached the second peak at 60 dph,while Gsdf gene showed an increasing trend with age.These results suggested that the time of the expression of sex-linked genes was earlier that the histological differentiation.Thus,we speculated that the key period of sex differentiation of female and male largemouth bass should be early than 20 dph and 30 dph,respectively.3.Effects of methyltestosterone-17α（MT）on growth and sex differentiation in Largemouth bassIn order to breed a all-female largemouth bass population,15 days old Largemouth bass with an average length of 15.1±0.09 mm were fed with a dietary supplementation of 0（MT0）,50（MT50）and 100 mg/kg（MT100）,respectively.After a continuous feeding of 60 days,the effects of MT on the growth,sex differentiation,hormone levels and gene expression variations in largemouth bass were analyzed,respectively.The body length and weight of Largemouth bass in MT50 and MT100 group were significantly lower than the MT0 group（P<0.05）.Both the male rate in the MT50 and MT100 group were 100%,which were significantly higher than that in the MT0 group（45%）（P<0.05）.Histological observation showed that the gonadal structures of physiological males induced by MT were similar to the wild males.Both the concentration of estradiol in the physiological males of MT50 and MT100 group were significantly lower than the wild females in the MT0 group（P<0.05）,and the concentration of testosterone in physiological males of MT50 group was significantly higher than the females in the MT100 and the MT0 group（P<0.05）.Additionally,in comparison to the wild females,the expressions of Dmrt1 and Gsdf in the gonads of physiological males in MT50 and MT100 group were up-regulated,while the expressions of Foxl2 and Cyp19a1a were down-regulated.In conclusion,dietary supplementation of 17α-methyltestosterone could induce the 15 days old female Largemouth bass turned into the physiological males successfully,and the suitable concentration was 50-100 mg/kg.4.Effects of oestradiol-17β（E₂）on growth and sex differentiation in Largemouth bassThe aim of this study was to study the effect of E₂on the sex differentiation of bass.The study was conducted on the Largemouth Bass（15.1±0.09 mm）after the film was taken as the study object,and 60 days were fed with feed containing 0（E0）,50 mg/kg（E50）,100 mg/kg（E100）and 200 mg/kg（E200）respectively.The effects of the above factors on sex differentiation,sex steroid hormone level and expression of gonadal development related genes were analyzed.The results showed that E₂could significantly inhibit the growth of the bass.In addition,the female rate of E50 group,E100 group and E200 group was 100%,which was significantly higher than that of control group（55%）.The results showed that the gonad structure of the female induced by E50 and E100 was similar to that of the female in the E0 group,while the gonads of the female in E200 group had no ovarian cavity structure.The results of sex steroid hormone determination showed that the content of estradiol in the serum of physiological female increased with the increase of E₂concentration,while the content of testosterone was opposite.In addition,compared with the male in the E0 group,the expression of Foxl2 and Cyp19a1a genes in the gonads of the female was significantly increased,while the expression of Dmrt1 and Gsdf genes was significantly down regulated.In conclusion,E₂can effectively induce the male Larch bass to be a female female,and the appropriate concentration is 50-100 mg/kg.