Study Of Sex-specific Molecular Markers Andsome Sex-related Genes In Large Yellow Croaker (Larimichthys Crocea)

Large yellow croaker,Larimichthys crocea,is one of the most economically important marine fish in China,which shows obvious sexual dimorphism in growth.But it is difficult to distinguish the gender by the morphological characters before sexual maturity,and there is no difference in karyotype.So far,a molecular marker that can effectively identify the genetic sex of large yellow croaker is still absent and the molecular mechanism of its sex determination remains unknown.In this study,several polymorphic markers,which were identified from the genome re-sequencing data and significantly related to the sex of large yellow croaker,were screened and validated,and two sets of primers were successfully exploited and used to identify the genetic sex of different growth stages of large yellow croaker.Four genes including Dmrt1,Gsdf,Amh and Foxl2 were cloned and their mRNA expression levels were characterized in the large yellow croaker.The main results are as follows:1.Development of sex-specific molecular markers of large yellow croaker.Based on the predicted sex determination region of large yellow croaker through high-density gene map previously constructed in our laboratory,six sets of re-sequencing data(two males,two females,one mixed male pool and one mixed female pool respectively with 50 individuals)were carefully mapped to the referenced whole-genome sequences.The results showed that there was a heterozygous 15 bp insert / deletion fragment in the male Dmrt1 gene.Utilizing this insert / deletion fragment,we developed and verified two pairs of primers,which could be used to accurately identify the genetic sex of large yellow croaker.Meanwhile,these two pairs of primers were used to identify the genetic sex of large yellow croaker from larvae to adult.2.The open reading frame(ORF)of the large yellow croaker Dmrt1 gene is 918 bp which encoding 305 amino acids with a DM domain.Quantitative Real-time PCR(qRT-PCR)analysis showed that the Dmrt1 gene of large yellow croaker was specifically expressed in adult testis,but not in ovary and other tissues.The expression of Dmrt1 gene was first detectable in the male gonad after 41 days post hatching(dph),and reached the highest level in 112 dph then decreasing after that.However,the Dmrt1 gene showed a significant sexual dimorphism expression and was barely expressed in the female gonad.In addition,the expression level of Dmrt1 gene in gonad of pseudomale,which was female induced with androgen,was significantly up-regulated than that in normal female gonad(p < 0.05).These results indicated that the Dmrt1 gene has an important relationship with the formation of the testis of large yellow croaker.3.The Gsdf gene was specifically expressed in the gonads of the adult large yellow croaker and was highly expressed in testis,but no expression in other tissues.The Gsdf gene mRNA expression was detected firstly in the gonad of male large yellow croaker at 41 dph,and increased to the highest in 123 dph.The expression level of this gene in female was significantly lower than that in male(P < 0.05),but showed a similar trend that increasing first and then decreasing to male.In addition,the expression of Gsdf gene in pseudo-male gonad was significantly up-regulated(p < 0.05)compared with normal female.4.The expression of Amh gene was mainly in the gonad and the expression level in testis was significantly higher than that in ovary(p < 0.05).Gsdf gene expression was detectable in the male gonad at 41 dph.The expression level was significantly up-regulated at 55 dph,and reached the highest peak at 123 dph,but then decreased after that.However,this gene barely expressed in ovary.The expression of Amh gene in the gonad of pseudo-male was significantly higher than that in normal female and close to the expression level in normal male.5.The expression profile of Foxl2 gene in large yellow croaker was analyzed by qRT-PCR.It was found that the Foxl2 gene was mainly expressed in the brain as well as the gonad of large yellow croaker,and the expression showed significant sexual-dimorphism in the gonad.Moreover,Foxl2 gene was almost expressed in all growth stages gonads,but mainly in the stage of gonad differentiation,especially in the stage of ovary differentiation.In addition,the expression level of this gene in the pseudo-male gonad was significantly lower than that in normal female(p <0.05).