Identification Of Sex-specific Molecular Marker And The Preliminary Study Of Sex Determination Mechanism In The Yellow Drum (Nibea Albiflora)

Fish serves as an evolutionary link between invertebrates and higher vertebrates.It is the most species-rich group of vertebrates and possesses an amazing variety of sex determination mechanisms,which endows fish with a great advantage in studying sex chromosome evolution and sex determination mechanisms.Numerous aquatic fish species have substantially different sexual dimorphism.It is important to identify a sex-specific marker at developing sex control breeding and in deciphering the sex determination machenisms.In this study,a sex-linked marker have been successfully detected which can be used to effectively identify genetic sex of the yellow drum.Furthermore,three sex related genes?Dmrt1,Gsdf and Amh?were cloned,and the expression patterns of which were characterized in the yellow drum.Meanwhile,we discussed the sex determination mechanism of the yellow drum based on the aforementioned results.The main research results were as follows:1.We uncovered a 45 bp indel fragment between X and Y chromosome located in the first intron of the Dmrt1 gene from the yellow drum through comparing the gene structure difference.Two sex-specific primer pairs were designed to identify the genetic sex based on the 45 bp deletion within the Y chromosome.Importantly,genotyping the deletion in different cohorts of yellow drum confirmed its male-specific property.This male-specific marker with a good stability and reliability could be used to identify the genetic sex of the yellow drum.2.The full-length cDNA of the Dmrt1 gene in the yellow drum was 2457 bp with an open reading frame?ORF?of 906 bp encoding 301 amino acids.In the 3'-UTR of the Dmrt1,the same as other species,an 11-bp protein-binding motif was identified.The Dmrt1 gene of the yellow drum was specifically expressed in adult testis?P=0.0016?.The expression of the Dmrt1 gene was obviously detectable in the male gonad after 49 days post hatching?dph?,and reached to the highest level on 104 dph then decreasing.The stable expression was about 20%of the highest level?104 dph?.During the whole process of the gonad development,no expression of the Dmrt1gene was detected at ovary.These results indicated that the Dmrt1 gene played a critical role in the differentiation and development of the testis in the yellow drum.3.The Gsdf mainly expressed in the testis of the yellow drum?P<0.001?.In the other tissues,a small amount of expression was detected but its expression level was much lower than the testis.The expression of the Gsdf in the genetic males was detected on 40 dph and reached to the maximum peak on 120 dph.During the whole process of the gonad development,no expression of the Gsdf gene was detected at ovary.These results suggested that the Gsdf was tightly related to the testis differentiation and development in the yellow drum.4.The Amh gene of the yellow drum was specifically expressed in adult testis(P=5.35×10^-6).The first and tiny expression of Amh gene was detected on 40 dph and reached to the highest level on 78 dph.The expression level has become stable after 390 dph,which was about 10%of the highest level?78 dph?.During the whole process of the gonad development,no expression of the Amh gene was detected at ovary.The Amh gene showed highly expression at the stage of the testis differentiation in the yellow drum,which indicated that participated in the process of the differentiation and development of the testis in the yellow drum.