Study On Sex Determination Mechanism And Sex Relation Marker In Oreochromis Niloticus

The Nile tilapia(Oreochromis niloticus) is one of most important species in worldwide fish aquaculture. Research on the sex determination system in O.niloticus has provided evidence for genetic sex determination(GSD) with an XX female and XY male sex determination system. Analysis of the hybridization of probes drived from X and Y chromosomes to different genotype has demonstrated that the sequence differents exist between the sex chromosomes of O.niloticus. The inability to rapidly determine the genotypic sex of an individual is a problem in aquculture, where precocious maruration and subsequent reproduction can be a significant problem under certain conditions. The article tested male and female O. niloticus to search for molecular markers associated with sex chromosome. The major research results of this study are as follows:1. the Dmrt genes of O. niloticus were amplified by using a pair of degenerated primers which were designed based on the conservative DM domain of different species. The amplification band of Dmrt gene familay was-observed in testis of O. niloticus whose length was 140bp. Then, PCR products were cloned. To detect the different clones, SSCP technique was used. Two different Dmrt genes were obtained and sequenced respectively. After sequence analysis, those two Dmrt genes are named as onDmrt1, onDmrt2 according to their high homology to corresponding Human DMRT genes because their identities to those human DMRT genes in the amino acid sequence are 89.1ï¼…, 100ï¼…respectively; This may be concluded that Dmrt genes are highly conserved in phylogenetic. To study the function of Dmrt1 and Dmrt2 in development, we designed specific primers according the sequence of onDmrt1 and onDmrt2 for amplification by RT-PCR of different tissues in O. niloticus Dmrt2 expression is detectable in the brain, eye, gill, heart, testis and ovary. Dmrt1 expression is only detectable in the testis. No expression was detected for other tissues. This indicated that Dmrt2 genes may only play roles in the development of organs and Dmrt1 mainly participates in sex determining and differentiation. Based on DM-domain of in Dmrt1 of O. niloticus, primer P5 and P6 were designed. A rapid amplification of cDNA ends(RACE) was used for isolation of the length cDNA of Dmrt1 from testis of Oreochromis niloticus. We amplified a fragment about 1108bp by 3'RACE-PCR, encoding 262 amino acid. Sequence analysis revealed that the homology of Dmrt1 in O. niloticus and O. aurea is 99ï¼…. The secondary structure of Dmrt1 protein was predicted by the methods of Gamier-Robson, Chou-Fasman and Karplus-Schulz based on the amino acid sequence of Dmrt1. And Hydrophilicity plot, Surface probability plot and Antigenic index for Dmrt1 protein were obtained by the methods of Kyte-Doolittle, Emini and Jameson-Wolf, respectively. Theses results are helpful for studies on sex control mechanism of Dmrt1 in O. niloticus2. In the part of studies, Using a pair of degenerate primers based on the conservative region, HMG-box, of Human SRY gene, the Sox genes of O. niloticus were amplified. Five DNA bands with the length 200bp, 400bp, 600bp, 800bp, 1200bp were observed in the PCR products of both male and female O. niloticus. To detect the different clones, SSCP technic was used. Five different Sox genes were obtained and sequenced respectively. After sequence analysis, those Five Sox genes are named afer O. niloticus as onSox1a, onSox1b, onSox3, onSox4 and onSox12, according to their high homology to corresponding Human SOX genes because their identities to those human SOX genes in the amino acid sequence are 97.2ï¼…,97.2ï¼…,94.4ï¼…,96ï¼…,88.2ï¼…respectively, This may be concluded that Sox genes are highly conserved in phylogeny. The Sox3 gene expression analysis of different tissues from O. niloticus was studied by using RT-PCR. An expression was observed in testis in male, ovary in female, brain, eye in both male and female, no expression in gill, heart in both male and female. Based on these results, we suggest that Sox3 should play a key role in central nervous system development and gonad of O. niloticus. This research provided molecular data for the sex-determining mechanism of O. niloticus and expression pattern of Sox gene.3. RAPD analysis was applied to study sex and genetic diversity in O. niloticus. A total of 20 samples, which are 10 males and 10 females, were used in the test. Of 80 random oligonucleotide primers for the amplification of O. niloticus genomic DNA, 17 could produce reproducible, distinctive and characteristic bands from 200-2000 bp. 127 sites were detected, 45 of which(35.43ï¼…) were polymorphic. The average genetic similarity index and the average genetic distance were 0.8688 and 0.1312 respectively. Genetic diversity quantified by Shannon index(H₀) was 0.1910(male) and 0.1797 (female) respectively, with an average of 0.1854 Partition of genetic variation indicated that 13.63ï¼…was distributed within groups and 86.37ï¼…among male and female groups.. Difference bands can not be detected between male and female from the electrophoresis profiles. 4. In this paper genomic DNA polymorphism of female and male O. niloticus were detected using amplified fragment length polymorphism(AFLP) technique with 49 primer combinations. It produced a total of 2694 and an average of 55 bands. 20 primer combina tions were selected for further analysis. One AFLP band about 150bp related to the male was obtained with primer E4/M5, which was present in all males and absent in 14 of the 15 females of F₁ and 13 of the 15 F₂, it could have relations with sex.. The male associateed fragment about 150bp was cloned and sequenced. In order to convert the AFLP marker into SCAR(Sequence Characterized Amplified Regions) marker, one pair of 20-mer specific primer was constructed and used for PCR amplifing. The male-linked dominant SCAR marker was obtained.