| Tilapia is one of the three fishes which the FAO introduced to the whole world, and now it’s cultivated all through the world. Nile tilapia is the most widely breeding species. Owing to the high growth speed of the male Nile tilapia which is40%faster than the female, the gender control breeding producing the all-male Nile tilapia will significantly improve the overall yield. The combination of genetically male tilapia (GMT) and gender specific molecular markers would significantly improve the all-male fish production process. In our research, AFLP molecule marker technology was used to screen the genomic DNA of XX, XY and YY genotype Nile tilapia. At the end, we got eight AFLP fragments associated with X chromosome, and transformed one fragment to a SCAR marker linked with X chromosome. The SCAR marker NTX is considered as the foundation of appraisal method of supermale Nile tilapia to speed up the all-male fish production process.The genomic DNA of XX, XY and YY genotype Nile tilapia were conducted following four steps:restriction of DNA by two enzymes (Pst I and Mse I), simultaneous ligation of adaptors, amplification of the resulted fragments with preselective primers and amplification of the diluted products with selective primers. After screening with256selective primers, specific bands linked with X chromosome were found in the result of eight primers (P2M2, P2M11, P5M14, P6M5, P11M5, P14M16, P15M8and P15M13) which named according to the primers. Then, based on the sequencing and genome walking, the flank sequence of four fragments (P2M2, P11M5, P15M8and P15M13) was obtained. In order to search the difference between X and Y chromosome, amplifications of part sequence of the four fragments and its flank sequence in XX and YY genotype individuals were carried out. In the result, a mutation site (C/T) was found in P15M8fragment. However, according to the analysis of P15M8fragment and its flank sequence in blastx of NCBI, none homologous sequence was found. Therefore, we considered it as part of a new gene. Then, the primer of NTX was designed and verified in XX, XY and YY genotype individuals, respectively. A109bp band was got in all individuals of XX and XY genotype, while only one individuals of YY genotype got the band. Therefore, the primer of NTX can be used as the foundation of an effective method in the all-male Nile tilapia breeding progress to identify the supermale fish at an early age, and speed up the process of Nile tilapia breeding.Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of glucagons superfamily, is able to stimulate the secretion of growth hormone, gonadotropin, prolactin and somatolactin, which has impact on the development of brain and reproduction. In the research, RT-PCR and RACE technology were used to clone PACAP of Nile tilapia. The full length of PACAP cDNA was1013bp, and a coding sequence of528bp. Using PCR-SSCP to detect SNPs in PACAP coding area, the primer of P2revealed3genotypes which were recoded as AA, AB and BB. One SNP (G42A) was confirmed in exon2of PACAP, and no amino acid was changed. The total length, standard length, body depth, body width, caudal fin depth and body weight of individuals with AA genotype were significantly different from those of individuals with AB genotype in male population. Therefore, genotype AA has a positive influence on the growth traits of male Nile tilapia, while genotype AB is opposite. SNP (G42A) can be used in marker assisted selection of male Nile tilapia breeding in the future.Myogenic factor6(MYF6), a member of myogenic regulatory factors (MRFs) family, abundantly expressed in many adult muscle fibres. MRFs family also contains Myf4, Myf5and MyoD. Myf6and Myf4are involved in differentiation of myotubes, while Myf5and MyoD are required for the determination of skeletal myoblasts. In the research, RT-PCR and RACE technology were used to clone MYF6of Nile tilapia. The full length of MYF6cDNA was990bp, and a coding sequence of678bp. Using PCR-SSCP to detect SNPs in MYF6coding area, the primer of M2revealed3genotypes which were recoded as AA, AB and BB. One SNP (G137T) was confirmed in exon1of MYF6, and no amino acid was changed. The body depth of individuals with AA genotype was significantly different from those of individuals with AB genotype in male population (P<0.05). The total length, standard length, body depth and body weight of individuals with AA genotype were significantly different from those of individuals with AB genotype (P<0.05). Therefore, genotype AA has a positive influence on the growth traits of Nile tilapia, while genotype AB is opposite. SNP (G137T) can be used in marker assisted selection of Nile tilapia breeding in the future. |
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