Cloning and Application of Sex-specific DNA Fragments from Ostriches and Columbidae Birds

• Main Authors: Chean-Ping Wu, 吳建平
• Other Authors: 黃木秋
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/99093067610740860401

博士 === 國立中興大學 === 畜產學系所 === 94 === Cloning and Application of Sex-specific DNA Fragments from Ostriches and Columbidae Birds Abstract Random primers were used for random amplified polymorphic DNA fingerprinting to sexing Ostrich and Streptopelia orientalis. The OPAJ-13 primer produced a sex-specific fragment in all females but no males for Ostrich. Moreover, the OPAJ-13 primer produced a novel roughly 750bp DNA fragment found only in tested females of Ostrich. The OPAV-17 primer produced an approximately 900 bp sex-specific band in all S. orientalis females but not in males. These sex-specific DNA fragments of ostrich and S. orientalis were isolated and constructed into pCRII-TOPO vector for nucleotide sequencing. Two novel 760 bp and 902 bp sex-specific DNA sequences were obtained from female ostrich and S. orientalis. Two primers of OstSexOPAJ13-F and R were designed based on the cloned female-specific sequence of ostriches. The genomic DNA samples of ostriches were amplified using these two primers by PCR. The sex-specific fragment in the gel appeared in all ostrich females but not in males. A pair of primer (TurSexOPAV17F&R) was designed based on the cloned female-specific sequence to amplify the female-specific band by PCR for S. orientalis sexing. Sex-specific bands in the gel of PCR products were represent in females but not males. This sex-specific marker was also amplified from genomic DNA samples of Columbidae S. chinensis and S. livia. Sequence analysis showed that this novel sex-specific marker is highly conserved among these three bird species. In contrast, the PCR product was not amplified from male DNA from these species, or from either sex of the S. chinensis formosa bird. Therefore, our experiments show that our novel marker can be used to rapidly and accurately identify the sex of birds from three species of Columbidae. The Columbidae sexing identification problem can be solved using these two novel primers by PCR. In conclusion, resoult showen RAPD was the ways of proceduce for screening unknowen birds sex-specific gene with different primers RAPD from several known sex ostrich and Columbidae S. orientalis genomic DNA, and screening sex-specific fragments, sequenceing, designing primers and used for ostrich and Columbidae birds sex identification. The sex-specific sequences of birds obtained from these trails in this study were all novel sequences. Two pairs of primers were designed according to the cloned sex-specific sequences for sexing. OstSexOPAJ13-F&R primers could be used for sex determination in ostrich. The sex identified of Columbidae S. orientalis, S.chinensis and S. livia could be performed easily with TurSexOPAV17F&R primers by PCR.