Analysis of CHD Gene Polymorphism as a Model Object for Molecular Sexing of Eurasian Eagle-Owl (Bubo bubo)
2021
Kulibaba, R. O. | Liashenko, Yu. V.
CHD gene polymorphism as a model object for molecular sex determination in Bubo bubo (Eurasian eagle-owl) was studied. Classical PCR methods for amplification of target regions of the CHD gene using P2/P8, 2550F/2718R, and 123L/1272H marker systems and restriction (PCR-RFLP) and heteroduplex methodological approaches were analyzed. It was shown that the use of the P2/P8 marker system within classical PCR is efficient for avian sexing only under electrophoretic separation of the fragments in polyacrylamide gel. The application of 2550F/2718R and 123L/127H markers demonstrated their lack of efficiency both in agarose and polyacrylamide gels because it was not possible to differentiate between genotypes CHDZZ and CHDZᵂ. The presence of a polymorphic restriction site for HaeIII in CHDZ and CHDᵂ fragments amplified with primers P2/P8 was proven. No polymorphic site was detected for DdeI restriction endonuclease. High efficiency of the heteroduplex analysis based on the P2/P8 markers for sexing individuals under electrophoretic separation of the amplified fragments both in agarose and polyacrylamide gels was proven. In case of experimental samples containing CHDZ and CHDᵂ amplicons, additional fragments of heteroduplex DNA were found, the presence of which makes it possible to accurately determine the genotype of an avian.
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