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Vol. 48 No. 5, 2009
Sex Determination by PCR-RFLP in the Oriental White Stork Ciconia boyciana
Jae-Ik Han1, Hye-Jin Jang1, Seokhwan Cheong2, Sukyung Kim2, Shi-Ryong Park2, and Ki-Jeong Na1,*
1Veterinary
Laboratory Medicine, Department of Veterinary Medicine, College of
Veterinary Medicine, Chungbuk National University, Gaeshin-dong,
Sungbong-ro 410, Cheongju 361-763, South Korea
2CKorea Institute of Oriental White Stork Rehabilitation
Research, Korea National University of Education, Cheongwon 363-892,
South Korea
Jae-Ik Han, Hye-Jin Jang, Seokhwan Cheong, Sukyung Kim, Shi-Ryong Park, and Ki-Jeong Na (2009) The Oriental White Stork (Ciconia boycaian)
is difficult to sex based on phenotype alone, and thus alternative
methods capable of producing an unambiguous result are necessary.
Griffiths’ polymerase chain reaction (PCR) method based on intronic
length polymorphism in the chromo-helicase DNA-binding (CHD) protein
gene is widely used in birds; however, we were able to show that it is
not appropriate for sexing this species of stork, as it resulted in a
single band of approximately 380 bp in both males and females on a 2%
agarose gel. However, Griffiths’ sexing method can be applied to
many birds, and we made applicable that method to this stork using a
restriction enzyme. Sequencing of the male’s CHD-Z PCR product
revealed a 377 bp fragment with several specific restriction sites,
including HaeIII. A restriction fragment length polymorphism (RFLP) analysis of the female PCR product with HaeIII
and subsequent sequencing showed that the 380 bp band was, in fact,
composed of 2 fragments, one of 377 bp (CHD-Z) and a larger fragment of
384 bp (CHD-W), specific to females. A number of restriction
enzymes specific for CHD-W were also identified. Consequently, we
demonstrate that a combination of PCR together with RFLP analysis
(PCR-RFLP) is a simple and rapid method for sexing this stork species.
Key words: Oriental White Stork, Chromo-helicase-DNA-binding (CHD) protein, PCR, Restriction fragment length polymorphism (RFLP), HaeIII.
*Correspondence: Fax: 82-43-2613224. E-mail:sigol@cbnu.ac.kr
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