Sex determination by PCR-RFLP in the oriental white stork Ciconia boyciana

The Oriental White Stork (Ciconia boyciana) 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.