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That most Columbidae birds have no conspicuous sexual dimorphism often makes it difficult to identify their sex on the basis of external morphology. In the present study, we report a novel sex-specific DNA marker in Columbidae birds. DNA was extracted from one member of this bird group, Streptopelia orientalis (S. orientalis, oriental turtle dove), and used to identify a female-specific DNA marker using a random amplified polymorphic DNA (RAPD) fingerprinting. One hundred and sixty random primers were used for the RAPD-PCR reactions. When using the OPAV17 primer, a novel 902 bp sex-specific PCR product was amplified from known female birds. This fragment of DNA was cloned and sequenced. Two primers, TurSexOPAV17-F and TurSexOPAV17-R, were designed from the cloned sex-specific sequence, and were successfully used to amplify a 777 bp female-specific fragment using PCR from S. orientalis DNA. This sex-specific marker was also amplified from genomic DNA samples of two other female Columbidae, S. chinensis and Columba livia. Sequence analysis showed that this novel sex-specific marker was highly conserved amongst these three bird species. In contrast, the PCR product was not amplified from male DNA of these species, nor from either sex of the S. chinensis formosa birds. Therefore, we concluded that our novel marker can be used to rapidly and accurately identify the sex of birds from three species of Columbidae.

The absence of conspicuous sexual dimorphism in pigeons often makes it difficult to determine their sex on the basis of external morphology. We identified a novel female-specific DNA marker in pigeons, presenting the possibility of pigeon gender determination using a PCR-based method. One-hundred and twenty random primers were used for RAPD fingerprinting in order to find any sex-specific fragments in pigeons. One of these primers, OPC-20, produced a female-specific band in the DNA fingerprints. This DNA fragment was isolated from the gel and inserted into a vector for nucleotide sequencing. A novel female-specific 732 bp sequence was obtained. A pair of primers (DoveOPC20F & R) was designed, based on the cloned sequence, for amplifying the female-specific band by PCR for pigeon gender determination. Sex-specific bands in the gel were observed in all females but not in males. The PCR products in the gel were then transferred onto nylon membranes and hybridized with a DIG-labeled probe of the cloned female-specific DNA fragment. Clear hybridization signals were found only in all of the female pigeons; the same result was obtained from dot blot hybridization. This demonstrates that the sex of pigeons can be accurately and rapidly identified by PCR.