A . - Determining the sex of a bird on the basis of its morphological characteristics is difficult in% of bird species; in chicks, this rate is even higher. Since males have two identical sex chromosomes (ZZ), while females are heterogametic (ZW), the development of a DNA-based sexing method has been extensively investigated in several species. Gri ﬃ ths et al. reported a simple DNA test using intronic size variation; however, this method cannot differentiate birds that have small or no size variations. We present a novel and simple DNA test for avian sex determination using a novel primer (P) specific for the unique sequence of the chromium-helicase (CHD) -W DNA-binding gene. Use of a multiplex polymerase chain reaction (PCR) and simple agarose gel electrophoresis with P and with the primers P and P described by Griﬃths et al. revealed that the combination P - P could amplify the partial CHD-W gene alone.
Consequently, we found that P could be used to identify only the CHD-W gene. We anticipate that this multiplex PCR will be useful for universal sexing of birds, regardless of intron size variation. She receivedO ctob er accepted April . Keywords: CHD gene, CHD-W specific primer, sex differentiation, universal sexing. S is important not only in behavioral ecology but also in evolutionary biology, genetics, and conservation biology, especially for captive breeding of endangered species (Ito et al. ). However, it is difficult to identify sex in many bird species from phenotypic characteristics alone. It has been estimated that adults cannot be phenotypically sexed in> % of bird species, and sexing by phenotype is even more difficult in chicks (Grifths et al. ). Since males have two identical sex chromosomes (ZZ), while females are heterogametic (ZW), several methods have been described using polymerase chain reaction (PCR) primers to amplify homologous sections of two junction genes. a conserved chromohelicase (CHD) DNA, located on sex chromosomes of all birds (Gri ﬃ ths et al. , Kahn et al. , Fridolfsson and Ellegren , Wang et al. , Bantock et al. ).
The primers amplify homologous sections of the two genes, incorporating introns, which usually vary in size, allowing the sex of the bird to be determined. However, since these methods cannot differentiate sex in species in which introns have little or no size variation, the development of other methods is necessary (Gri ﬃ ths et al. , Ito et al. . , Reddy et al. Al. , Bantock et al. , Huang and Huang ). Here is a simple and improved DNA test for avian sex determination. To overcome the limitation of the sexing method of Gri ﬃ ths et al. (), we designed a primer (P) specific for the unique sequence of the CHD-W gene of domestic chicken