Identification of the sex of a wide range of carinatae birds by PCR using primer sets selected from chicken EE0.6 and its related sequences

A 0.6 kb EcoRI fragment (EE0.6), cloned from the W chromosome of chickens, is a nonrepetitive sequence and contains an exonlike sequence, ET15, which is likely a part of a pseudogene. The EE0.6 sequence is conserved in all species of birds examined both in Carinatae and Ratitae. A counterpart sequence of EE0.6 is present on the Z chromosome. The extent of diversity between the W- and Z-linked sequences are variable among species. The W- and Z-linked EE0.6 sequences, cloned from 12 different species, were compared and four forward and three reverse primers were selected to amplify parts of the EE0.6 sequence by polymerase chain reaction (PCR). By choosing a suitable combination of primers for EE0.6 and a set of primers for a Z/W-common sequence, as an internal control, the sex of 36 species belonging to 16 different orders of Carinatae could be determined clearly by PCR. The sex of two other species representing different orders could be determined by Southern blot hybridization using ET15 as a probe. For the two Ratitae species, emu and ostrich, EE0.6 sequences on W and Z chromosomes could not be distinguished either by, PCR or Southern blotting.