A simple and universal method for molecular sexing of non-ratite birds

Molecular sexing is an attractive means to determine the sex of sexually mo nomorphic birds, e.g, chicks of mast species. A universal approach for mole cular sexing of birds would require that a conserved W chromosome-linked se quence could be analysed, but no single gene has previously been known from any avian W chromosome, The recent discovery of the CHD1W gene, apparently W-linked in all non-ratite birds, has opened new possibilities in this dir ection, although there is a problem in that the gene also exists in a very similar copy on the Z chromosome (CHD1Z), Here we describe a universal meth od for molecular sexing of non-ratite birds which is based on the detection of a constant size difference between CHD1W and CHD1Z introns. Using highl y conserved primers flanking the intron, PCR amplification and ag-arose ele ctrophoresis, females are characterised by displaying one (CHD1W) or two fr agments (CHD1W and CHD1Z), while males only show one fragment (CHD1Z) clear ly different in size from the female-specific CHD1W fragment, With one part icular pair of primers (2550F and 27181R) we applied this test to 50 bird s pecies from 11 orders throughout the avian phylogeny, successfully sexing 4 7 of the species, Using an alternative pair of primers, the three failing s pecies could be reliably sexed. This means that a simple, rapid and cheap u niversal system for molecular sexing of non-ratite birds is non available.