A Simple and Universal Method for Molecular Sexing of Non-Ratite Birds

TLDR

Molecular sexing of sexually monomorphic birds requires a conserved W‑linked sequence, but until the discovery of the CHD1W gene—present in all non‑ratite birds—no single W‑chromosome gene had been identified. The authors present a universal method for sexing non‑ratite birds based on detecting a size difference between CHD1W and CHD1Z introns. Using conserved primers flanking the intron, PCR amplification and agarose gel electrophoresis distinguishes females (two fragments) from males (one fragment) by size. The method, tested with primers 2550F/2718R on 50 species from 11 orders, successfully sexed 47 species, and an alternative primer pair allowed reliable sexing of the remaining three, demonstrating a simple, rapid, and inexpensive universal system.

Abstract

Molecular sexing is an attractive means to determine the sex of sexually monomorphic birds, e.g. chicks of most species. A universal approach for molecular sexing of birds would require that a conserved W chromosome-linked sequence 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 direction, 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 method for molecular sexing of non-ratite birds which is based on the detection of a constant size difference between CHD1W and CHD1Z introns. Using highly conserved primers flanking the intron, PCR amplification and agarose electrophoresis, females are characterised by displaying one (CHD1W) or two fragments (CHD1W and CHD1Z), while males only show one fragment (CHD1Z) clearly different in size from the female-specific CHD1W fragment. With one particular pair of primers (2550F and 2718R) we applied this test to 50 bird species from 11 orders throughout the avian phylogeny, successfully sexing 47 of the species. Using an alternative pair of primers, the three failing species could be reliably sexed. This means that a simple, rapid and cheap universal system for molecular sexing of non-ratite birds is now available.