Long-term stability of sex chromosome gene content allows accurate qPCR-based molecular sexing across birds.

Embryos, juveniles, and even adults of many bird species lack pronounced external sexually dimorphic characteristics. Accurate identification of sex is crucial for research (e.g., developmental, population, and evolutionary studies), management of wildlife species, and captive breeding programmes for both conservation and poultry. An accurate molecular sexing method applicable across the entire bird radiation is theoretically possible thanks to the long-term stability of their ZZ/ZW sex chromosomes, but current methods are not applicable in a wide range of bird lineages. Here, we developed a novel molecular sexing method based on the comparison of gene copy number variation by quantitative real-time PCR (qPCR) in conserved Z-specific genes (CHRNA6, DDX4, LPAR1, TMEM161B, VPS13A), i.e. genes linked to Z but absent from W chromosomes. We tested the method across three paleognath and 70 neognath species covering the avian phylogeny. In addition, we designed primers for four Z-specific genes (DOCK8, FUT10, PIGG and PSD3) for qPCR-based molecular sexing in three paleognath species. We have demonstrated that the genes DOCK8, FUT10, PIGG and PSD3 can identify sex in paleognath birds and the genes CHRNA6, DDX4, TMEM161B, and VPS13A can reveal sex in neognath birds. The gene LPAR1 can be used to accurately identify sex in both paleognath and neognath species. Along with outlining a novel method of practical importance for molecular sexing in birds, our study also documents in detail the conservation of sex chromosomes across the avian phylogeny.