Feasibility of universal CHD1 sexing markers in various bird orders

SUMMARY Background: Defining the sex of individual birds can be crucial for scientific studies and captive breeding, as well. However, many bird species (and almost all nestling) can only be sexed via molecular methods. Universal diagnostic primers that can sex most birds are used widely, most commonly those that are based on amplifying differently sized fragments of the CHD1 (Chromohelicase DNA binding protein 1) gene on the sex chromosomes. Still, little is known about their reliability in several avian orders or in different tissue samples. objectives: The aim of this research was to test four frequently used universal bird sexing markers in 13 Neognathae bird orders and in different sample types. These markers (P2/P8, 2550F/2718R, CHD1-i16 and CHD1-i9) amplify fragments of intronic regions of the CHD1-Z and CHD1-W genes. Samples of more than 60 bird species and various tissue types (feathers of different sizes, dried and fluid blood) were tested. Amplified CHD1 fragments were visualized with UV light, following agarose gel electrophoresis.Results and Discussion: Our results confirm the universality of these primer pairs in most avian orders, but their application needs some consideration. 21% of the small size feathers gave no detectable results. The marker CHD1-i9 did not work in 14.5% of the feather samples. Such error occurred only in 4% of blood samples. The marker P2/P8 needed the longest electrophoresis time, but did not yield visible sex-specific bands even after 120 minutes in nine species. Similarly, no sex-specific bands were detected with 2550F/2718R in seven species. In CHD1-i16, aspecific bands making sexing diagnosis difficult were common, and even after improving the protocol, four species gave inconsistent results. In conclusion, these four markers can be assumed as near universal, easy-to-use tools for molecular sexing of Neognathae birds, but they all have some limitations. When choosing a marker for molecular sexing, not only the species, but also the tissue sample should be taken into consideration. We suggest to avoid the CHD1-i9 marker when using small or degraded feather samples. P2/P8 is not recommended when the size difference between the two sex chromosome-linked fragments is too small