# Molecular Sexing in Owls (Aves, Strigiformes) and the Unique Genetic Structure of the Chromodomain Helicase DNA-Binding Protein 1 (CHD1) Gene on Chromosome W

## TL;DR

This paper explains how owl sex can be determined using DNA and reveals a unique genetic feature in owls that affects current methods.

## Contribution

The study identifies a unique 600 bp insertion in the CHD1-W gene of owls and explains why existing primers fail in some species.

## Key findings

- A unique 600 bp insertion in intron 17 of the CHD1-W gene reverses the usual PCR product length relationship.
- Mutations in the 2550F primer binding site of the CHD1-Z gene in some owl species prevent successful amplification.
- A new primer set was developed to amplify both Z- and W-derived CHD1 products across owl species.

## Abstract

Background: The accurate determination of bird sex is crucial in various biological fields, including ecology, behavioral research, and conservation. However, this task remains challenging in species in which males and females exhibit similar external morphologies, such as owls. Although polymerase chain reaction (PCR)-based molecular sexing techniques that target the chromodomain helicase DNA-binding protein 1 gene found on sex chromosomes Z (CHD1-Z gene) and W (CHD1-W gene) are widely used, we encountered atypical banding patterns when applying the previously reported primers 2550F and 2718R to four wild owls of unknown sex. This study aims to reveal the owl-specific genetic structure of the CHD1 gene. Methods: We developed a new primer set and determined the nucleotide sequences—including the binding sites for the primers 2550F and 2718R—within both the CHD1-Z and CHD1-W genes. Results: Sequencing analysis, conducted using a newly developed primer set that successfully amplified both Z- and W-derived CHD1 products across various owl species, revealed a unique genetic insertion of approximately 600 bp in intron 17 of the CHD1-W gene. This insertion reversed the usual length relationship between PCR products from the chromosomes Z and W. Additionally, mutations identified in the 2550F primer binding site of the CHD1-Z gene in certain owl species may explain the failure to amplify CHD1-Z-derived PCR products. Conclusion: These findings provide valuable insights for improving molecular sexing in owls.