# Fast and Simple Molecular Test for Sex Determination of the Monomorphic Eudromia elegans Individuals

## TL;DR

This paper presents a new, fast, and simple molecular test to determine the sex of the monomorphic bird species Eudromia elegans using DNA from blood or feather samples.

## Contribution

A novel, reliable, and efficient PCR-based method for sex determination specifically tailored for Eudromia elegans.

## Key findings

- The PCR method successfully amplified two DNA fragments in females and one in males of E. elegans.
- The test uses the CHD1 gene and sex-specific primers to distinguish between ZZ (male) and ZW (female) chromosomes.
- The method is accurate, fast, and suitable for routine use with non-invasive samples like blood and feathers.

## Abstract

A lot of avian species are morphologically monomorphic, and thus, sex determination is difficult if it is based only on their external examination. Molecular sex identification has become a crucial tool in various fields of biology, including ornithology. This non-invasive method used to determine the genetic sex of birds of any age, in our case, Eudromia elegans (Paleognathae, Tinamiformes), can proceed with regard to the differences between avian male (ZZ) and female (ZW) chromosomes. According to our experience, the other molecular methods failed in the sex determination of this exact species. The new test was designed to be reliable, fast, and simple to determine the sex of the individuals of E. elegans. Molecular sexing has significant implications for understanding various aspects of its biology, including mating systems, population genetics, and conservation strategies. By accurately determining the sex of individuals within a population, researchers can investigate sex ratio patterns, reproductive success, and gene flow.

Sex determination based just on morphological traits such as plumage dichromatism, sexual size dimorphism, behavior, or vocalizations is really challenging because of the sexual monomorphism present in more than half of avian species. Currently, a lot of them can be tested through DNA-based procedures, but they do not fit all the avian species, such as Eudromia elegans. The aim of this study was to design a new molecular method suitable for routine sex determination for that species that is fast, simple, and cost- and time- effective. DNA was isolated from dry blood stain and/or chest feather samples of E. elegans species. We used two sets of sex-specific primers (ZF/ZR and WF/WR) to amplify the expected fragments localized on the highly conserved CHD1 gene to distinguish between sexes due to the W-specific DNA sequence present only in females. We confirmed the accuracy and consistency of the PCR-based method based on length differences to distinguish between the sexes of E. elegans, which amplified two fragments in females and one fragment in males.