A molecular genetic approach for sex determination on helmeted hornbill ( Rhinoplax vigil ) casque: a forensic casework

Helmeted Hornbill (Rhinolax vigil) is the only hornbill that equipped with solid casque made from keratin for both males and females. The demand for casque in the black market was huge and resulted in IUCN status leaped up from vulnerable to critically endangered. We received a total of 68 confiscated helmeted hornbill casques. As part of the casework and the objectives of the study, we determined to reveal the sex status of those casques and the best methods to work with keratinous material. Molecular methods to determining sex in birds rely on the CHD gene located on male and female chromosomes ZZ and ZW, respectively. We optimized laboratory protocols for genetic sexing using three independent sets of primers P2/P8, 2550F/2718R, and CHD1F/CHD1R to amplify regions of the sexlinked CHD-Z and CHD-W genes. The CHD1F/CHD1R determined sex 80.88% of samples. The 2550F/2718R were quite successful, sexing 51.47% of samples. In contrast, the P2/P8 only identified the sex around 20.58% of samples. These results showed that CHD1F/CHD1R works the most effective for sexing the casques with 52.9% females, 27.9% males, and 19.1% unidentified. Therefore, the most accurate and suitable primers are CHD1F/CHD1R, 2550F/2718R, and P2/P8, respectively for keratinous samples.