Comparative evaluation of five primer sets and validation of CHD1LF/CHD1LR primers for molecular sexing of the critically endangered Straw-headed Bulbul (Pycnonotus zeylanicus)
The Straw-headed Bulbul (Pycnonotus zeylanicus) (Gmelin, 1789), locally known as “Cucak Rowo” in Indonesia, is a conservation-focused songbird in Southeast Asia that is currently listed as Critically Endangered on the IUCN Red List due to overharvesting. Sustainably managed captive breeding programs are critical for ensuring its long-term survival. However, like many other avian species, P. zeylanicus exhibits monomorphism, rendering external morphological sex determination unreliable. Accurate sex identification is essential for successful captive breeding management. Molecular sexing techniques, specifically Polymerase Chain Reaction (PCR) amplification targeting the Chromodomain Helicase DNA-binding (CHD) gene, provide a reliable and efficient method for sex identification in birds. This study aimed to evaluate the effectiveness of the CHD1LF/CHD1LR primer set for the molecular sex determination of P. zeylanicus and compare the results with breeder-reported sex assignments based on external morphology. We also tested four other commonly used CHD primer sets: P2/P8, 1237L/1272H, 2550F/2718R, and CHD1F/CHD1R. A total of ten P. zeylanicus samples were collected from two breeders in Central Java and Yogyakarta, Indonesia. The results demonstrated that the CHD1LF/CHD1LR primer set can be used for sex identification in P. zeylanicus. Males produced a single PCR amplicon of approximately 474 bp, whereas females showed two distinct bands at approximately 474 bp and 319 bp, respectively. In contrast, the four other primer sets were ineffective for this species because their amplification patterns, in terms of band size and number, did not match those reported in established references. A notable discrepancy was observed between breeder-reported sex and PCR results, with breeders achieving only 70% accuracy, suggesting that the molecular method is more accurate than morphological observation. The CHD1LF/CHD1LR primers provide a promising preliminary method for sex identification in P. zeylanicus. These findings also emphasize the importance of species-specific primer validation for accurate molecular sex identification.
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