Utility of Sanger Sequencer Based Fragment Analysis for Sex Identification in Vultures
S. M. Kolangath *
Wildlife Research & Training Centre, Gorewada, Maharashtra Animal & Fishery Sciences University, Nagpur, India.
S. V. Upadhye
Wildlife Research & Training Centre, Gorewada, Maharashtra Animal & Fishery Sciences University, Nagpur, India.
Priyanka Mulmule
Wildlife Research & Training Centre, Gorewada, Maharashtra Animal & Fishery Sciences University, Nagpur, India.
Shruti Patwa
Wildlife Research & Training Centre, Gorewada, Maharashtra Animal & Fishery Sciences University, Nagpur, India.
M. D. Pawshe
Wildlife Research & Training Centre, Gorewada, Maharashtra Animal & Fishery Sciences University, Nagpur, India.
V. M. Dhoot
Wildlife Research & Training Centre, Gorewada, Maharashtra Animal & Fishery Sciences University, Nagpur, India.
B. K. Bhadane
Wildlife Research & Training Centre, Gorewada, Maharashtra Animal & Fishery Sciences University, Nagpur, India.
M. S. Patil
Nagpur Veterinary College, Seminary Hills, Nagpur, 440001, India.
P. K. Bhangale
Wildlife Research & Training Centre, Gorewada, Maharashtra Animal & Fishery Sciences University, Nagpur, India.
R. M. Kolangath
Independent Researcher, 29-A, Telephone Nagar, Mhow, Indore, India.
*Author to whom correspondence should be addressed.
Abstract
Background: Accurate sex determination is fundamental to avian ecology, conservation genetics, and ex-situ breeding programs. Sex differentiation is altogether difficult among monomorphic bird species owing to high resemblance and limited morphological distinguishing features. Molecular methods are the last resort as morphological and behavioural traits fail to distinguish sexes in monomorphic species.
Aim: The study aimed to explore about Utility of Sanger Sequencer based Fragment Analysis for sex identification in Vultures.
Methods: The ZW-ZZ sex determination pattern in aves provides an opportunity to utilise the CHD (Chromodomain Helicase DNA-binding) gene as a reliable marker for the sex determination of birds. In this study, fluorescently labelled P2–P8 primers were used to amplify the CHD gene in Indian Vultures (Gyps indicus) and White-rumped Vultures (Gyps bengalensis) to identify the sex and were resolved by Capillary Gel Electrophoresis (CGE) on a Sanger Sequencer.
Results: The males (ZZ); whereas females (ZW) sex chromosome composition. The limit of resolution of the Agarose Gel Electrophoresis (AGE) is 10 bp, and in certain species of monomorphic birds, the existing slender difference between the CHD-Z & CHD-W is beyond the resolution capacity of the AGE. The conventional PCR-AGE failed to allocate sex identities to birds, whereas the Sanger Sequencer-based fragment analysis identified the CHD-Z and CHD-W fragments sized at 381.09 ± 0.04 bp and 385.75 ± 0.06 bp, respectively, revealing a 4 bp difference sufficient for accurate sexing. Males displayed a single CHD-Z peak, while females exhibited both peaks. The assay’s precision was further validated in Sarus Crane (Grus antigone) and Common Crane (Grus grus) samples, where a 1 bp difference between fragments was successfully resolved.
Conclusion: The Sanger-based CGE fragment analysis is a robust, sensitive and high-throughput approach for sexing in birds, with significant implications for conservation breeding and population management.
Keywords: Fragment analysis, P2-P8 primers, sanger sequencer, sex identification, vulture