Molecular sexing of threatened Gyps vultures: an important strategy for conservation breeding and ecological studies

Abstract

During the last two decades populations of three resident species of Gyps vulture have declined dramatically and are now threatened with extinction in South Asia. Sex identification of vultures is of key importance for the purpose of conservation breeding as it is desirable to have an equal sex ratio in these monogamous species which are housed together in large colony aviaries. Because vultures are monomorphic, with no differences in external morphology or plumage colour between the sexes, other methods are required for sex identification. Molecular methods for sex identification in birds rely on allelic length or nucleotide sequence discrimination of the chromohelicase-DNA binding (CHD) gene located on male and female chromosomes ZZ and ZW, respectively. We characterized the partial sequences of CHD alleles from Gyps indicus, Gyps bengalensis, Gyps himalayensis and Aegypius monachus and analysed the applicability of five molecular methods of sex identification of 46 individual vultures including 26 known-sex G. bengalensis and G. indicus. The results revealed that W-specific PCR in combination with ZW-common PCR is a quick, accurate and simple method, and is ideal for sex identification of vultures. The method is also suitable to augment ecological studies for identifying sex of these endangered birds during necropsy examinations especially when gonads are not apparent, possibly due to regression during non-breeding seasons.

Figure 1

Sequence alignment ofCHD-ZandCHD-Wallele sequences amplified by Griffith’s universalCHDprimer pair P2/P8 in four species of vultures (Gyps bengalensis,Gyps indicus,Gyps himalayensis,Aegypius monachus) by ClustalW (MegAlign DNAstar). The sequences of primers P2, P8, NP, MP, ZW common primer and probe, W-specific primer and probe recognition sites; BamHI and RsaI restriction sites are boxed. (−−) Dashed lines indicate sequence similarity. A.) Alignment of CHD-Z sequences. B.) Alignment of CHD-W sequences.

Figure 2

Gel view for molecular method for sex identification ofGyps bengalensisusing Griffith’s universal CHD primer pair P2/P8 PCR based methods: P2/P8 PCR (L1), BamHI digest of P2/P8 amplicon (L2). RsaI digest of P2/P8 amplicon (L3), ARMS PCR (L4), P2/ZW-common PCR (L5), and P2/W-Specific PCR (L6). M: 100 bp DNA ladder; NTC: No template control. The PCR products and restriction digests were resolved in run 3% agarose gel electrophoresis and stained with ethidium bromide A. Anatomically confirmed female (P10). B. Anatomically confirmed male (P33).

Figure 3

Real-time PCR curve for sex identification of known -sexGyps bengalensis(male P33, female P10) andGyps indicus(male P16, female P31) using TaqMan probes. W and ZW indicated the positive signals of TaqMan probes for CHD-W specific (FAM labelled) and CHD-ZW-common (HEX labelled) regions, respectively. ZW alone and W/ZW represented the male and female birds, respectively. A, B, C, D - Amplification plots with X-axis: PCR Cycle number, Y-axis: Fluorescence (dR). E - Dual color scatter plot with X- axis: Ct-HEX (dR), Y- axis Ct- FAM (dR).

Figure 4

Gel view for molecular methods for sex identification using post-mortem and live bird samples of vultures by Griffith’s universal CHD primer P2/P8 based PCR methods- PCR- RFLP using BamHI (A), PCR-RFLP using RsaI (B), ARMS-PCR (C), P2/ZW-Common PCR (D), P2/W-Specific PCR (E). M: 100 bp DNA ladder; NTC: No template control. The PCR products and restriction digests were resolved on 3% agarose gel and stained with ethidium bromide.

Figure 5

Real-time PCR curves and scatter plot for sex identification of field necropsy specimen ofGypsvultures using TaqMan probes.W and ZW indicate the positive signals of TaqMan probes for CHD-W specific (FAM-labelled) and CHD-ZW-common (HEX-labelled) regions, respectively.