Conservation Genetics Resources

, Volume 4, Issue 2, pp 299–301

Molecular sexing of tigers, Panthera tigris

Authors

    • Royal Zoological Society of Scotland
  • K. Ouitavon
    • DNP Wildlife Forensic Science Unit, Wildlife Conservation OfficeDepartment of National Parks, Wildlife and Plant Conservation
  • J. J. Rovie-Ryan
    • Wildlife Genetic Resource Bank (WGRB) LaboratoryDepartment of Wildlife and National Parks
  • Wulansari
    • Eijkman Institute for Molecular Biology
  • F. T. Sitam
    • Wildlife Genetic Resource Bank (WGRB) LaboratoryDepartment of Wildlife and National Parks
  • R. Ogden
    • Royal Zoological Society of Scotland
Technical Note

DOI: 10.1007/s12686-011-9529-x

Cite this article as:
McEwing, R., Ouitavon, K., Rovie-Ryan, J.J. et al. Conservation Genet Resour (2012) 4: 299. doi:10.1007/s12686-011-9529-x

Abstract

We report the development of a fast and reliable PCR-based method for sex identification of tiger DNA designed to be incorporated into fluorescent short tandem repeat (STR) profiling. A single primer pair, consisting of a fluorescently-labelled forward primer and an unlabelled reverse primer, is used to co-amplify homologous fragments of a zinc finger (ZF) protein intron which exhibits size polymorphism between the X and Y chromosomes. The ZFX and ZFY amplicons differ in size by 12 bp and can thus be differentiated by capillary electrophoresis.

Keywords

Molecular sex identificationTigerZinc finger proteinForensicsFelids

Introduction

Tiger parts are highly sought after, primarily as an ingredient in traditional Asian medicine and as ornamental skins. The demand for tiger parts along with habitat destruction was the principle cause for the decline in tiger numbers during the twentieth century, and despite international legislation and domestic bans on tiger part trade, poaching and illegal activity continue to threaten the world’s tigers today (Linkie et al. 2003). Six extant subspecies of tiger are currently recognized, Panthera tigris tigris, Panthera tigris corbetti I, Panthera tigris corbetti II (jacksoni/malayensis), Panthera tigris altaica, Panthera tigris sumatrae and Panthera tigris amoyensis, and all are listed as ‘endangered’ or ‘critically endangered’ on the IUCN Red List of Threatened Species (IUCN 2010). Population monitoring of these species, including sex ratio data, is therefore vital to provide information for their conservation and management. Although the sex of adult tigers can be determined by an experienced observer, field observations may be hampered by distance, position relative to the animal or by vegetation. Furthermore, juvenile tigers are difficult to sex, and illegally traded parts and derivatives of tigers have no sexual characters.

Here, we report the development of a non-invasive molecular sexing technique for tiger DNA. In this study, we exploit a size difference (~12 bp) between ZFX and ZFY homologs, elucidated by capillary electrophoresis, to determine the sex of tiger samples. The current research forms part of a larger project to develop genetic markers for linking tiger parts to their individual source animal, in order to help investigate illegal poaching and trade of tigers, and their parts.

Methods

Samples

Samples of blood or hair were collected from zoo tigers of known sex (male = 15, female = 17) from four of the six extant subspecies; no samples from P.t. amoyensis or P.t. corbetti II were available for testing. Ten additional tiger samples of unknown sex were provided from zoos or from seizures of tiger meat confiscated in South East Asia. All shipped samples were subject to CITES licence permits.

DNA extraction

DNA was extracted from blood, tissue and hair samples using the Qiagen DNeasy Blood and Tissue kit following the manufacturer’s instructions for each sample type. Total extracted DNA was quantified by absorbance using a NanoDrop® ND-1000 UV–Vis Spectrophotometer and normalized to 5 ng/μl.

PCR amplification

A single primer pair was designed on a consensus sequence generated from ZFX and ZFY sequences from Panthera tigris obtained from GenBank (see Table 1). The fluorescently labelled forward primer PTZFf (5′-FAM–GTACAGWYASMCTGAAT-3′) and the unlabelled reverse primer PTZFr (5′-AAGTGTGYGTTCTGAACAT-3′) target a region which exhibits a 12 bp difference between the X and Y homologs. PCR mixtures (total volume 10 μl) used 5 ng of DNA and the Qiagen Type-it microsatellite mastermix, selected so future multiplexing with microsatellite loci could be achieved.
Table 1

The region amplified by the primers PTZFf and PTZFr

https://static-content.springer.com/image/art%3A10.1007%2Fs12686-011-9529-x/MediaObjects/12686_2011_9529_Tab1_HTML.gif

The region amplified relates to bp 638–793 of GenBank sequence AB211419

Amplifications were carried out using a BioRad Dyad thermocycler with the following conditions: initial denaturation at 95°C for 5 min; followed by 28 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 90 s, and extension at 72°C for 30 s; followed by a final extension step at 60°C for 30 min. All amplifications included a negative control without template DNA, and a positive control containing 5 ng of P. tigris DNA. Amplification products were visualised under UV using stained agarose gels, then diluted 20-fold in ABI Hi-Di Formamide before capillary electrophoresis on an Applied Biosystems Inc. 3130xl Genetic Analyzer (ABI). Alleles were sized against an internal standard Genescan™ 500LIZ® and scored using GeneMapper software v4.0.

Results and discussion

Female P. tigris individuals were characterised by two identically sized X chromosome amplicons 150 bp in length. Males were characterised by the 150 bp X chromosome amplicon, and an additional Y chromosome amplicon 138 bp in length, making them easily distinguishable from females (see Fig. 1). A shorter Y chromosome fragment is preferred for sex determination using PCR as this prevents false female positives being identified due to degraded DNA where the Y chromosome fragment fails to amplify. Although other felid sex DNA primers have been described (Pilgrim et al. 2005) with a utility to correctly sex tiger DNA, the primers described here solely for tiger studies provide a shorter X and Y fragment length making them more suitable for compromised DNA samples and pooling alongside published microsatellite markers.
https://static-content.springer.com/image/art%3A10.1007%2Fs12686-011-9529-x/MediaObjects/12686_2011_9529_Fig1_HTML.gif
Fig. 1

Electropherogram image showing results produced by a female and b male tiger samples

Sex was correctly assigned for all control tiger samples (male = 15, female = 17) and determined for the samples of unknown sex (male = 4, female = 6). This technique could be combined with other molecular markers for more detailed population analyses. Additionally, sex identification alleles can be very powerful forensic markers for matching trace evidence to individuals, as their roughly equal population frequency allows 50% of potential source animals to be immediately excluded. Work is ongoing in our laboratories to incorporate the sex identification test into a forensic STR-based assay for the protection of tigers.

Acknowledgments

This work is part of a collaboration funded by the Darwin Initiative with the purpose of developing DNA techniques to monitor and enforce against the illegal trade of protected species in South East Asia. The authors thank the zoo organisations for reference tiger samples, and in particular to Sarah Christie (ZSL). Thanks are also due to the Programme Coordination Unit of ASEAN WEN and TRAFFIC SE Asia.

Copyright information

© Springer Science+Business Media B.V. 2011