TECHNICAL NOTE

A triple-primer PCR method for sexing endangeredcaprine species

Bo Zeng Æ Liu Xu Æ Bisong Yue Æ Fangdong Zou

Received: 13 October 2008 / Accepted: 13 January 2009 / Published online: 11 February 2009

� Springer Science+Business Media B.V. 2009

Abstract Molecular sexing is becoming an essential tech-

nique in understanding the sexual structure and dynamics of

natural populations. Herein, we report on a triple-primer PCR

method based on the last introns of the ZFX/Y alleles for sex

identification in Bovidae, and its successful application to five

endangered caprine species. The male samples generated a

*230 bp ZFX-specific fragment and a *140 bp ZFY-spe-

cific fragment, and the female samples only generated the

*230 bp fragment. This method is very sensitive to the

Y-linked fragment, thus effectively avoiding false negatives.

Genomic DNA extracted from well preserved tissues, non-

invasive samples and smoked meat are all usable for analysis

with this method.

Keywords Molecular sexing � Sex-linked zinc

finger alleles � Caprinae � Triple-primer PCR

Introduction

Estimation of the sex ratios in wild populations is impor-

tant for understanding the population dynamics and

structure of endangered mammalian species. For a number

of species in the Bovidae family, the sex ratio varies in

different seasons and environmental conditions (Schaller

1998). Field observation is a basic and direct way to

identify male and female individuals, but it is often difficult

to determine the sex of all individuals in a population

despite the presence of phenotypic sexual dimorphism. For

example, surveys on both for the extremely dimorphic blue

sheep (Pseudois nayaur) and nearly monomorphic takin

(Budorcas taxicolor) have failed to sex half the individuals

in the populations (Wang et al. 2000; Zeng et al. 2002). As

a formerly supplementary method, molecular sexing is

becoming an essential technique in ecological and con-

servation studies. Non-invasive samples, such as hair and

feces, can be obtained from the field without injuring

animals and used for genetic analyses. Using these sam-

ples, molecular sexing, combined with mitochondrial DNA

and microsatellite analyses to distinguish different indi-

viduals, could accurately reflect the sex ratios in the natural

populations.

Amelogenin (AMEL) and the sex-linked zinc finger

protein (ZF) are the two loci widely used in mammalian

sex identification. An AMELX/Y assay has been developed

for Bovidae species and can clearly distinguish the sex of

several Bovinae species and domestic sheep and goat

(Weikard et al. 2006). However, the authors did not test

this method on any non-invasive samples or any wild

Caprinae species in their study. As another candidate, the

ZFX/Y alleles have been employed in sex identification for

ruminants (forest musk deer, Qiao et al. 2007; white-tailed

deer, Lindsay and Belant 2008). The methods developed in

these two studies are both designed to be species-specific,

and as in other mammals (Wilson and Erlandsson 1998),

the amplified fragments were relatively longer ([250 bp)

than which was expected for highly degraded DNA

(Villesen and Fredsted 2006). Herein, we designed a novel

PCR method for the sex identification of bovid species with

B. Zeng � L. Xu � B. Yue � F. Zou (&)

Sichuan Key Laboratory of Conservation Biology

on Endangered Wildlife, Key Laboratory of Bio-resources

and Eco-environment (Ministry of Education),

College of Life Sciences, Sichuan University,

610064 Chengdu, People’s Republic of China

e-mail: fundzou@yahoo.com.cn

L. Xu

College of Bioengineering, Southwest Jiaotong University,

610031 Chengdu, People’s Republic of China

123

Conserv Genet (2009) 10:1609–1612

DOI 10.1007/s10592-009-9807-2

short products from the ZFX/Y alleles, and tested its

applicability to several endangered caprine species.

Materials and methods

Samples and DNA extraction

In total we used a panel of 32 samples from six bovid

species (Table 1), which covered five types of samples

collected from the wild, museums and local residents in

natural reserves. Genomic DNA from tissues stored in 95%

ethanol and smoked meat was isolated using the standard

proteinase K digestion and phenol/chloroform extraction

procedures. The DNA from dry skin and hair was isolated

following the method used for hair follicle DNA extraction

by Zou et al. (2005) with minor revisions to the pretreat-

ment of skin samples.

Primer design and PCR amplification

The last introns within the ZFX/Y alleles of a male blue sheep

were simultaneously amplified using the primers LGL-335

(50-AGACCTGATTCCAGACAGTACCA-30) and LGL-331

(50-CAAATCATGCAAGGATAGAC-30) (Cathey et al.

1998). The two amplified fragments with about a 120 bp size

difference were separately purified from 1% agarose gel and

then cloned into pMD 18-T vectors (TaKaRa, Dalian, China)

and sequenced with the universal M13 primers. The

sequences were aligned with homologous sequences from

cattle (Bos taurus) and sheep (Ovis aries) available in Gen-

Bank (accession numbers AF241271-AF241274). All the

sequences were checked with the program RepeatMasker

(www.repeatmasker.org) to exclude interspersed repetitive

elements which may reduce PCR specificity when the primers

were designed annealing to these regions. Considering the

distribution of conserved domains within the ZFX/Y

sequences, a triple-primer PCR system was designed, which

included one forward primer specific to the ZFX conserved

region, one forward primer specific to the ZFY conserved

region and one reverse primer located in the ZFX/Y con-

served region. The sequences of these primers are as follows:

ZF-X 50-AAAGGATTGTTAAGCAGCAAGTAG-30, ZF-Y

50-GCTTGTAAATGTGTTCAGAACTCAC-30 and ZF-R

50-CAAGGATAGACAGTCAAGGGATG-30. The combi-

nation of ZF-X and ZF-R could amplify a *180 bp fragment

for cattle and a *230 bp fragment for sheep and blue sheep.

The combination of ZF-Y and ZF-R resulted in a 141 bp

fragment for all three species. This system was applied to the

samples from the six bovid species described in Table 1 to

confirm its reliability. The PCR was carried out in a 25 ll total

volume reaction mixture consisting of 18.0 ll Milli-Q water,

2.5 ll 10 9 PCR buffer, 1.5 ll MgCl2 (25 mM), 0.8 ll

dNTP (2.5 mM each), 0.33 ll each primer (25 lM), 1.0 ll

genomic DNA (about 100 ng), and 0.2 ll rTaq polymerase

(5 u/ll, TaKaRa, Dalian, China). Thermal conditions were an

initial denaturation at 95�C for 3 min followed by 30 cycles

of 94�C for 30 s, 55�C for 30 s and 72�C for 20 s. Three

independent PCRs for each sample were carried out to

investigate repeatability and reliability of the results. Resul-

tant products were electrophoresed on 2% agarose gel

containing ethidium bromide (10 mg/ml) and visualized

under an ultraviolet light.

Results and discussion

Amplified fragments of the ZFX/Y introns from blue sheep

were 977 bp and 852 bp in length. Such a size difference is

enough to distinguish male individuals from females, but

these fragments that are nearly 1 kb length are too long to

be amplified from highly degraded DNA, which makes it

not applicable to non-invasive samples. The RepeatMasker

program did not detect any interspersed repetitive elements

Table 1 Information on the bovid samples used in this study

Species Sample type Sample size

I II III IV V VI

Blue sheep (Pseudois nayaur) 6#, 3$ 1# 1# 1#, 1$ 13 (9#, 4$)

Takin (Budorcas taxicolor) 3#, 2$ 3#, 1$ 9 (6#, 3$)

Chinese goral (Nemorhaedus caudatus) 1#, 2$ 1#, 1$ 5 (2#, 3$)

Sumatran serow (Capricornis sumatrensis) 1# 1$ 2 (1#, 1$)

Chiru (Pantholops hodgsonii) 2# 2#

Yak (Bos grunniens) 1# 1#

Total sampling 32 (21#, 11$)

The Roman numerals I–VI indicate the sample types as follows: (I) muscle or liver in 95% ethanol; (II) skin with hair stored in 95% ethanol;

(III) dry skin with hair from museum specimen; (IV) skin peeled off from rotten and air-dried skulls in the wild; (V) hair stored at room

temperature; (VI) the internal portion from the smoked meat

1610 Conserv Genet (2009) 10:1609–1612

123

in the analyzed sequences of cattle, sheep and blue sheep,

so it would be more specific to design primers annealing to

this region. PCR amplification for the 32 tested samples

confirmed this speculation, in which no unexpected product

band was found. All the samples were correctly sexed with

males exhibiting the *140 bp ZFY-specific band and

females exhibiting a single *230 bp ZFX-specific band

(as represented by Fig. 1). However, and this was unex-

pected, for some highly degraded genomic DNA (most

fragments \250 bp), the simultaneous amplification of the

ZFX/Y alleles was intensively biased to the shorter ZFY

fragment and led to the absence of a ZFX-specific band for

male samples (for example, the chiru in Fig. 1). We

speculated that it was caused by the preferential amplifi-

cation of the shorter fragment by PCR and could not be

easily avoided by adjusting the reaction conditions. So an

additional control was set to amplify the ZFX fragment

from these DNA samples with only ZF-X and ZF-R

primers added. By comparing the results of the triple- and

double-primer PCR (Fig. 2), the sex of the samples could

be determined objectively due to the distinct sizes of the

ZFX and ZFY fragments. During the repetitive PCR for

each male sample, the ZFY fragment could always be

amplified without affecting by the qualities of template

DNA. This indicated our primers were very sensitive to the

male samples and thus could avoid false negative results

effectively.

Our results showed that the triple-primer system based

on ZFX/Y alleles was reliable for sex identification in

caprine species. The samples used in this study could rep-

resent the four generally recognized tribes within Caprinae:

Caprini (blue sheep), Ovibovini (takin), Rupicaprini (goral

and serow), and Pantholopini (chiru) (Schaller 1998; Her-

nandez-Fernandez and Vrba 2005). Therefore, from an

evolutionary perspective, our method has the potential to

successfully work on most (if not all) caprine species. This

assay is also theoretically applicable to Bovinae due to the

conservation of primer annealing regions. We obtained a

*180/141 bp band pattern for a male yak in the experi-

ment, which is much better than the *280/217 bp pattern

for seven bovine species by Weikard et al. (2006). The

advantage of this method is its wide applicability not only

for a large group of bovid species but also for various

sample types. Since the amplified fragments are very short,

highly degraded DNA from non-invasive samples can be

used for analysis. Furthermore, a novel type of samples was

described and utilized in this study, which was the smoked

meat made from two gorals. In southwestern China, local

residents in the mountains used to hunt wild deer, blue

sheep and gorals and smoke their meat for long-term

preservation. Although hunting for these animals has been

forbidden by law, the poaching still exists in some areas.

The smoked meat is very easy to collect from the villagers

but not easy to determine its origin by appearances and

smell. The successful analysis of DNA extracted from the

smoked meat will be helpful to investigate the poaching

motivation of local residents and provide public awareness

for wildlife conservation, and can be used in the forensic

identification of illegally harvested animal products.

Acknowledgments We would like to acknowledge Guo Cai for

collecting samples, and Emily King for critical reading and improving

of the manuscript. This research was supported by Sichuan Youth

Science and Technology Foundation (09ZQ026-044) (Zou F.).

References

Cathey JC, Bickham JW, Patton JC (1998) Introgressive hybridization

and nonconcordant evolutionary history of maternal and paternal

lineages in North American deer. Evol Int J Org Evol 52:1224–

1229. doi:10.2307/2411253

Fig. 1 Gel photograph for the PCR amplification of the ZFX/Y

alleles in six bovid species. The male samples are recognized by the

appearance of *140 bp ZFY-specific band and the females are found

to generate a single *230 bp ZFX-specific band

Fig. 2 The preferential amplification of the shorter ZFY-specific

fragment by PCR. Lane 1, DNA ladder; lane 2, amplification of ZFX

allele from a male chiru sample with primers ZF-X and ZF-R; lane 3,

amplification of ZFX/Y alleles from a male chiru sample with primers

ZF-X, ZF-Y and ZF-R; lane 4 and 5, amplification of ZFX allele with

primers ZF-X and ZF-R, and ZFX/Y alleles with primers ZF-X, ZF-Y

and ZF-R from a male goral sample

Conserv Genet (2009) 10:1609–1612 1611

123

Hernandez-Fernandez M, Vrba ES (2005) A complete estimate of the

phylogenetic relationships in Ruminantia: a dated species-level

supertree of the extant ruminants. Biol Rev Camb Philos Soc

80:269–302. doi:10.1017/S1464793104006670

Lindsay AR, Belant JL (2008) A simple and improved PCR-based

technique for white-tailed deer (Odocoileus virginianus) sex

identification. Conserv Genet 9:443–447. doi:10.1007/s10592-

007-9326-y

Qiao Y, Zou F, Wei K, Yue B (2007) A rapid sex-identification test

for the forest musk deer (Moschus berezovskii) based on the

ZFX/ZFY gene. Zoolog Sci 24:493–495. doi:10.2108/zsj.24.493

Schaller GB (1998) Wildlife of the Tibetan Steppe. University of

Chicago Press, Chicago

Villesen P, Fredsted T (2006) Fast and non-invasive PCR sexing of

primates: apes, old world monkeys, new world monkeys and

Strepsirrhines. BMC Ecol 6:8. doi:10.1186/1472-6785-6-8

Wang XM, Peng JT, Zhou HM (2000) Preliminary observations on the

distribution and status of dwarf blue sheep Pseudois schaeferi.Orxy 34:21–26. doi:10.1046/j.1365-3008.2000.00089.x

Weikard R, Pitra C, Kuhn C (2006) Amelogenin cross-amplification

in the family bovidae and its application for sex determination.

Mol Reprod Dev 73:1333–1337. doi:10.1002/mrd.20554

Wilson JF, Erlandsson R (1998) Sexing of human and other primate

DNA. Biol Chem 379:1287–1288

Zeng ZG, Zhong WQ, Song YL, Li JS, Guo F (2002) Group size,

composition and stability of golden takin in Shaanxi Foping

Nature Reserve, China. Folia Zool (Brno) 51:289–298

Zou F, Yue B, Xu L, Zhang Y (2005) Isolation and characterization of

microsatellie loci from forest musk deer (Moschus berezovskii).Zoolog Sci 22:593–598. doi:10.2108/zsj.22.593

1612 Conserv Genet (2009) 10:1609–1612

123