A sexing protocol for wild ruminants based on PCR amplification of amelogenin genes

Based on the sequences of the bovine amelogenin genes, we have designed a protocol for sexing DNA samples of wild ruminants. Basically the protocol consists on the co-amplification of two specific fragments, one from Ychromosome and one for the X chromosome, making the use of a PCR control unnecessary. It has been demonstrated to be useful for sex identification in a total of 164 samples belonging to six different wild ruminant species. We propose adding to the census procedure commonly based in faecal groups counting, the faecal sampling and application of the protocol design here, to estimate the sex ratio.


Introduction
Sex identification of unknown samples is useful for natural population managing, allowing the knowledge of the sex ratio.In mammals females are the homogametic sex and have two copies of the X chromosome.Males are the heterogametic sex and have only one copy of the X chromosome and one copy of the Y chromosome.Different approaches, based on PCR methods, have been used in order to determine the sex.Some of the existing protocols are only based on the PCR-detection of Y chromosome specific sequences, such as genes: SRY (TAKAHASHI et al., 1998;MARA et al., 2004), and TSPY (LEMOS et al., 2005); or repeated sequences (SCHROEDER et al., 1990;BREDBACKA and PEIPPO, 1995;KAGEYAMA et al., 2004).The presence of no signal does not necessarily mean that the sample has a female origin, because experimental errors can also lead to negative results.Then, sexing protocols also need a PCR product being out of the Y chromosome, as a positive control of template (DNA) in the sample, or an X chromosome specific fragment.
In mammals, the amelogenin (AMEL) genes are present on both X and Y chromosomes.Amelogenin genes have been used for PCR sex typing test in humans Bovines and deers, because of the difference in length of PCR products from the X and Y chromosomes (SULLIVAN et al., 1993;ENNIS and GALLAGHER, 1994;YAMAUCHI et al., 2000;PFEIFFER and BRENIG, 2005).In this work, we tried to design a protocol based on the simultaneous amplification of amelogenin genes as a method to assign sex to unknown samples of any ruminant species.
DNA isolation A piece of muscle or skin was collected from hunted animals with known sex, and stored in 96% ethanol.Total blood was collected from domestic ruminants.Total DNA was isolated from, both muscle and skin, following the manufactured instruction of the Nucleo Spin © (Macherey-Nagel) commercial kit.

Simultaneous PCR amplification of amelogenin genes
We used a single pair of primers based on the bovine sequences previously described in ENNIS and GALLAGUER (1994).Amelogenin amplification was carried out in 10 µl containing 25 ng of DNA, 2 mM of each dNTP, 0.2 µM of the primers amel-up ( 5'-Cy5-CAGCCAAACCTCCCTCTGC-3') and amel-dn (5'-CCCGCTTGGTCTTGTCTGTTGC-3'), 2 mM MgCl 2 , 10x buffer (Biotools, SA), and 0.2 u Taq polymerase (Biotools, SA).The PCR protocol consisted on a first denaturing step at 95ºC for 5 min, followed by 35 cycles of 95ºC for 30 sec, 60ºC for 30 sec, and 72ºC for 30 sec.Final extension was at 72ºC for 10 min.One of these primers (amelup) was labelled in 5' with a Cy5 fluorocrome.Once the PCR method was performed, the product was electrophoresed in an AlfExpress II semi-automated sequencer system.In order to confirm sexing results we also used a common protocol based on the SRY gene (see as examples: TAKAHASHI et al., 1998;PHUA et al., 2003).Primers SRYup (5'-CTGCTATGTTCAGAGTATTG-3') and SRY-dn (5'-TCAATATTGAACATAAGCGC-3') were designed based on the ovine SRY sequence (accession number AY604733, MEADOWS et al., 2004).SRY amplification was carried out in 10 µl containing 25 ng of DNA, 2 mM of each dNTP, 0.5 µM of the primers SRY-up and SRY-dn, 2 mM MgCl2, 10x buffer (Biotools, SA), and 0.2 u Taq polymerase (Biotools, SA).The PCR protocol consisted on a first denaturing step at 95ºC for 5 min, followed by 35 cycles of 95ºC for 30 sec, 50ºC for 30 sec, and 72ºC for 30 sec.Final extension was at 72ºC for 10 min.

Results
As expected from the bovine sequence, a 271 bp product, representing amplification from the X chromosome amelogenin, was detected for the cow.For the bull, we detected the 271 bp, representing X chromosome, and a new 211 bp band, representing specific Y chromosome amelogenin amplification.Samples with known sex from some domestic (sheep and goat) and several wild ruminants (roe deer, Spanish ibex, chamois, fallow deer, red deer, mouflon) were used under the same conditions of amplification.All female samples showed one product and all male samples showed two bands.In this case, we found that the presumable Xproduct was 255 bp long, instead of 271 bp long as in the cow.Differences in size of the X-product have already been referenced in sika deer in other studies (YAMAUCHI et al., 2000).The presumable Y-product was 211 bp long in all the tested species except for red deer, which PCR-product was 205 bp long (Fig. ).The protocol presented here was used to correctly assign sex to 164 samples of different species of wild ruminants with unknown sex.We obviously detected Xspecific product in all the samples, and 102 of them also showed another band representing the Y-specific product.These samples were assigned the male sex.These results were consistent with those of the SRY protocol (not shown).Results of sexing by species are shown in the Table.Discussion We have established a simple and accurate method to determine the sex of unknown wild ruminant samples based on a PCR method, using bovine amelogenin primer sequences.Moreover, this method can also be applied to other domestic ruminant species other than bovine, whose sequence was used to design this protocol.The major advantage of this method is the co-amplification, in a single tube, of two specific fragments, one from Y-chromosome and one for the X chromosome, using a single primer pair, and making the use of a PCR control unnecessary.This protocol has been demonstrated to be useful in all the species tested, and has already been used in recent works for roe deer sexing (ROYO et al., 2007).In this work we have used samples from muscle or skin from all the wild ruminants hunted in the Iberian Peninsula, either natives like roe deer, fallow deer, red deer, chamoise and Spanish ibex, or reintroduced like mouflon.Then, this protocol can be used as a tool for sex identification in population managing in game preserves, at least, in most Mediterranean areas.The sex ratio knowledge is an important parameter for managing of natural populations.One of the most used method to estimate population number is the faecal counting method (AULAK and BABINSKA-WERKA, 1990).The development of this protocol, allow us to propose adding to the common faeces counting pellet groups procedure for census, the faecal sampling and after DNA isolation, the application of the protocol.This can lead to the inclusion of the estimation of the sex ratio into the census method.