One of the key tools for determining the social structure of wild …

• Abstract
• Background
• Results
• Discussion
• Conclusion
• Methods
• References
• Figures
• Table 1

The ability to identify the sex of a DNA sample is an important tool in molecular ecology and conservation genetics. The optimal marker would work on small amounts of non-invasive samples that are likely to include highly degraded DNA and be applicable in many species.

Molecular sex identification normally works by PCR amplification of sex specific regions that differ in length. The PCR products can then be visualized using standard electrophoresis, revealing females as homozygotes (XX) and males as heterozygotes (XY). Generally, in order to detect the male sex, a Y chromosomal fragment must be amplified. This can be done by (a) amplification of a homolog region on X and Y with a length difference, (b) a triple primer PCR with a common primer X/Y and a Y specific primer (short fragment) and X specific primer (longer fragment) or (c) a multiplex PCR with a Y region and an positive control (autosomal or X region).

Several loci have been used for sex identification in humans and closely related species, e.g. the amelogenin system [1-3] the zinc-finger protein [4,5], the SRY locus [6] or a combination [7]. Detection of sex-specific restriction patterns [8] requires some pre-analysis development (i.e. sequencing to identify restriction sites) and enzyme restriction of PCR products, which is time consuming. The SRY locus requires co-amplification of external control regions [6,7], which may be unreliable for non-invasive DNA samples with DNA of low quality.

The widely used amelogenin system [1] has recently been found to provide ambiguous results in humans such as null-alleles, primer mutations or amplification failure, resulting in erroneous gender determination [9-11]. It works in closely related apes [2], but not orang-utans [6], baboons or more distantly related species [12]. Also, the Sullivan amelogenin system amplifies a very small X-Y size difference (6 bp), which does not consistently resolve well on agarose gels. Therefore this system needs more time-consuming and expensive acrylamide gel- or capillary electrophoresis. Recently, a new general multiplexing method was published, using the amelogenin locus, the SRY locus and group specific primers and suitable for non-invasive samples [7]. Non-identification of males may result from non-amplification of the Y fragment and it has been argued [13] that all "females" (XX or XY with no Y amplification) should be verified with a second independent sex test. This calls for the development of multiple independent tests that can be carried out in parallel.

We have previously developed a new primer pair for a different region of the amelogenin gene suitable for sexing lemurs and humans and therefore possibly most primate species [3]. However, like the zing-finger protein system [4], the resulting fragments are too long (> 250 bp) for non-invasive samples, which often contain highly degraded DNA. Alternatively, we then designed primers for a small region of the DEAD-BOX gene, which are able to sex apes and monkeys, but these primers do not work in prosimians – probably due to primer region mutations [14].

At present, genomic sequence information is only available for a few primates (human, chimpanzee, macaque) and rodents (mouse, rat) or even more distantly related species. So far, we find that it is impossible to identify suitable homolog XY regions with the desired degree of conservation for a standard two-primer PCR design that will work in all primates.

The objective of this study was to identify a suitable region for a triple primer PCR design with a shared XY primer in combination with an X- and Y-specific primer. The primers should amplify a short region and be widely conserved through primate evolution, such that the method would work on non-invasive samples from all primate species.