A rapid genetic method for sex assignment in non-human primates Anthony Di Fiore 1,2, * 1 Department of Anthropology and Center for the Study of Human Origins, New York University, 25 Waverly Place, New York, NY, 10003, USA; 2 New York Consortium in Evolutionary Primatology (NYCEP), New York, NY, USA (*Corresponding author: Phone: +1-212-998-3813; Fax: +1-212-995-4014; E-mail: anthony.difiore@nyu.edu) Received 4 January 2005; accepted 26 January 2005 Key words: amelogenin, molecular ecology, non-invasive sample, PCR-based sex-typing, SRY The field of primate molecular ecology is growing rapidly and holds promise for providing insights into aspects of primate social structure that are difficult to address though observational studies. Recent molecular studies have used samples col- lected non-invasively from wild animals to shed light on mating systems, dispersal behavior, pop- ulation structure, and patterns of relatedness in a variety of non-human primates (Di Fiore 2003). Indeed, studies utilizing such samples may be the only tractable way for primate biologists and conservation geneticists to gain critical informa- tion about taxa that are highly endangered, elu- sive, or difficult to habituate. In order to take full advantage of these kinds of samples for investi- gating a number of features of primate social systems (e.g., population sex ratios, sex biases in dispersal patterns), researchers must be able to confidently and reliably identify the sex of sampled individuals. Several molecular methods have been developed for sex assignment in humans, most of which rely on fixed polymorphisms between the X- and Y-borne copies of the nuclear gene amelogenin (e.g., Akane et al. 1991; Nakahori et al. 1991; Sullivan et al. 1993; Faerman et al. 1995; Haas-Rochholz and Weiler 1997). Over a decade ago, Sullivan et al. (1993) developed a simple PCR-based human sex test that uses a single primer pair to amplify homologous fragments of amelogenin X and Y that differ in size by 6 bp. Male (XY) samples thus pro- duce two amplicons, while females (XX) produce just one. Although this basic assay is now the stan- dard used in forensic work (Cotton et al. 2000) and is effective for determining sex in several apes (e.g., gorills, chimpanzees, gibbons) (Bradley et al. 2001; Ensminger and Hoffman 2002), the procedure, unfortunately, is not broadly applicable across the rest of the primate order. For example, the assay is ineffective for sex-typing orangutans, lemurs, ba- boons, and many platyrrhines (Ensminger and Hoffman 2002; Steiper and Ruvolo 2003, Di Fiore, unpublished data). Here, I present a new PCR-based sexing assay that is effective across most primates. The proce- dure uses a single, multiplex PCR to simulta- neously amplify fragments of the amelogenin X gene and the Y-linked sex-determining region (SRY) gene. The amelogenin locus is expected to amplify in all samples containing sufficient nuclear DNA and thus serves as a positive PCR control, while the SRY locus should amplify only if a Y chromosome template is present and thus is used to assign sex. The two amplicons differ in size by 35 bp and are easily separated and visualized using benchtop procedures. Importantly, the target fragments are short and are expected to amplify reliably even from degraded DNA tem- plates recovered from non-invasive samples. Primate-specific PCR primers for amelogenin X and SRY were designed based on sequence data available in GenBank. The primers AMEL-F1: 5¢-ACCACCAGCTTCCCAGTTTA-3¢ and AMEL- R1: 5¢ -GCTGGGWTAGAACCAAGCTG-3¢ amplify Conservation Genetics (2005) 6:1053–1058 Ó Springer 2006 DOI 10.1007/s10592-005-9086-5