Sex identification assay useful in great apes is not diagnostic in a range of other primate species.код для вставкиСкачать
American Journal of Primatology 56:129–134 (2002) BRIEF REPORT Sex Identification Assay Useful in Great Apes Is Not Diagnostic in a Range of Other Primate Species AMANDA L. ENSMINGER AND SUSAN M.G. HOFFMAN* Department of Zoology, Miami University, Oxford, Ohio The ability to identify the sex of individuals from noninvasive samples can be a powerful tool for field studies. Amelogenin, a nuclear gene proximate to the pseudoautosomal region of mammalian sex chromosomes, has a 6 base-pair (bp) size difference between human X and Y chromosomes that can be PCR-amplified and sized to distinguish male from female DNA. We examined whether this test can be used to identify sex from different DNA sources across a number of nonhuman primate taxa. Using human amelogenin primers, we were able to amplify diagnostic products from the four great ape species tested, but products from five other primate species were not sexually dimorphic. Am. J. Primatol. 56:129–134, 2002. © 2002 Wiley-Liss, Inc. Key words: amelogenin; sex identification; noninvasive samples; primates INTRODUCTION Identifying the sex of primates in the field can be difficult because of subject inaccessibility, lack of habituation, or ambiguous morphological cues. However, individuals can be sexed from field samples such as hair, feces, and partially chewed foodstuffs by PCR amplification of divergent sequences from homologous portions of the X and Y chromosomes. In mammals, both the sex-determining SRY gene [Griffiths & Tiwari, 1993; Taberlet et al., 1993] and the enamel protein gene amelogenin [Bradley et al., 2001; Nakahori et al., 1991] have been used for sex identification. Amelogenin has been used successfully to identify the sex of cattle embryos from blastomeres [Chen et al., 1999], wild sika deer from feces [Yamauchi et al., 2000], and ancient human remains from bone samples [Stone et al., 1996]. Amelogenin is a single-copy nuclear gene that lies proximal to the pseudoautosomal region on the short arms of both mammalian sex chromosomes, and includes a 6 base-pair (bp) insertion in the third intron of the Y gene vs. the homologous X gene in humans [Salido et al., 1992]. The Y-specific allele remains unique to males, since the location of the gene rarely allows for recombination. A single primer pair (Amel-A/B) designed from intron 3 of the human amelogenin sequence [Sullivan et al., 1993] generates 106 and 112 bp products from the genes on the X and Y chromosomes, respectively. Amplification of a female (XX) sample Contract grant sponsor: Miami University Summer Field Research Workshop. *Correspondence to: Susan Hoffman, Department of Zoology, Miami University, Oxford, OH 45056. E-mail: firstname.lastname@example.org Received 3 April 2001; revision accepted 17 October 2001 © 2002 Wiley-Liss, Inc. DOI 10.1002/ajp.1069 130 / Ensminger and Hoffman results in products of one length, while a male (XY) sample results in products of two different lengths, creating a diagnostic sex test. Tests using loci that are unique to the Y chromosome, such as SRY, do not yield any product when amplifying female samples, and thus cannot distinguish female samples from failed amplifications. To correct for this problem, a second primer set can be used as a positive control, but this is unnecessary with amelogenin since every successful PCR reaction produces bands (in both sexes). Recently this assay was shown to work reliably on fecal samples from gorillas and chimpanzees [Bradley et al., 2001]. In this study, we test the utility of the amelogenin assay across additional primate species to determine the range of its application as a diagnostic sex test. METHODS We amplified an intron 3 amelogenin segment in nine primate species, ranging from great apes to prosimians, and in the domestic cat. DNA sources were varied (Table I). DNA was extracted from Felis, Gorilla, and female Homo blood samples using the QIAamp DNA Blood Kit (Qiagen Inc., Valencia, CA), and from Papio tissue and Saimiri blood using the DNeasy Tissue Kit (Qiagen Inc., Valencia, CA). Female Homo and all Pan paniscus plucked hair samples were extracted using a Chelex extraction protocol [Gagneux et al., 1997]. Pan troglodytes and Papio sp. fecal samples were extracted using the QIAamp DNA Stool Kit (Qiagen Inc., Valencia, CA), with modifications as described in Morin et al. . All other DNA extract samples were provided by other researchers. We used the previously published Amel-A/B primers [Sullivan et al., 1993] designed from the human sequence of the amelogenin gene, which flank a 6-bp insertion in the third intron of the Y gene vs. the homologous X gene. (The numbering of exons and introns in the amelogenin gene has varied between studies, due to its unusual structure; here we follow the designations of Salido et al., 1992). PCR amTABLE I. Species and Source Material of DNA Samples (the Number of Individuals Analyzed Is Shown in Parentheses), and the Number of Products Resulting From Amplification With Amel-A/B Primers for Each Sex Tested* Source material (n) Species Homo sapiens Pan troglodytes Pan paniscus Gorilla gorilla Papio spp. Ateles chamek Saimiri sciureus Saguinus oedipus Lemur macaco Felis catus Y samples a a Blood (1) , placenta (1) Blood (1)a, feces (11)b,c Hair (4) Cell culture (2)a Blood (8)a, skin tissue (1), feces (2)b,d Blood (1)a Blood (8), hair (2)a Muscle tissue (1)a Muscle tissue (1)a Blood (1) Number of alleles X samples Y X Blood (1), hair (1) Blood (1)a, feces (43)b,c Hair (2) Blood (1) Blood (12)a, feces (3)b,d 2 2 2 2 1 1 1 1 1 1 (No samples available) Hair (2)a Muscle tissue (1)a Muscle tissue (1)a Blood (4) 1 1e 1 1 1 – 1 1 1 1 *Unless otherwise indicated, samples were from individuals of known sex. a Samples of DNA extracts were gifts from other researchers. b Fecal extractions done by A.L. Ensminger at the Max Planck Institute for Evolutionary Anthropology (Leipzig); all other extractions done at Miami University. c Sex as determined by the amelogenin test described below. d Sex assignments tentatively made at the time of collection. e Amplification of one sample resulted in two products, while nine samples resulted in only one product. Amelogenin Assay for Primates / 131 plification was carried out in a total volume of 25 µl consisting of 1X PCR buffer [10 mM Tris-HCl, pH 9.0; 50 mM KCl; 0.1% Triton® X-100], 1 mM MgCl2, 200 µM each dNTP, 0.5 µM each primer, 0.75 units of Taq polymerase (Promega, Madison, WI), and 0.5–2 µl template DNA. Amplification conditions on an MJ Research (Waltham, MA) PTC-100 thermocycler were: 35 cycles of 1 min at 94°C, 1 min at 58°C, and 1 min at 72°C. Each sample was amplified at least twice. Fecal extracts were amplified in triplicate with a modified protocol, as described in Morin et al. . All products were initially run on 4% NuSieve agarose gels for 90 min at 90V; they were then resolved on either 8% polyacrylamide gels with ethidium bromide for 2.5 hr at 70V (EagleEye Imaging System, Stratagene, La Jolla, CA), or by capillary electrophoresis on an ABI 310 PRISM with Gene Scan 2.0 software (Perkin-Elmer Applied Biosystems, Foster City, CA). RESULTS Invasive samples amplified more consistently with the Amel-A/B primers than did noninvasive samples. Amplification products from invasive samples could usually be resolved on agarose gels, while products from noninvasive sample amplifications often faded during agarose electrophoresis. The best resolution was achieved by using polyacrylamide gels for hair extract amplifications, and capillary electrophoresis for fecal extract amplifications. Two bands that differed in size by 6 bp could be reliably amplified by the Amel-A/B primers from human male genomic DNA and from DNA of males of three great ape species (Pan paniscus, P. troglodytes, and Gorilla gorilla). For all of these species, a single strong band was obtained from female DNA samples, of the same size as the smaller of the two male bands (Fig. 1). One set of bonobo female hair extracts showed faint second bands of approximately the same length as the Y chromosome product (Fig. 1). These second bands could be distinguished from male Y chromosome bands because they were very faint relative to the X Fig. 1. Selected male (M) and female (F) primate samples amplified with primer pair Amel-A/B and run on 8% polyacrylamide gels. SS = size standard (BRL 100 bp ladder). Data not shown for all species. The male Saimiri sciureus shown is the exceptional (apparently heterozygous) individual. The female Pan paniscus amplification shown is from the first set of extractions, and thus shows the faint higher band. 132 / Ensminger and Hoffman chromosome bands (mean density of Y:X = 0.13 vs. 0.79). These second bands were not present in amplifications of another set of extractions from female bonobo hairs, suggesting that they were due to contamination of the initial extracts. The Amel-A/B primer pair was tested on five additional primate species (Table I), including one Old World monkey species (Papio spp.), three New World monkey species (Saimiri sciureus, Saguinus oedipus, and Ateles chamek), and one prosimian species (Lemur macaco), as well as the domestic cat. A single Amel-A/B product of approximately 106 bp was obtained from both males and females for all of these species. The only exception was a single Saimiri male (out of 10 males tested); two independent amplifications from this individual each gave a two-band pattern similar to that of the male great apes (Fig. 1). We could not obtain additional DNA from this individual male, so we were unable to confirm whether the second band was due to contamination or to a real polymorphism. DISCUSSION The amelogenin gene can be amplified from both fecal and hair samples, and used to identify sex in some primates. The 6-bp insertion in intron 3 of the Y chromosome amelogenin gene, however, does not appear consistently in any of the primate species tested other than the African great apes. The presence of faint second bands in amplifications from some female bonobo extracts, presumably due either to contamination or to “false allele” artifacts of PCR [Taberlet et al., 1996], could be discounted by quantifying their densities. Allelic dropout, which is frequently a problem with DNA extracted from noninvasive samples, could cause males to be misidentified as females using this test. Multiple extractions from each sample and multiple amplifications from each extract, as done in this study when possible, can usually resolve this problem [Taberlet et al., 1996]. Our results for hair and fecal samples are consistent with those of Bradley et al. , who found that this assay, using the same protocols, detects the Y band in known gorilla and chimpanzee males about 97% of the time. Misidentification of males can also occur due to the loss of the Y chromosome amelogenin allele, since its location outside the pseudoautosomal region makes it vulnerable to deletion mutations. Such losses of the Y chromosome allele can create heterogeneity between males within a species. This is potentially an explanation for our results for Saimiri sciureus, and also may account for some of the 2–3% identification error seen by Bradley et al. . Overall, however, such mutations seem to occur at an acceptably low rate in the species examined to date. More significantly, in the genus Papio, the Southern-blot analysis of Nakahori et al.  suggests that the Y chromosome allele has been entirely deleted from all males: their amelogenin probe detected only a single band for male Papio hamadryas, of a lesser intensity than the single female band. This explanation was supported by Huang et al. , who were unable to amplify a portion of the amelogenin Y-specific allele in Papio cynocephalus. Regardless of mechanism, however, any time a significant fraction of males shows a one-band pattern, this assay cannot be reliably used; across the range of primates tested, it is valid only for African great apes. Southern blots of samples from additional primate species (Cebus apella, Macaca fascicularis, M. fuscata, and M. mulatta) have shown other sex-specific size differences when probed with a human genomic fragment containing the amelogenin gene [Nakahori et al., 1991]. Bailey et al.  amplified another Amelogenin Assay for Primates / 133 region of the amelogenin gene and found a 184-bp size difference between X and Y alleles in the great apes (including the orangutan), Macaca mulatta, and M. fascicularis. For great ape species, this sex identification test has both advantages and disadvantages. Products amplified from less invasive samples require more elaborate resolution techniques, increasing the time and cost involved. Amplification products from another region of the amelogenin gene that contains a larger bp difference between alleles [Bailey et al., 1992] would be more easily resolved on agarose gels. However, since noninvasive materials, such as feces and hair, are likely to contain highly fragmented DNA, the short fragments produced by the Amel-A/B primers are more likely to amplify than longer fragments. Overall, while this set of primers may provide the best means available for sex-typing noninvasive great ape samples, primatologists interested in using this kind of test to sex-type other species should look elsewhere in the amelogenin gene for diagnostic polymorphisms. ACKNOWLEDGMENTS We thank Susan Cropp, Evan Eichler, Linda Marchant, Bill McGrew; Pat Parrie and the Tulane Regional Primate Research Center; Jeff Rogers and the Southwest Foundation for Biomedical Research; Jack Vaughn; the Cincinnati Zoo; and the Columbus Zoo for blood, fecal, hair, and DNA samples. We are indebted to Christophe Boesch and everyone in the Department of Primatology at the Max Planck Institute for Evolutionary Anthropology (Leipzig), particularly Brenda Bradley, Karen Chambers, Heike Siedel, and Linda Vigilant, for their instruction, resources, and assistance. We are especially grateful to Doug Meikle and his students, and to Linda Marchant, Bill McGrew, and Tom Gregg for all their help. 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