Microsatellite primers for the wild brown capuchin monkey Cebus apella

Molecular ecological studies are not common in primatological research. Part of the problem with monkeys from Central and South America (New World) is the difficulty of obtaining DNA from the animals, given their arboreal life. This problem can now be overcome with the new methods developed to extract DNA from fecal material, food wedges and urine. The second problem is the lack of microsatellite primers that can be used to amplify the small amounts of DNA found in the fecal samples. Here, I report the characterization of five microsatellite primers isolated from Cebus apella, and tv\ro primers isolated from Lagothrix lagotricha. The use of these primers in other neotropical primate species is also reported. Five micrograms of genomic DNA from Cebus apella and Lagothrix lagotricha were digested with Sau3AI and fragments in the range of 400-1000 bp were selected for cloning into pBluescript-SK cut with Bamííl. Eight-hundred colonies of each species were screened with (GT)jg and (CT)jg DIG-labeUed probes and positive clones were detected using a DIG detection kit (Boehringer Mannheim). Positive clones were sequenced on an Applied Biosystem ABI 373A automated sequencer, and analysed using Perkin Elmer DNA Sequencing Software (version 2.1.1). Out of the five clones sequenced from the Lagothrix library, four contained microsatellites, and out of the 13 clones sequenced from the Cebus hbrary, nine contained microsatelhtes. Primers were designed using GeneRun software. Seven of the 14 pairs of primers designed are reported in Table 1. PCR conditions were optimized on Cebus DNA extracted from blood using standard phenol-chloroform extractions. DNA amplifications were performed in a total volume of 25 |xL (10 mM Tris-HCl (pH 8.9), 50 mM KCl, 20 |XM of each dNTP, 0.5 U of native Taq polymerase (Perkin-Ehner/Cetus) and 4 pmol of each of the appropriate primer). One primer of each pair was labelled with a fluorescent dye (either TET or HEX). The optimum MgClj and BSA concentrations as well as optimum annealing temperatures for the different primers are shown in Table 1. The thermal cychng (on a PTC-IOOTM Programmable thermal controller, MJ Research Inc.) was performed at the foUow^ing conditions: 2 min at 92 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at the selected annealing temperature and 30 s at 72 °C and an extra extension time of 10 min at 72 °C. PCR products were visualized on 3% agarose gels stained with ethidium bromide. One microlitre of each PCR product was mixed with 2 |J.L of formamide, and 0.5 |xL of the size standard (GeneScan TAMRA-500, Apphed Biosystems) and the TAMRA buffer, denatured at 95 °C for 2 min, and loaded on 5% 'Sequagel' (National Diagnostics) with Ix TBE buffer, in the Apphed Biosystems 373A automated sequencer. Alíele sizes were estabhshed using the GeneScan software. The primers, both those derived from the Cebus Hbrary and those from the Lagothrix hbrary, were tested in three populations of Cebus apella: the colony at the animal centre of the NIH; a group from Parque Natural Nacional Tinigua, Macarena Colombia; and a group from Parque Nacional de Iguazú in Argentina. DNA from the last two populations were extracted from fecal samples using the Constable et al. (1995) protocol, while the NIH samples were extracted from blood (and some individuals from both blood and faeces as a control for the fecal amphfications) following standard protocols. In addition, the primers were tested in DNA samples extracted from tissue of other neotropical primer species using the same PCR conditions as before but with an annealing temperature of 45 °C. The results are shown in Table 2.