Isolation of 10 microsatellite markers for mongoose lemurs (Eulemur mongoz)

The lemurs represent a prosimian group endemic to Madagascar. The mongoose lemur is one of five species in the genus Eulemur, which belongs to the family Lemuridae. E. mongoz occurs in three geographically isolated populations; on two Comorian islands (Anjouan and Mohéli) and in the northwest of Madagascar (Tattersall 1982). It is one of only two lemur species occurring outside Madagascar, and was probably introduced to the Comores by humans (Mittermeier et al. 1994; Pastorini et al. 2003). The species is listed in Appendix 1 of CITES, and is protected by law in Madagascar and on the Comores (Harcourt & Thornback 1990). In the wild, mongoose lemurs live in small family groups, consisting of an adult pair and associated offspring (Curtis & Zaramody 1998). Currently, there are more than 100 mongoose lemurs in captivity. While microsatellite markers have been developed for other lemur species, their application to E. mongoz has not been reported. The microsatellite loci described here will allow collection of genetic data that can guide management of captive E. mongoz (Bettinger 2000) and contribute to our understanding of group dynamics and breeding systems in wild populations.

Genomic DNA was extracted from an E. mongoz tissue sample using a standard phenol-chloroform extraction (Sambrook et al. 1989). The DNA was digested with the restriction enzyme Sau3AI. After treatment with Calf Intestinal Phosphatase (New England Biolabs) the DNA fragments were ligated into BamHI-digested LITMUS 29 phagemid vector (New England Biolabs) using T4 DNA Ligase (New England Biolabs). One Shot® Max Efficiency DH5α^TM-T1^R (Invitrogen) competent cells were used for the transformation. The transformation was plated on agar containing ampicillin and X-gal. Insert-bearing clones were grown in liquid LB and plated onto nylon membranes. In total, 5088 clones with inserts were screened for microsatellites using four [γ³²P]-dATP labelled oligonucleotide probes (CA)₁₆ (TC)₁₆ (GC)₁₆, and (AT)₁₆. A total of 373 positive clones were identified and were then PCR amplified and sequenced using universal primers to determine the repeat length and the flanking DNA sequences. Of the 373 positive clones, 109 contained a motif with four or more repeats. From these 109, primers were designed for 16 loci (Em1–Em16).

Primer pairs were first used to amplify and sequence each locus to ensure it contained the expected repetitive motif. Microsatellite polymorphism was assessed on four distantly related individuals of E. mongoz. Three loci showed no variability. For the 13 polymorphic loci the PCR was optimized for analysis on an ABI PRISM 377XL automated DNA sequencer. Forward primers were labelled with one of three fluorescent dye markers TET, FAM or HEX. Approximately 10–100 ng template DNA was amplified in 20 µL reactions using 0.06 m Tris, 0.015 m (NH₄)₂SO₄, 1.5 mm MgCl₂, 0.78 m DMSO, 0.025 mm each dNTP, 1 mm each primer, and 0.5 U Thermophilus aquaticus (Taq) polymerase (Promega) in a GeneAmp PCR System 9700 (Applied Biosystems) thermal cycler. The following protocol was used for amplification: 25–35 cycles of denaturing at 95 °C for 30 s, primer annealing at 50–65 °C for 60 s, and extension at 72 °C for 60 s. The cycles were followed by a final extension for 5 min at 72 °C. Amplification conditions were optimized by changing the number of cycles and/or the annealing temperature (results in Table 1). The amplification products were electrophoresed on 5.3% 0.2 mm urea-acrylamide gels together with an internal size standard (TAMRA, Applied Biosystems) in an ABI PRISM 377XL DNA sequencer and allele sizes were analysed using genescan 3.1.2 (Applied Biosystems) software. For three of the 13 polymorphic loci the PCR could not be satisfactorily optimized.

To verify Mendelian inheritance, samples of 13 offspring and their five known pairs of parents from the captive colony were genotyped for the 10 optimized loci. For one locus, one allele of an offspring was absent in the genotypes of both its parents. Therefore, that locus was removed from the data set. Alleles at the other nine loci segregated according to Mendelian expectations.

Two of the remaining nine loci in E. mongoz deviated from Hardy–Weinberg proportions (Em5, Em15; P < 0.05). However, when the captive and wild populations were tested separately, all loci followed the Hardy–Weinberg principle (P > 0.20).

Thus, nine microsatellite loci derived from the E. mongoz library were found to be promising for population genetic analyses of mongoose lemurs. One additional suitable polymorphic marker was derived from a Lemur catta library (Lc1). The genotypes of up to 54 captive and 34 wild animals from Anjamena, northwest Madagascar, were determined at the 10 prospective marker loci. The number of alleles and their size ranges as well as the expected and observed heterozygosities found at each locus in E. mongoz are given in Table 1. The high polymorphism and Mendelian inheritance of the microsatellite markers presented here makes them a suitable tool for genetic studies of E. mongoz.