Development of eight microsatellite loci from the endangered huemul ( Hippocamelus bisulcus ) and cross-species amplific
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TECHNICAL NOTE
Development of eight microsatellite loci from the endangered huemul (Hippocamelus bisulcus) and cross-species amplification in six other ungulate species Aaron B. A. Shafer • Paulo Corti • David W. Coltman Juan Carlos Marı´n • Corey S. Davis
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Received: 16 December 2011 / Accepted: 17 December 2011 / Published online: 5 January 2012 Ó Springer Science+Business Media B.V. 2011
Abstract We developed eight polymorphic microsatellite loci for the huemul (Hippocamelus bisulcus) and tested for cross-species amplification in six other ungulate species. Microsatellite loci were typed in 58 individuals from the Lago Cochrane National Reserve, Chile. Two to five alleles were observed per locus (mean 3.4) and observed heterozygosity ranged from 0.29 to 0.60 (mean 0.53). One to five loci amplified in other ungulate species. These markers will be used to refine studies of population structure and allow for more accurate pedigree reconstruction in the huemul. Keywords Microsatellites Huemul Andean deer Hippocamelus bisulcus
The huemul (Hippocamelus bisulcus), or Andean deer, is endemic to the mountains of southern Chile and Argentina. The huemul is considered endangered with fewer than 2,500 mature individuals thought to exist in the wild (Jime´nez et al. 2008). Microsatellite loci developed in other ungulates have been analyzed in the huemul and showed low genetic variability (Corti et al. 2011). Species-specific microsatellite markers would help moot concerns over A. B. A. Shafer D. W. Coltman C. S. Davis (&) Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada e-mail: [email protected] P. Corti Instituto de Ciencia Animal, Programa de Investigacio´n Aplicada en Fauna Silvestre, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile J. C. Marı´n Departamento de Ciencias Ba´sicas, Facultad de Ciencias, Universidad del Bı´o-Bı´o, Chilla´n, Chile
ascertainment bias and improve the accuracy of population genetic and parentage studies. Here, we report the isolation and characterization eight polymorphic microsatellite loci from the huemul. Variability was assessed in 58 individuals from the Lago Cochrane National Reserve (LCNR) and six other ungulate species. A library was constructed from huemul genomic DNA and enriched for GT microsatellites using the method of Hamilton et al. 1999. Seventy-two clones were sequenced on an ABI 3730 DNA analyzer using the BigDye v3.1 sequencing kit (Applied Biosystems). Microsatellite primers were designed for 29 loci using the software Primer3 (Rozen and Skaletsky 2000) and amplified in eight individuals using PCR and M13 fluorescent labeling (Schuelke 2000). 15 ll PCRs consisted of 19 PCR buffer (10 mM Tris pH 8.8, 0.1% Triton X-100, 50 mM KCl, 0.16 mg/ml BSA), 1 U of Taq polymerase (isolated as in Engelke et al. 1990), 2.4 mM MgCl2, 0.2 lM of each dNTP, 0.08 lM M13(-21) tailed forward primer, 0.32 lM reverse primer, 0.32 lM fluorescently labelled M13(-21) primer and approximately 25 ng of tem
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