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Volume 65, Issue 6


Multiplex PCR amplification of 13 microsatellite loci for Aquila chrysaetos in forensic applications

Marcela Bielikova
  • Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynska dolina B2, SK-84215, Bratislava, Slovakia
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/ Andrej Ficek
  • Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynska dolina B2, SK-84215, Bratislava, Slovakia
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/ Danka Valkova
  • Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynska dolina B2, SK-84215, Bratislava, Slovakia
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/ Jan Turna
  • Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynska dolina B2, SK-84215, Bratislava, Slovakia
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Published Online: 2010-10-15 | DOI: https://doi.org/10.2478/s11756-010-0112-9


The golden eagle (Aquila chrysaetos) is an endangered raptor, which is threatened mainly by illegal egg and nestling robbery. Here we describe a fluorescently labeled, multiplex PCR method using 13 microsatellite markers, which provides a powerful tool for the individual identification and parentage testing of the Golden eagle. This test should be applicable to both forensic analysis and population studies. Fifteen polymorphic loci from A. chrysaetos were cross-amplified. Subsequent PCR condition optimization led to the successful co-amplification of 13 different loci in a single PCR reaction. Fifty samples from wild-living individuals and 89 samples from captive-bred individuals were examined. The results indicated that both populations have similar levels of moderate inbreeding, unsurprising in a small population. This probability of excluding a random individual in parentage analysis was 0.9912 for the wild population and 0.9932 in the captive-bred one in the case that both the individual and its mother were examined together. The probability of identity was estimated to be 3 × 10−8 for the wild and 4 × 10−8 for the captive-bred populations. Given the size of the Slovak golden eagle population, this test should therefore be sufficient to reliably identify individual raptors and assess parentage in both conservation studies and forensic analysis.

Keywords: Aquila chrysaetos; golden eagle; microsatellites; multiplex-PCR; parentage assessment

  • [1] Amos W., Wilmer J., Fullard K., Burg T.M., Croxall J.P., Bloch D. & Coulson, T. 2001. The influence of parental relatedness on reproducive success. Proc. R. Soc. Lond., Ser. B: Biol. Sci. 268: 2021–2027. http://dx.doi.org/10.1098/rspb.2001.1751CrossrefGoogle Scholar

  • [2] Balloux F., Amos W. & Coulson T. 2004. Does heterozygosity estimate inbreeding in real populations? Mol. Ecol. 13: 3021–3031. http://dx.doi.org/10.1111/j.1365-294X.2004.02318.xCrossrefGoogle Scholar

  • [3] Bayle P. 1999. Preventing birds of prey problems at transmission lines in western Europe. J. Raptor Res. 33: 43–48. Google Scholar

  • [4] Bonnet A., Thévenon S., Maudet F. & Maillard J.C. 2002. Efficiency of semi-automated fluorescent multiplex PCR’s with eleven microsatellite markers for genetic studies of deer populations. Anim. Genet. 33: 343–350. http://dx.doi.org/10.1046/j.1365-2052.2002.00873.xCrossrefGoogle Scholar

  • [5] Bourke B.P. & Dawson D.A. 2006. Fifteen microsatellite loci characterized in the golden eagle Aquila chrysaetos (Accipitridae, Aves). Mol. Ecol. Notes 6: 1047–1050. http://dx.doi.org/10.1111/j.1471-8286.2006.01429.xCrossrefGoogle Scholar

  • [6] Bourke B.P., Frantz A.C., Lavers C.P., Davison A., Dawson D.A. & Burke T.A. 2010. Genetic signatures of population change in the British golden eagle (Aquila chrysaetos). Conserv. Genet. (in press); DOI: 10.1007/s10592-010-0076-x. CrossrefWeb of ScienceGoogle Scholar

  • [7] Brook B.W., Tonkyn D.W., O’Grady J.J. & Frankham R. 2002. Contribution of inbreeding to extinction risk in threatened species. Conserv. Ecol. 6: art. No. 16. Google Scholar

  • [8] Bruford M.W., Hanotte O., Brookfield J.F.Y. & Burke T. 1998. Multi-locus and single-locus DNA fingerprinting, pp. 287–336. In: Hoelzel A.R. (ed.), Molecular Genetic Analysis of Populations: A Practical Approach. Oxford University Press, New York. Google Scholar

  • [9] Busch J.D., Katzner T.E., Bragin E. & Keim P. 2005. Tetranucleotide microsatellites for aquila and haliaeetus eagles. Mol. Ecol. Notes 5: 39–41. http://dx.doi.org/10.1111/j.1471-8286.2004.00823.xCrossrefGoogle Scholar

  • [10] Coulson T., Albon S., Slate J. & Pemberton J. 1999. Microsatellite loci reveal sex-dependent responses to inbreeding and out-breeding in red deer calves. Evolution 53: 1951–1960. http://dx.doi.org/10.2307/2640453CrossrefGoogle Scholar

  • [11] Danko Š., Darolová A. & Krištín A. 2002. Birds Distribution in Slovakia. Veda, Bratislava, 688 pp. (In Slovak) Google Scholar

  • [12] Edwards M.C. & Gibbs R.A. 1994. Multiplex PCR: advantages, development, and applications. Genome Res. 3: 65–75. http://dx.doi.org/10.1101/gr.3.4.S65CrossrefGoogle Scholar

  • [13] Frankham R. 1998. Inbreeding and extinction: island populations. Conserv. Biol. 12: 665–675. http://dx.doi.org/10.1046/j.1523-1739.1998.96456.xCrossrefGoogle Scholar

  • [14] Genlous S. & Björn S. 2003. Microsatellite variability and heterozygote deficiency in the arctic-alpine Alaskan wheatgrass (Elymus alaskanus) complex. Genome 46: 729–737. http://dx.doi.org/10.1139/g03-052CrossrefGoogle Scholar

  • [15] Hailer F., Gautschi B. & Helander B. 2005. Development and multiplex PCR amplification of novel microsatellite markers in the White-tailed Sea Eagle, Haliaeetus albicilla (Aves: Falconiformes, Accipitridae). Mol. Ecol. 5: 938–940. http://dx.doi.org/10.1111/j.1471-8286.2005.01122.xCrossrefGoogle Scholar

  • [16] Hille S.M., Nesje M. & Segelbacher G. 2003. Genetic structure of kestrel populations and colonization of the Cape Verde archipelago. Mol. Ecol. 12: 2145–2151. http://dx.doi.org/10.1046/j.1365-294X.2003.01891.xCrossrefGoogle Scholar

  • [17] Kendall M. & Stewart A. 1977. The Advanced Theory of Statistics. Vol. 1., MacMillan, New York. Google Scholar

  • [18] Lande R. & Barrowclough G.F. 1987. Effective population size, genetic variation, and their use in population management, pp. 87–123. In Soulé M.E. (ed.), Viable Populations for Conservation. Cambridge University Press, New York. http://dx.doi.org/10.1017/CBO9780511623400.007CrossrefGoogle Scholar

  • [19] Marshall R., Buchanan K. & Catchpole C. 2003. Sexual selection and individual genetic diversity in a songbird. Proc. R. Soc. Lond., Ser. B: Biol. Sci. 270: 248–250. http://dx.doi.org/10.1098/rsbl.2003.0081CrossrefGoogle Scholar

  • [20] Martínez-Cruz B., David V.A., Godoy J.A., Negro J.J., O’Brien S.J. & Johnson W.E. 2002. Eighteen polymorphic microsatellite markers for the highly endangered Spanish imperial eagle (Aquila adalberti) and related species. Mol. Ecol. Notes 2: 323–326. http://dx.doi.org/10.1046/j.1471-8286.2002.00231.xCrossrefGoogle Scholar

  • [21] McRae S.B. & Amos W. 1999. Can incest within cooperative breeding groups be detected using DNA fingerprinting? Behav. Ecol. Sociobiol. 47: 104–107. http://dx.doi.org/10.1007/s002650050655CrossrefGoogle Scholar

  • [22] Nesje M. & Roed K.H. 2000. Microsatelite DNA markers from the gyrfalcon (Falco rusticolus) and their use in other raptor species. Mol. Ecol. 9: 1433–1449. http://dx.doi.org/10.1046/j.1365-294x.2000.00991.xCrossrefGoogle Scholar

  • [23] Nesje M., Roed K.H., Lifjeld J.T., Lindberg P. & Steen O.F. 2000. Genetic relationships in the peregrine falcon (Falco peregrinus) analysed by microsatellite DNA markers. Mol. Ecol. 9: 53–60. http://dx.doi.org/10.1046/j.1365-294x.2000.00834.xCrossrefGoogle Scholar

  • [24] O’Grady J.J., Brook B.W., Reed D.H., Ballou J.D., Tonkyn D.W. & Frankham R. 2006. Realistic levels of inbreeding depression strongly affect extinction risk in wild populations. Biol. Conserv. 133: 42–51. http://dx.doi.org/10.1016/j.biocon.2006.05.016CrossrefGoogle Scholar

  • [25] Ortego J., González E.G., Sánchez-Barbudo I., Aparicio J.M. & Cordero P.J. 2007. Novel highly polymorphic loci and cross-amplified microsatellites for the Lesser kestrel Falco naumanni. Ardeola 54: 101–108. Google Scholar

  • [26] O’Toole L., Fielding A.H. & Haworth P.F. 2002. Re-introduction of the golden eagle into the Republic of Ireland. Biol. Conserv. 103: 303–312. http://dx.doi.org/10.1016/S0006-3207(01)00141-0CrossrefGoogle Scholar

  • [27] Padilla J.A., Parejo J.C., Salazar J., Martínez-Trancón M., Rabasco A., Sansinforiano E. & Quesada A. 2008. Isolation and characterization of polymorphic microsatellite markers in lesse kestrel (Falco naumanni) and cross-amplification in common kestrel (Falco tinnunculus). Conserv. Genet. 10: 1357–1360. http://dx.doi.org/10.1007/s10592-008-9711-1CrossrefGoogle Scholar

  • [28] Pedrini P. & Sergio F. 2001. Golden eagle Aquila chrysaetos density and productivity in relation to land abandonment and forest expansion in the Alps. Bird Study 48: 194–199. http://dx.doi.org/10.1080/00063650109461218CrossrefGoogle Scholar

  • [29] Raymond M. & Rousset F. 1995. GENEPOP (Version 1.2): population genetics software for exact tests and ecumenicism. J. Hered. 86: 248–249. Google Scholar

  • [30] Read M.M. 2006. Focus on DNA fingerprinting research. Nova Science Publishers, Inc., New York. Google Scholar

  • [31] Reed D. & Frankham R. 2003. Correlation between population fitness and genetic diversity. Conserv. Biol. 17: 230–237. http://dx.doi.org/10.1046/j.1523-1739.2003.01236.xCrossrefGoogle Scholar

  • [32] Rychlik I., Kubícek O., Holcák V., Bárta J. & Pavlík I. 1994. DNA fingerprinting in falconidae. Vet. Med. 39: 111–116. Google Scholar

  • [33] Slate J., Marshall T. & Pemberton J. 2000a. A retrospective assessment of the accuracy of the paternity inference program CERVUS. Mol. Ecol. 9: 801–808. http://dx.doi.org/10.1046/j.1365-294x.2000.00930.xCrossrefGoogle Scholar

  • [34] Slate J., Kruuk L., Marshall T., Pemberton J. & Clutton-Brock T. 2000b. Inbreeding depression influences lifetime breeding success in a wild population of red deer (Cervus elaphus). Proc. R. Soc. Lond., Ser. B: Biol. Sci. 267: 1657–1662. http://dx.doi.org/10.1098/rspb.2000.1192CrossrefGoogle Scholar

  • [35] Suchentrunk F., Haller H. & Ratti P. 1999. Gene pool variability of a golden eagle (Aquila chrysaetos) population from Swiss Alps. Biol. Conserv. 90: 151–155. http://dx.doi.org/10.1016/S0006-3207(99)00025-7CrossrefGoogle Scholar

  • [36] Taylor A.C., Sherwin W. & Wayne R. 1994. Genetic variation of microsatellite loci in a bottlenecked species: the northern hairynosed wombat Lasiorhinus krefftii. Mol. Ecol. 3: 277–290. http://dx.doi.org/10.1111/j.1365-294X.1994.tb00068.xCrossrefGoogle Scholar

  • [37] Tingay R.E., Dawson D.A., Pandhal J., Clarke M.L., David V.A., Hailer F. & Culver M. 2007. Isolation of 22 new Haliaeetus microsatellite loci and their characterization in the critically endangered Madagascar fish-eagle (Haliaeetus vociferoides) and three other Haliaeetus eagle species. Mol. Ecol. Notes 7: 711–715. http://dx.doi.org/10.1111/j.1471-8286.2007.01690.xCrossrefGoogle Scholar

  • [38] Valiére N. 2002. GIMLET: a computer program for analysing genetic individual identification data. Mol. Ecol. Notes 2: 377–379. http://dx.doi.org/10.1046/j.1471-8286.2002.00228.xCrossrefGoogle Scholar

  • [39] Waits L.P., Luikart G. & Taberlet P. 2001. Estimating the probability of identity among genotypes in natural populations: cautions and guidelines. Mol. Ecol. 10: 249–256. http://dx.doi.org/10.1046/j.1365-294X.2001.01185.xCrossrefGoogle Scholar

  • [40] Weir B.S. & Cockerham C.C. 1984. Estimating F-statistics for the analysis of population structure. Evolution 38: 1358–1370. http://dx.doi.org/10.2307/2408641CrossrefGoogle Scholar

  • [41] Weir B.S. & Hill W.G. 2002. Estimating F-statistic. Annu. Rev. Genet. 36: 721–750. http://dx.doi.org/10.1146/annurev.genet.36.050802.093940CrossrefGoogle Scholar

  • [42] Witkowski Z.J., Król W. & Solarz W. (eds) 2003. Carpathian List of Endangered Species. WWF and Institute of Nature Conservation, Polish Academy of Sciences, Vienna-Krakow, 84 pp. Google Scholar

About the article

Published Online: 2010-10-15

Published in Print: 2010-12-01

Citation Information: Biologia, Volume 65, Issue 6, Pages 1081–1088, ISSN (Online) 1336-9563, ISSN (Print) 0006-3088, DOI: https://doi.org/10.2478/s11756-010-0112-9.

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© 2010 Slovak Academy of Sciences. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

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