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Cross-species amplification of seventeen polymorphic microsatellite loci in
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the endangered crowned eagle (Harpyhaliaetus coronatus)
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J. H. Sarasola1-2-3, D. Canal4, C. Solaro1-2, M. A. Galmes1-3, J. I. Zanón-Martínez1-2 & J. J.
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Negro4
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Universidad Nacional de La Pampa – CONICET, Avda. Uruguay 151, 6300 Santa Rosa, La
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Pampa, Argentina.
Centro para el Estudio y Conservación de las Aves Rapaces en Argentina (CECARA),
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Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET), Avda. Uruguay
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151, 6300 Santa Rosa, La Pampa, Argentina.
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The Peregrine Fund, 5668 West Flying Hawk Lane, Boise, ID 83709 U.S.A.
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Department of Evolutionary Ecology, Estación Biológica de Doñana-CSIC, Avda. Américo
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Vespucio s/n, 41092 Seville, Spain.
Instituto de las Ciencias de la Tierra y Ambientales de La Pampa (INCITAP), Consejo
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Keywords: crowned eagle, Harpyhaliaetus coronatus, cross-species amplification,
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microsatellite, population genetics.
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Corresponding author: José Hernán Sarasola, Phone: +54 2954 430157. Email:
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sarasola@exactas.unlpam.edu.ar
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Running title: Microsatellite markers for the crowned eagle
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Abstract
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The crowned eagle (Harpyhaliaetus coronatus) is one of the most severely threatened birds of
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prey in the world for which genetic markers have not been developed. We examined the cross
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amplification of thirty seven microsatellite loci in this endangered eagle. Seventeen loci were
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polymorphic and hence valuable as tools for population genetic studies. The number of alleles
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per locus ranged from 2 to 9, and the average number of alleles across all polymorphic loci
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was 4.4. The markers tested provide a valuable resource for research in population genetics
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and the conservation of this species. The success of cross-species amplification suggests that
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these microsatellites will be useful for studies in a broad range of raptor species.
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The crowned eagle (Harpyhaliaetus coronatus) is one of the rarest and most severely
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threatened birds of prey in the world. Its range extends from southern Brazil to northern
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Patagonia, where it inhabits a variety of forested habitats, including woodlands and other
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savanna-like landscapes (Fergusson-Lees & Christie 2001). The species is listed as
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endangered under the IUCN Red List with a declining world population estimated at less than
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1,000 individuals. Crowned eagles are considered extinct in Uruguay where no records have
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been recorded since 1930 (BirdLife International 2008). Human persecution and other
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anthropogenic factors seem to be the main threat for the species in semiarid habitats of central
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and western Argentina (Sarasola & Maceda 2006, Sarasola et al. 2010). Current population
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status and population trends make the development of molecular tools that can be used to aid
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the management and conservation of this species crucial.
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Microsatellite loci are one of the best and more powerful classes of molecular markers
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for studies of genetic processes in natural populations (Frankham et al. 2004). However, their
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development maybe costly and a time consuming task. As flanking sequences of
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microsatellites are conserved region (Dawson et al. 2006, Meglécz et al. 2007) cross-species
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amplification has been proved, across a range of taxa (insects: Augustinos et al. 2011;
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amphibians: Hendrix et al. 2010; birds: Dawson et al. 2006), as a cost effective approach to
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obtain microsatellites markers in additional species. Here we communicate the cross-species
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amplification of polymorphic microsatellite loci between a phylogenetically diverse group of
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raptors and the crowned eagle. These microsatellite loci will enable further studies in
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population genetic structure and genetic diversity to be undertaken on this endangered raptor
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species.
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Thirty eight samples of crowned eagles were analyzed in the study: eighteen feathers
and twenty blood samples obtained from wild individuals from La Pampa province,
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Argentina. Genomic DNA was extracted by a standard phenol-chloroform method (Sambrook
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et al. 1989). Thirty-seven polymorphic microsatellite loci isolated for seven raptor species
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(order Falconiformes) were chosen for testing amplification in the crowned eagle. All primers
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were tested for amplification in a preliminary screening with a subset of six blood samples of
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the species. PCRs were performed in 25-µL reaction volumes containing 1x buffer, 2.0 mM
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MgCl2, 0.2 mM of each dNTP, 0.5 U Taq Polymerasa, 0.2 µM of each primer and 25 ng of
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DNA as template. PCR amplifications consisted of initial denaturation (2 min at 94°C)
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followed by 35 cycles of 30 s at 94°C, 30 s at a annealing temperature between 50ºC and 60
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ºC and 30 s at 72°C, plus a final extension of 10 min at 72°C. The entire PCR product was run
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on a 2% agarose gel, and amplicon sizes were determined using a 100-bp-size standard
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marker (BIOLINE Hyperladder). As some loci either failed to amplify or showed problems in
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the amplification (i.e. weak or nonspecific bands in gel) we ran new PCRs in order to
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optimize amplification conditions. One primer of the 35 pairs that apparently amplified a
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single amplicon was tagged with VIC, FAM, PET or NED fluorescent labels (Applied
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Biosystems). Fluorescent products were analysed on an ABI377 automated sequencer using
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Genescan 500-LIZ internal size standard. Polymorphism and alleles sizes were determined
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with Genemapper 4.0 software (Applied Biosystems). Thirteen loci were monomorphic and
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four showed unspecific amplifications that, despite new attempts to optimized PCR
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conditions, could not be eliminated. The remained 18 loci were sequenced to ensure that the
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homologous microsatellite region was being amplified. This step revealed that locus Hf-C1D2
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have lost the repetition motif as a consequence of both a deletion in the number of repetitions
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and substitution of “GA” by “GG” and was therefore discarded for further analyses. Feathers
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were genotyped three times to evaluate the frequency of genotyping errors (Horváth et al.
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2004) but no discrepancies among repetitions were found. Genotyped obtained from samples
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confirmed that each one belonged to different individuals.
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We developed a multiplexing protocol for the polymorphic markers (Table 1). For
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multiplex PCR reactions we used Quiagen multiplex PCR Kit following the supplier’s
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protocol with an annealing temperature of 56ºC. Allele sizes of markers from single locus
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amplification and multiplex reactions were compared to ensure reliability of multiplexing
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amplifications. This protocol allowed amplifying 18 loci in five reactions reducing
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significantly laboratory cost and time. The number of alleles (N), observed and expected
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heterozygosities (HO and HE) were calculated using CERVUS version 3.0.3 (Marshall 1998).
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Probability of deviation from Hardy–Weinberg equilibrium and linkage disequilibrium were
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tested using Genepop 4.0 (Raymond & Rousset 1995).
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Of the multiple loci tested from seven raptor species, at least some loci amplified from
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six of the seven species tested. The number of alleles per locus ranged from 2 to 9 with an
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average of 4.4 alleles per locus (Table 1). All loci but Hal04 and BswD107 conformed to
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Hardy-Weinberg equilibrium and no pairs of loci showed significant linkage disequilibrium
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after Bonferroni correction (Table 1). The success of cross amplification in crowned eagles
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was high in the case of primers described for Aquila heliaca (two of two primers, 100%)
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followed by Buteo buteo (83% two of three primers, monomorfic locus: Bbu51), Haliaaetus
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albicilla (60 % three of five; locus Hal03 was monomorfic and Hal01 gave non specific
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amplification), Falco rusticolus (50% one of two, locus NVHfr195–2 failed to amplify), B.
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swainsoni (49% six of thirteen; monomorfic loci: BswB220w, BswA204w, BswA303w,
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BswD327w, BswA312w; loci that failed: BswB221w and BswA110w) and Hieraeetus
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fasciatus (43% three of seven; monomorfic: Hf-C7G4, Hf-C7E1 and Hf-C1D2; non specific
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amplification: Hf-C1E6; ). No polymorphic markers were obtained from five primers
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designed by Martínez-Cruz et al. (2002) for Aquila adalberti (monomorphics: Aa11, Aa26,
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Aa36 and Aa43; non specific amplification: Aa57). The success of amplification of a locus in
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cross-species strategy in avian species could be higher when the genetic distance between
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source and target species is small (Primmer et al. 2005). Primers designed for the two Buteo
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species, which are included in the same sub-family with Harpyhaliaetus, showed a relative
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good success of amplification considering the large number of primers tested. Primers
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designed for Falco genus, however, showed low to moderate success as expected for species
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included in a different family (Falconidae) than the target species (Accipitridae).
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The microsatellite markers tested here provide a powerful tool for management and
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conservation of crowned eagles, allowing further population genetic studies, unambiguous
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individual identification and paternity assessment in this endangered species.
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Acknowledgements
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This work was supported by the Agencia Española de Cooperación Internacional para el
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Desarrollo (AECID, Spain), the Peregrine Fund (USA) and the Universidad Nacional de La
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Pampa (Argentina).
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References
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Augustinos AA, Asimakopoulou AK, Papadopoulos NT, Bourtzis K. (2001) Cross-amplified
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microsatellites in the European cherry fly, Rhagoletis cerasi: medium polymorphic-
125
highly informative markers. Bulletin of Entomological Research, 101, 45-52.
126
127
BirdLife International (2008) Harpyhaliaetus coronatus. In: IUCN 2010. IUCN Red List of
Threatened Species. Version 2010.2. www.iucnredlist.org
Page 6 of 11
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129
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Molecular Ecology Resources
Busch JD, Katzner TE, Bragin E, Keim P (2005) Tetranucleotide microsatellites for aquila
and haliaeetus eagles. Molecular Ecology Notes, 5, 39-41.
Dawson DA, Burke T, Hansson B, Pandhal J, Hale MC, Hinten GN, Slate J (2006) A
131
predicted microsatellite map of the passerine genome based on chicken- passerine
132
sequence similarity. Molecular Ecology, 15, 1299-1320.
133
134
135
136
Fergusson-Lees J, Christie DA (2001) Raptors of the world. Helm Identification Guides,
London, UK.
Frankham R, Ballou JD, Briscoe DA (2004) A primer of conservation genetics. Cambridge
University Press.
137
Hailer F, Gautschi B, Helander B (2005) Development and multiplex PCR amplification of
138
novel microsatellite markers in the White-tailed Sea Eagle, Haliaeetus albicilla (Aves,
139
Falconiformes, Accipitridae). Molecular Ecology Notes, 5, 938-940.
140
Hendrix R, Hauswaldt JS, Veith M, Steinfartz S (2010) Strong correlation between cross-
141
amplification success and genetic distance across all members of ‘True Salamanders’
142
(Amphibia: Salamandridae) revealed by Salamandra salamandra-specific microsatellite
143
loci. Molecular Ecology Resources, 10, 1038–1047.
144
Hull JM, Tufts D, Topinka JR, May B, Ernest HB (2007) Development of 19 microsatellite
145
loci for Swainson’s hawks (Buteo swainsoni) and other buteos. Molecular Ecology
146
Notes, 7, 346–349.
147
Horváth MB, Martínez-Cruz B, Negro JJ, Kalmàr L, Godoy JA (2004) An overlooked DNA
148
source for non-invasive genetic analysis in birds. Journal of Avian Biology, 36, 84–88.
149
Johnson PCD, Fowlie MK, Amos W (2005) Isolation of microsatellite loci from the common
150
buzzard, Buteo buteo (Aves, Accipitridae). Molecular Ecology Notes, 5, 208-211.
Molecular Ecology Resources
151
152
153
Marshall TC, Slate J, Kruuk L, Pemberton JM (1998) Statistical confidence for likelihood
based paternity inference in natural populations. Molecular Ecology, 7, 639–65.
Martínez-Cruz B, David VA, Godoy JA, Negro JJ, O’Brien SJ, Johnson WE (2002) Eighteen
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polymorphic microsatellite markers for the highly endangered Spanish imperial eagle
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(Aquila adalberti) and related species. Molecular Ecology Notes, 2, 323-326.
156
Meglécz, E., Anderson, A., Bourguet, D., Butcher, R., Caldas, A., Cassel-Lundhagen, A.,
157
Cœur d’Acier, A., Dawson, A.D., Faure, N., Fauvelot, C., Franck, P., Harper, G.,
158
Keyghobadi, N., Kluetsch, C., Muthulakshmi, M., Nagaraju, J., Patt, A., Péténian, F.,
159
Silvain JF., Wilcock, HR (2007) Microsatellite flanking region similarities among
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different loci within insect species. Insect Molecular Biology, 16, 175-185
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165
166
167
168
169
170
171
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Mira S, Wolff K, Cancela ML (2005) Isolation and characterization of microsatellite markers
in Bonelli’s eagle (Hieraaetus fasciatus). Molecular Ecology Notes, 5, 493-495.
Nesje M, Røed KH (2000) Microsatellite DNA markers from the gyrfalcon (Falco rusticolus)
and their use in other raptor species. Molecular Ecology, 9, 1438-1440.
Primmer CR, Painter J N, Koskinen MT, Palo JU, Merilä J (2005) Factors affecting avian
cross microsatellite amplification. Journal of Avian Biology, 36, 348-360.
Raymond M, Rousset F (1995) Genepop (version 1.2), population genetics software for exact
tests and ecumenicism. Journal of Heredity, 86, 248-249.
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Sarasola JH, Maceda JJ (2006) Past and current evidence of persecution of the endangered
Crowned Eagle Harpyhaliaetus coronatus in Argentina. Oryx, 40, 347–350.
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Sarasola JH, Santillán MA, Galmes MA (2010) Crowned eagles rarely prey on livestock in
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central Argentina: persecution is not justified. Endangered Species Research, 13, 207-
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213.
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Table 1. Summary of the polymorphic microsatellite loci developed for six different raptor species and amplified successfully in crowned eagles:
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original and new Gene Bank Accession Number, annealing temperature in simplex PCR (Ta), alleles per polymorphic locus (N), observed
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heterozygosity (HO), expected heterozigocity (HE) and source.
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Specie
Buteo swainsoni
Locus
S1
BswB234w
S4
BswB111aw
Gene Bank
Number
New Gene
bank Number
Ta (ºC)
N
HO
HE
Source
DQ988163
JQ309945
56
6
0.393
0.423
Hull et al. (2007)
DQ985713
JQ309946
60
2
0.328
0.341
BswD220w
S3
DQ985722
JQ309947
56
5
0.754
0.795
BswD107w
S3
DQ985716
JQ309948
56
8
0.793
0.865**
BswA317w
S4
DQ985712
JQ309960
56
4
0.333
0.319
BswA302w
S1
DQ985709
JQ309961
56
2
0.316
0.292
AF200207
JQ309958
56
3
0.517
0.497
Nesje & Røed (2000)
Busch et al. (2005)
S5
Falco rusticolus
NVHfr206
Aquila heliaca
IEAAAG04
S4
AY631063
JQ321581
56
6
0.756
0.716
IEAAAG15
S4
AY631070
JQ309959
56
2
0.052
0.051
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Haliaeetus albicilla
Buteo buteo
Hieraaetus fasciatus
Molecular Ecology Resources
Hal04S1
AY817043
JQ309957
56
7
0.518
0.633
Hal09
S1
AY817048
JQ309956
56
2
0.667
0.538
Hal10
S2
AY817049
JQ309955
56
3
0.375
0.449
Bbu42
S5
AJ715912
JQ309954
56
9
0.793
0.708
Bbu46
S3
AJ715916
JQ309953
56
7
0.766
0.709
AY823587
JQ309952
53
3
0.241
0.592**
Hf-C1E8S5
Hf-C3F2
S2
AY823596
JQ309951
56
4
0.500
0.493
Hf-C5D4
S3
AY823597
JQ309950
56
2
0.286
0.319
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** indicates Hardy-Weinberg disequilibrium at p< 0.01.
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Multiplex set reactions are denoted by S1, S2, S3, S4 and S5.
Hailer et al. (2005)
Johnson et al. (2005)
Mira et al. (2005)