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Polymorphic microsatellite markers isolated from a southern European population of
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pied flycatchers (Ficedula hypoleuca iberiae)
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David Canal1,José A. Dávila2, Pedro J. G. de Nova2, and Jaime Potti1
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Vespucio s/n, 41092 Sevilla, Spain
Estación Biológica de Doñana – CSIC, Department of Evolutionary Ecology, Américo
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Ronda de Toledo s/n 13005 Ciudad Real, Spain
Instituto de Investigación en Recursos Cinegéticos, IREC (CSIC-UCLM-JCCM),
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Address correspondence to David Canal.
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Vespucio s/n, 41092 Sevilla, Spain
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Telephone number: +34954466700 ext 1406.
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E-mail: davidcanal@ebd.csic.es
Estación Biológica de Doñana – CSIC, Department of Evolutionary Ecology, Américo
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Running title: New microsatellites for the pied flycatcher
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Keywords: Ficedula hypoleuca iberiae, microsatellites, molecular marker, primers.
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Abstract
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Nine polymorphic microsatellite loci for the pied flycatcher (Ficedula hypoleuca),
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from a wild population in Spain are isolated and their variability described on 70
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individuals. The number of alleles per locus ranged from 6 to 41 and observed
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heterozygosity ranged from 0.75 to 0.98. These markers are being used to study mating
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strategies in Ficedula hypoleuca iberiae.
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Introduction
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Three subspecies are currently recognized for the pied flycatcher (Ficedula hypoleuca): F.
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h. hypoleuca breeding from southern France to northern Europe, F. h. sibirica distributed
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across southwest Siberia and F. h. iberiae in the Iberian Peninsula (Sætre et al. 2001,
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Dickinson 2003).
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Over time, pied flycatcher subspecies have been exposed to different pressures and
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responded in different ways, diverging phenotypically and genetically due to selection,
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genetic drift and mutation. Thus, the three subspecies present differences in traits as
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plumage colour, size of the forehead patch (Lundberg and Alatalo 1992) or song (Haavie et
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al. 2004) whereas at the genetic level Spanish pied flycatchers differ significantly from
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Czech and Norwegian ones at both microsatellite loci and mtDNA sequences (Haavie et al.
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2000). Therefore, as a consequence of evolutionary processes members of different
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populations, subspecies or species will show higher degrees of genetic divergence with
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increasing time since isolation.
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In a recent study of paternity in Spanish pied flycatchers (Canal et al. submitted)
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most of the six microsatellites already published by Scandinavian researchers (Ellegren
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1992, Primmer et al. 1996) showed some type of problem. Null alleles were detected in
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FhU1 and FhU3 whereas FhU4 showed weak polymerase chain reaction (PCR)
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amplifications. Regarding FhU5 and FhU6, PCR amplified a high number of unspecific
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bands, preventing their optimization. In addition, FhU1, FhU2, and FhU3 exhibited
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moderate levels of polymorphism (3, 6, and 7 alleles in 80 individuals, respectively).
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Therefore, development of additional, more informative microsatellite markers is required
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even in this relatively well-studied species. Recently, a new set of markers from a Finnish
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(F. h. hypoleuca) population has been published (Leder et al. 2008). Here we describe the
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development and optimization of a new set of highly variable, polymorphic microsatellite
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DNA loci isolated from a population of F. h. iberiae.
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Blood samples were collected from a wild population in La Hiruela (central Spain)
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and stored at room temperature in 100% ethanol. Genomic DNA was extracted from blood
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by a standard phenol-chloroform method (Sambrook et al. 1989). An enriched
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microsatellite genomic library was constructed following procedures modified from
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Hamilton et al. (1999). Briefly, 50 ug of DNA were digested with 100 U each of NheI,
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BsuRI (HaeIII) and RsaI (Fermentas). Restriction fragments of 300 – 1000 base pairs were
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excised from agarose gel, blunt ended, dephosphorylated and ligated to SNX linkers.
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Microsatellite enrichment was carried out by hybridization capture of repeat sequences
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using biotinylated (CAT)8, (GAT)8, (GACA)7, (GATA)7, (TCCA)7 and (TGGA)7
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oligonucleotides and streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin,
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Invitrogen) and subsequently amplified by PCR with SNX-F primer. Amplified DNA was
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ligated into the XbaI site of pUC19 (Fermentas) and plasmid constructs were used to
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electroporate
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(Stratagene). Positive recombinants were replated on LB agar medium and lifted onto nylon
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membranes, probed with DIG-labelled microsatellite motives used in enrichment. Seventy
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positive clones were sequenced in ABI 3130xl Sequencer (Applied Biosystems).
ElectroTen-Blue
electroporation-competent
Escherichia
coli
cells
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Sequences were edited in BIOEDIT 7.0.5.2 (Hall 1999) and primers for those
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sequences containing microsatellites were designed by eye. PCRs were performed in 10-µ L
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reaction volumes (BIOTOOLS: 1x standard reaction buffer, 2.0 mM MgCl2, 0.2 mM of
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each dNTP and 0.5 U Taq Polymerase) using 0.5 µ M of each primer and containing 50 ng
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of 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 annealing temperature (Table 1) and 30 s at
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72°C, plus a final extension of 10 min at 72°C. One primer of each pair that reliably
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amplified a polymorphic locus was tagged with VIC, FAM, PET or NED fluorescent labels
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(Applied Biosystems). Loci from seventy individuals were genotyped on ABI PRISM
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3130xl sequencer (Applied Biosystems). Allele sizes were determined with Genescan 600-
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LIZ internal size standard and Genemapper 4.0 software (Applied Biosystems).
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Observed and expected heterozygosities were calculated using ARLEQUIN
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(Excoffier et al. 2005), which was also used to analyse linkage disequilibrium and assess
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the significance of deviations from Hardy–Weinberg equilibrium (100 000 Markov chains).
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Nine loci remained after others were discarded because of sequence redundancy,
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lack of microsatellite repeat, insufficient flanking sequence for primer design,
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monomorphism or non-specific PCR products. The nine loci were highly variable: all
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except one had 10 or more alleles per locus and, remarkably, two of them displayed more
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than 40 alleles per locus. Observed heterozygosity ranged from 0.75 to 0.98. All loci
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conformed to Hardy-Weinberg equilibrium and no pairs of loci showed significant linkage
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disequilibrium (Table 1).
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Microsatellites here developed have been successful in revealing high rates of extra-
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pair paternity in relation to patterns of sexual selection on male secondary sex traits in our
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study population (Canal et al., submitted). Overall, these markers increase genetic resources
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for the species and, due to their high variability, are a useful tool for a wide variety of
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purposes, as studies of genetic diversity, breeding strategies or population structure.
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Acknowledgements
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Funded by project PAC05-006-1 (to JAD). DC was supported by a grant from Ministerio
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de Educación
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References
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Canal D, Potti J, Dávila JA (2008) Evidence for a link between extra pair fertilizations and
choice of good genes in pied flycatchers. Submitted
Dickinson, EC. (ed.). (2003). The Howard and Moore Complete Checklist of the Birds of
the World, Revised and enlarged 3rd Edition. Christopher Helm, London
Ellegren H (1992) Polymerase-chain-raction (PCR) analysis of microsatellites- a new
approach to studies of genetic relationships in birds. The Auk, 109, 886-895
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Excoffier L, Laval G, Schneider S (2005) ARLEQUIN (version 3.0): An integrated
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software for population genetics data analysis. Evolutionary Bioinformatics Online, 1,
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47–50
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Hall TA (1999) Bioedit: a user-friendly biological sequence alignment editor and analysis
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program for Windows 95/98/ NT. Nucleic Acids Symposium Series, 41, 95–98
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Hamilton MB, Pincus EL, Di Fiore A, Fleischer RC (1999) Universal linker and ligation
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procedures for construction of genomic DNA libraries enriched for microsatellites.
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BioTechniques, 27, 500–507
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Haavie J, Sætre G-P, Moum T (2000) Discrepancies in population differentiation at
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microsatellites, mitochondrial DNA and plumage colour in the pied flycatcher –
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inferring evolutionary processes. Molecular Ecology, 9, 1137-1148.
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Haavie J, Borge T, Bures S et al. (2004) Flycatcher song in allopatry and sympatry--
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convergence, divergence and reinforcement. Journal of Evolutionary Biology, 17,
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227-237
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Leder EH, Naraiskou N, Primmer R (2008) Seventy new microsatellites for the pied
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flycatcher, Ficedula hypoleuca and amplification in other passerine birds. Molecular
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Ecology Resources, 8, 874-880
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Lundberg A, Alatalo RV (1992) The pied flycatcher. London: Poyser
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Primmer G, Anders M, Ellegren H (1996) New microsatellites from the pied flycatcher
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Ficedula hypoleuca and the swallow Hirundo rustica genomes. Hereditas, 124, 281-
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283
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Sætre, G-P, Borge T, Moum T (2001) A new bird species? The taxonomic status of the
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'Atlas Flycatcher' assessed from DNA sequence analysis. The Ibis, 143, 494–497.
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Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd
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ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
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Table 1 Summary data for nine microsatellite loci isolated from the pied flycatcher with primer sequences, number of individuals
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genotyped (N), annealing temperature in PCR (Ta), repeat motif of the cloned microsatellite, GenBank Accession number, number of
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alleles (A.), size range (base pairs) of observed alleles, observed heterozygosity (HO), expected heterozygosity (HE) and P value of the
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test to detect significant departure from Hardy-Weinberg equilibrium. N (NED), F (FAM), V (VIC) and P (PET) indicate the
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fluorescent label used. Polymorphism data are based on adults from a single population in central Spain.
Locus
Primer sequence (5'-3')
Fhy 1-25
FF: TGGCAGGAGTAACCCAGATG
R: CAAACATCCACACCTGACTG
FP: TTCTTTACGGCTCTGCATTG
R: CAGGAAAGTGCCCAGCAATC
FV: GTGACAACTGAGCAAGAATTCC
R: TGCTGCTCTCAGATGGTTCTTC
FN: ATGTGGACACAAGAACATGG
R: TGTGTATGTGTCCATCTCAG
FF: ACTAGTTCCGGCAGGGTATCCA
R: CAATGTCCTGCACATGAAATGG
FF: GTTTTCTGTCTCCCTCAGGAC
R: GGGTGTGACAAGTGTGTACAT
FN: AGCCCCAGACATTGAGATG
R: TGATGCATGCCAGTGAATC
FP: GATCACAAGTTGGACTTGATG
R: CACCACATCTATTGCTGACAG
FN: TGCAGGGATTCAGCAGGACT
R: CCAATAACTGCAAGCACTGG
Fhy 3-60
Fhy 3-85
Fhy 4-95
Fhy 5-75
Fhy 6-126
Fhy 9-98
Fhy 14-41
Fhy B4-7
6
N
Ta (ºC)
Repeat motif
GenBank no.
A
Size range
HO
HE
P
70
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(CTGT)10
FJ389732
6*
136 - 169
0.75
0.70
0.96
70
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(CCAT)16
FJ389733
19*
181 - 273
0.98
0.92
0.99
70
63
(GGAT)14
FJ389734
17
219 - 315
0.92
0.86
0.66
70
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(GGAT)15
FJ389735
14
152 - 216
0.81
0.90
0.25
70
63
(CCAT)12
FJ389736
25
134 - 278
0.85
0.93
0.49
70
60
(TATC)43
FJ389737
34*
137 - 316
0.98
0.96
0.58
70
60
(CAT)16
FJ389738
14
121 - 166
0.93
0.91
0.21
70
60
(TATC)16
FJ389739
10
184 - 228
0.84
0.87
0.59
70
65
(TGA)97
FJ389740
41
295 - 667
0.93
0.96
0.25
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* These loci have been used in a recent study of paternity (Canal et al., sumbited). In this study, the number of alleles at F1-25, F3-60
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and f6-126 loci increased to 7, 21 and 42, respectively, as a result of a larger genotyped sample size (746 individuals).