Molecular Ecology Resources (2008) 8, 977–979
doi: 10.1111/j.1755-0998.2008.02121.x
PERMANENT GENETIC RESOURCES Blackwell Publishing Ltd
Isolation and characterization of 21 microsatellite markers in the barn owl (Tyto alba) R . B U R R I ,* S . A N T O N I A Z Z A ,* F. S I V E R I O ,† Á . K L E I N ,‡ A . R O U L I N § and L . F U M A G A L L I * *Laboratory for Conservation Biology, Department of Ecology and Evolution, Biophore, University of Lausanne, CH-1015 Lausanne, Switzerland, †Los Barros 21, E-38410 Los Realejos, Tenerife, Canary Islands, ‡Behavioural Ecology Group, Department of Systematic Zoology and Ecology, Eötvös Loránd University, Pázmány P. Sétány 1/c., H-1117 Budapest, Hungary, §Department of Ecology and Evolution, Biophore, University of Lausanne, CH-1015 Lausanne, Switzerland
Abstract We report 21 new polymorphic microsatellite markers in the European barn owl (Tyto alba). The polymorphism of the reported markers was evaluated in a population situated in western Switzerland and in another from Tenerife, Canary Islands. The number of alleles per locus varies between two and 31, and expected heterozygosity per population ranges from 0.16 to 0.95. All loci are in Hardy–Weinberg equilibrium and no linkage disequilibrium was detected. Two loci exhibit a null allele in the Tenerife population. Keywords: barn owl, cosmopolite, microsatellite, nocturnal raptor, population genetics, Tyto alba Received 4 November 2007; revision accepted 19 December 2007
The barn owl (Tyto alba, Tytonidae, Strigiformes) represents the most widespread member of the owl order, and is one of the most widespread bird species worldwide. The species occupies open habitat in temperate, subtropical and tropical zones throughout Africa, Europe, Southwestern and South Asia, Australia, South and North America (Del Hoyo et al. 2000). In pristine environment, barn owls usually occupy natural cavities, while in many places they benefit from cavities in human buildings like barns and church steeples. The deterioration of breeding sites and agricultural landscape over the last decades led to population declines in many regions where the species occurs commensally with man. Many issues about species conservation, population dynamics, phylogeography as well as evolution of lifehistory traits could gain important insights from population genetic approaches. Because no genetic markers for population studies in the barn owl are available yet, we isolated microsatellite markers for this species. Genomic DNA was extracted from the liver of a frozen Swiss barn owl road kill using the DNeasy Tissue Kit (QIAGEN). Microsatellite enrichment for the eight motifs Correspondence: R. Burri, Fax: +41 (0)21 692 41 65; E-mail:
[email protected] Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. EU220182– EU220197, EU220199, EU220200, EU220202, EU220205, EU220206) © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd
(CA)n, (GA)n, (AAC)n, (CAG)n, (AAAG)n, (CATC)n, (TACA)n and (TAGA)n was performed by Genetic Identification Services, as was cloning, sequencing of 100 clones and primer design on microsatellite-containing clones. Methods for DNA library construction, enrichment and screening were performed as described previously ( Jones et al. 2002). Genomic DNA was partially restricted with a cocktail of seven blunt-end cutting enzymes (RsaI, HaeIII, Bsr BI, PvuII, StuI, ScaI, EcoRV). Fragments in the size range of 300–750 bp were adapted and subjected to magnetic bead capture (CPG, Inc.), using biotinylated capture molecules. Libraries were prepared in parallel using biotin capture molecules in a protocol provided by the manufacturer. Captured molecules were amplified and restricted with HindIII to remove the adapters. The resulting fragments were ligated into the HindIII site of pUC19. Recombinant molecules were electroporated into Escherichia coli DH5alpha. Recombinant clones were selected at random for sequencing. Sequences were obtained on an ABI PRISM 377 sequencer (Applied Biosystems), using ABI PRISM Taq dye terminator cycle sequencing methodology. Primers were designed for 53 microsatellitecontaining clones using designer-pcr version 1.03 (Research Genetics), and tested for amplification success on one barn owl individual. Twenty-one primer pairs produced reproducible banding patterns, and their characteristics are listed in Table 1.
978 P E R M A N E N T G E N E T I C R E S O U R C E S Table 1 Characterization of 21 microsatellite loci within two barn owl populations (western Switzerland; Tenerife, Canary Islands) Locus and GenBank Accession no.
Primer sequences (5′–3′)
Ta-202 EU220182 Ta-204 EU220183 Ta-206 EU220184 Ta-207 EU220185 Ta-210 EU220186 Ta-212 EU220187 Ta-214 EU220188 Ta-215 EU220189 Ta-216 EU220190 Ta-218 EU220191 Ta-219 EU220192 Ta-220 EU220193 Ta-304 EU220194 Ta-305 EU220195 Ta-306 EU220196 Ta-308 EU220197 Ta-310 EU220199 Ta-402 EU220200 Ta-408 EU220202 Ta-413 EU220205 Ta-414 EU220206
F: TGGGTGACCTGAAACTGTG R: GCTCCACTTGCTGTCTGG F: TTGTGCAAGGACAGTCATATTC R: CAGGATTTAGCATTGTGGTACA F: TCGGTGAGAATCCATTTAATAG R: AGGTTCTTGGAAACTGAGACTT F: CAGTGCCAGGATTGGAGTT R: CAGCAGGTGAAGGAGAAGG F: AGCAGGAGACAGCACATTTC R: TTCATTTTTGCCACCTCTTC F: AGGGCTGTGCTTCCACTC R: TGCCAAAACACCATCACC F: TCTCCTTCTTCACCCCATTAC R: CAGTCACCCTTTTGTTGAGTG F: AGGATGGGCTCAGAAATAAG R: CCAAAGAAACCACAGTAGGTAG F: CAGGCTTCTTCTGAGGTCC R: GCATTGTGAAAGGGTTTACTG F: CGTTACACGCATACACACAAC R: CGATGCAACAATTATTCATGTC F: GCGGCTCCCTAATTCATC R: AAACCTCGGGTAAGCAGTG F: ACAGACGCACTGGAAGTATG R: GCATGTAACGGAAGGTTGTA F: GGCCCTGAACATCAGTGA R: GGCTGAGGACATCAAATTG F: TGGAAGGTGATGAACACATC R: TTCCCATTAAGTTGCACATTC F: TTTGAGCAATTCAGGTTTACAC R: GCATTTGCATATCATATTCTGC F: GGTTGGTGACTGGTGAGC R: ATGGGGGAAAGAATCAGG F: GCATTGTGCATCATTTGTCTT R: CATCCCAGTCCTATCTCGAAC F: CCAGTTCCACAGTCAGATGC R: CAAGCAAGCGGTGATGTC F: CAACTCCCACATCTTATGTCTC R: TGCTGGAAAGAAAGGAAAG F: CCTTCCTGTGATTCAAAGTTG R: AGAGGGACGGTGAGTGTG F: CCTCTTCTCTGCCAGTGG R: GGTGGGGGTTATTTACCTG
Repeat motif
Ta (°C)
Mg++ (mM)
(TG)13
58
(GT)12
nA
N
1
4
58
2
7
(CT)20
56
2
11
(CA)8TA(CA)9
58
1.5
3
(CA)7TA(CA)5
56
1
2
(CA)13
56
2
7
(CA)14
60
2.5
5
(CA)13
56
2
6
(TG)15
56
2
9
(CA)17
56
2.5
4
(CA)8
56
2
3
(CA)11
56
2
5
(CAA)7
58
1.5
2
(CAA)9
58
2
6
(CAA)7
58
2
2
(GTT)6TT(GTT)2GCT (GTT)2TT(GTT)5(ATT)7 (CAT)7T(ATT)7(GTT)7
56
2
5
58
1
7
(TAGA)16TAGG(TAGA)18
58
1
17
(CCAT)20
58
1.5
11
(TCCA)21
60
1
17
(TCCA)18CA(TCCA)TC CG(TCCA)11CA(TCCA)9
60
1.75
31
24 20 24 20 24 20 24 20 24 20 23 20 24 20 24 20 24 20 24 20 24 20 24 20 24 20 23 20 24 20 24 20 24 20 24 20 23 20 24 20 23 20
Size range (bp) 261–267 116–131 269–289 253–257 163–167 248–270 220–229 295–309 187–207 118–126 172–191 208–219 190–193 189–198 157–163 215–227 268–296 202–270 200–251 157–221 236–412
HO
HE
Null allele
0.50 0.30 0.67 0.55 0.83 0.95 0.67 0.50 0.17 0.30 0.83 0.60 0.67 0.30 0.50 0.85 0.71 0.80 0.71 0.50 0.25 0.20 0.63 0.60 0.17 mono 0.43 0.60 0.54 0.55 0.13 0.10 0.79 0.35 0.96 0.95 0.52 0.65 0.88 0.80 0.96 0.80
0.54 0.38 0.79 0.82 0.87 0.85 0.64 0.41 0.16 0.26 0.80 0.59 0.78 0.46 0.60 0.69 0.73 0.81 0.72 0.53 0.23 0.18 0.61 0.62 0.16 mono 0.43 0.80 0.49 0.50 0.20 0.19 0.70 0.58 0.90 0.89 0.51 0.72 0.91 0.89 0.95 0.86
— — — 0.14 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 0.14 — — — — — — — —
Ta, annealing temperature; nA, number of alleles; N, number of individuals genotyped per population; HO, observed heterozygosity; HE, expected heterozygosity; Null allele, frequency of null alleles where detected (Brookfield equation 2, Brookfield 1996); mono, monomorphic. Above numbers belong to the Swiss, below numbers to the Tenerife population.
To assess the variability of the isolated markers in natural populations, we genotyped 44 individuals from two geographically distinct populations, that is, 24 from western Switzerland (46°49′N, 06°56′E, approximate area 190 km2) and 20 from Tenerife, Canary Islands (28°17′N, 16°37′W,
approximate area 2034 km2). Genomic DNA was extracted from blood or muscle tissue using the DNeasy Tissue Kit (QIAGEN). Polymerase chain reactions (PCR) were performed in a final volume of 10 µL containing 1× PCR buffer, 1.0–2.5 mm MgCl2 (see Table 1) 0.2 mm each dNTP, 0.6 mm © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd
P E R M A N E N T G E N E T I C R E S O U R C E S 979 each fluorescent-labelled forward primer (6-FAM, HEX and NED) and nonlabelled reverse primer and 0.1 U Taq (QIAGEN). Two nanograms DNA were used as a template. PCR conditions included an initial denaturation step at 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 40 s, primer annealing at primer-specific annealing temperature (Table 1) for 40 s, and primer extension at 72 °C for 1 min. A final step at 72 °C for 5 min was used to complete primer extension. Fragment analysis was run on an ABI 3100 automated sequencer (Applied Biosystems), and allele sizes were assigned using genemapper 3.7 software (Applied Biosystems). To evaluate if the markers fulfil the requirements for population studies, we estimated observed and expected heterozygosities and tested for Hardy–Weinberg equilibrium and linkage-disequilibrium using arlequin 3.11 (Excoffier et al. 2005). Tests for the presence of null alleles were performed in micro-checker 2.2.3 (van Oosterhout et al. 2004). All loci were polymorphic, with two to 31 alleles detected per locus across populations and between one and 15 alleles per locus per population. Expected heterozygosities ranged from 0.16 to 0.95 (Table 1). All loci are in Hardy–Weinberg equilibrium and no linkage disequilibrium was detected. Loci Ta-204 and Ta-310 exhibit a null allele in the Tenerife population.
© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd
Acknowledgements We thank the Centro de Recuperación de Fauna Silvestre (Cabildo Insular de Tenerife) who provided samples from Tenerife, and Abraham Hernández and Manuel Siverio who helped with the tissue collection. This study was supported by Swiss National Science Foundation grants PPOOA-102913 to A.R. and 3100A0109852/1 to L.F. Á.K. was supported by the Hungarian Scholarship Board (No.39/2007). The Sheffield Molecular Genetics Facility kindly provided the facilities for some preliminary tests during the primer optimization procedure.
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