Molecular Ecology Notes (2007) 7, 833–835

doi: 10.1111/j.1471-8286.2007.01719.x

PRIMER NOTEBlackwell Publishing Ltd

Microsatellite markers isolated from barn swallows(Hirundo rustica)O L G A V . T S Y U S K O ,* M A U R E E N B . P E T E R S ,* C R I S H A G E N ,* T R A C E Y D . T U B E R V I L L E ,* T I M O T H Y
A . M O U S S E A U ,† A N D E R S P . M Ø L L E R ‡ and T R A V I S C . G L E N N *†*The University of Georgia, Institute of Ecology, Savannah River Ecology Laboratory, PO Drawer E, Aiken, SC 29802, USA,
†Department of Biological Sciences, University of South Carolina, Columbia, SC 29802, USA, ‡Laboratoire de Parasitologie Evolutive,Université Pierre et Marie Curie, FR-75252 Paris, Cedex 05, FranceAbstract
Fifteen polymorphic microsatellite DNA loci were isolated from barn swallow (Hirundorustica) and optimized for future studies of radiation-induced mutations in populations
from Ukraine. The loci were screened for variability among 25 individuals from two populations. The primers amplified loci with relatively high numbers of alleles ranging
from five to 32 alleles per locus and polymorphic information content from 0.481 to 0.951.
Observed heterozygosity varied from 0.458 to 0.960. None of the loci showed deviations
from Hardy–Weinberg equilibrium in either population.
Keywords: Hirundo rustica, microsatellites, mutation, PCR, primer, radiation
Received 2 December 2006; revision accepted 8 January 2007
Microsatellites from barn swallows (Hirundo rustica) havebeen used in several classic studies, including putative
radiation-induced germline mutations in wildlife near
Chornobyl (Ellegren et al. 1997). Given the heterogeneous
responses in the germline mutation rates observed between
two microsatellite loci used in previous barn swallow
studies (Ellegren et al. 1997; Brohede et al. 2002), further
work in this species requires additional loci to be developed.
Here we describe the development and optimization ofpolymorphic microsatellite DNA loci that can be used to
study barn swallows and the many other species from which
their primers are known to work (e.g. Primmer et al. 1996).
Blood was collected using heparinized capillaries, whichwere immediately transferred to cryovials with a citratebuffer, and then stored at −70 °C. DNA from two individuals
was extracted from blood using a DNeasy Kit (QIAGEN)
and was serially enriched twice for microsatellites using
three probe mixes [mix 2: (AG)12, (TG)12, (AAC)6, (AAG)8,(AAT)12, (ACT)12, (ATC)8; mix 3: (AAAC)6, (AAAG)6,(AATC)6, (AATG)6, (ACAG)6, (ACCT)6, (ACTC)6, (ACTG)6;
mix 4: (AAAT)8, (AACT)8, (AAGT)8, (ACAT)8, (AGAT)8]
following Glenn & Schable (2005). Briefly, the DNA wasdigested with restriction enzyme RsaI (New England

Correspondence: Olga Tsyusko, Fax: 803 725 3309;
E-mail: tsyusko@srel.edu© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing LtdBiolabs) and simultaneously ligated to double-stranded
SuperSNX linkers (SuperSNX24 forward 5′-GTTTAAGGCCTAGCTAGCAGCAGAATC and SuperSNX24 reverse
5′-GATTCTGCTAGCTAGGCCTTAAACAAAA). Linkerligated DNA was denatured and hybridized to biotinylated
microsatellite oligonucleotide mixes, which were thencaptured on magnetic streptavidin beads (Dynal). Unhybridized DNA was washed away and the remaining DNA
was eluted from the beads, amplified in polymerase chain
reactions (PCR) using the forward SuperSNX24 as a primer,and cloned with TOPO-TA Cloning Kits (Invitrogen). The
positive white clones with inserts were detected using
β-galactosidase gene. A total of 192 positive clones wereisolated by transferring each colony to an individual wellof a deep-well plate containing LB broth and the inserts
were amplified with M13 forward and reverse primers.
Total of 96 plasmids were sequenced using the BigDye
Terminators version 3.1 (Applied Biosystems) and ABI
3130xl capillary sequencer. Sequences from both strands
were assembled and edited in sequencer 4.1 (Genecodes)
and exported to ephemeris 1.0 (available at http://www.uga.edu/srel/DNA_Laboratory/programs.htm) for
microsatellite searching. Ninety-two out of the 96
sequenced clones contained microsatellites. Twenty-four
PCR primers were designed using oligo 6.67 (MolecularBiology Insights) and one primer in each pair was modified
on the 5′ end with an engineered sequence (CAG tag834 P R I M E R N O T E
Table 1 Characterization of 15 polymorphic microsatellite loci for Hirundo rustica. Size indicates the range of observed alleles in bp; k isnumber of alleles; n is the number of the genotypes obtained; Ho and He are expected and observed heterozygosity and PIC is polymorphic
information content; null alleles are estimated from cervus 2.0. A total of 25 individuals from two populations (eight from Dityatki and 17from Boryspil, Ukraine) were genotyped. Initial annealing temperature was 55 °C for all amplifications
Locus
Accession no.

Primer sequence 5′−3′

Repeat motif

Size (bp)

kn

HOHE

PIC

Null alleles

Hir 4
EF120606
Hir 5
EF120607
Hir 6
EF120608
Hir 7
EF120609
Hir 8
EF120610
Hir 10EF120611
Hir 11
EF120612
Hir 15
EF120613
Hir 16
EF120614
Hir 17
EF120615
Hir 19EF120616
Hir 20EF120618
Hir 21
EF120617
Hir 22
EF120619
Hir 24
EF120621

U: FAMGCAAGGACCGATCACA
L: AAAAACCCAAACAATCAAC
U: GTGTGCTGAAAAGTGTAT
L: FAMACAGGCATGTGTGAGT
U: FAMGACGGCCTGGGGGTAGA
L: AAGAGCATGACCACCAGAGAT
U: FAMCTTGCGCAGAAAGTAT
L: GCTCTGGGATCTCTAG
U: TAAAAGCAAGGAAGATCT
L: FAMGACACCGTCACAGTAGA
U: FAMGGACAAGGGGAGTCTTL: ATTCAGCCAGCCTCTAAT
U: AACACCTGAAAACCTACAC
L: FAMCTTTGAGCAAAATGAGTG
U: NEDTTGTGGTGGCTGACTT
L: CTGACGAGATGCAATTAC
U: NEDCTTCAGGCAGGTAAG
L: GAAAACCATTCCTGTTAA
U: NEDATGCCATGCTTCAGAT
L: CTGTCATGCCTAAGTATCA
U: NEDGCTCACAACCAGCTAGAC
L: ATAGCCACAGGGAAAGTCTU: GAAGTTGGAGAAAGATTAG
L: NEDTTATTGCTCTGGGTATGTU: AAATTGCCAATTTTGTGTC
L: FAMTGGTGGCGAAACATTAA
U: NEDATCCGCACCTAATGT
L: GATTCACATATCCCATCTAG
U: NEDCTCACCAAAGCGTGTGA
L: ACTCCACGGTTTCAGAGA(GTTT)10286–310

8240.458

0.828

0.786

0.270

(GTTT)5226–246

623

0.652

0.713

0.653

0.045

(TCTA)16202–230

9250.720

0.804

0.758

0.051

(CT)14247–284

18

250.760

0.942

0.918

0.098

(CTT)11CTA(CTT)14218–367

24240.792

0.952

0.928

0.082

(GTTT)9180–206

8240.917

0.868

0.832

− 0.039(GATA)14208–248

10250.960

0.850

0.812

− 0.076(ATGT)7236–252

7240.750

0.706

0.647

− 0.066(ATGT)6 … (GT)11301–329

12240.542

0.892

0.861

0.237(CTAT)10TTAT(CTAT)3215–267

16

250.920

0.935

0.910

−0.003(ATGG)4ACGG(ATGG)15(ATAG)14153–202

10240.875

0.848

0.809

−0.029259–28711250.800

0.860

0.826

0.016(CTTT)19CCTT(CTTT)8273–426

32

240.667

0.973

0.951

0.180(ATCT)7267–303

14240.750*

0.868

0.836

0.076(AGTG)12218–247

13250.520

0.895

0.865

0.256

*Significant deviations from Hardy–Weinberg equilibrium are indicated at P < 0.05. Primers with CAG tag (5′-CAGTCGGGCGTCATCA-3′)
are indicated with superscript FAM or NED, which was used as the fluorescent dye for genotyping.

5′-CAGTCGGGCGTCATCA-3′; see http://www.uga.edu/srel/DNA_Laboratory/protocols.htm) to allow use of athird primer in the PCR (CAG) that is fluorescently labelled
for detection on the ABI 3130xl.
The 24 PCR primer pairs were tested for amplification
using eight individuals across four different Ukrainian
populations. PCR amplifications were performed in a25-µL volume [10 mm Tris pH 8.4, 50 mm KCl, 25.0 µg/mL
BSA, 0.4 µm unlabelled primer, 0.04 µm tag-labelledprimer, 0.36 µm universal dye-labelled primer, 3 mm MgCl2,
0.15 mm dNTPs, 0.5 U JumpStart Taq DNA Polymerase(Sigma), and 50 ng DNA template] using an Applied
Biosystems thermalcycler (GeneAmp PCR System 9700).
Touchdown thermal cycling programs (Don et al. 1991)
encompassing a 10 °C span of annealing temperatures,ranging between 65–55 °C, 60–50 °C or 55 – 45 °C, wereused for the amplification. Cycling parameters were 21cycles at 96 °C for 20 s, highest annealing temperature(decreased 0.5 °C per cycle) for 20 s, and 72 °C for 30 s; and15 cycles of 96 °C for 20 s, lowest annealing temperaturefor 20 s, and 72 °C for 30 s. PCR products were run on anAB-3130xl sequencer and sized with Naurox size standard
prepared as described in DeWoody et al. (2004). Results
were analysed using genemapper version 4.0 (AppliedBiosystems). Fifteen of the tested primer pairs successfully
amplified PCR product of high quality.
Blood samples from 25 individuals from two populations were used to test for microsatellite variability acrossthe 15 loci. The PCRs were performed in 11.5 µL volumeand touchdown 55 program encompassing a 10 °C span ofannealing temperatures between 55 °C and 45 °C wasused. Characteristics of the 15 working primer pairs are© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd

P R I M E R N O T E 835
given in Table 1. We estimated the number of alleles perlocus (k), observed and expected heterozygosity (HO andHE), and polymorphic information content (PIC) andtested for deviations from Hardy–Weinberg equilibrium(HWE) using cervus version 2.0 (Marshall et al. 1998).
There was one locus (Hir 22) that deviated significantly
from HWE only when all samples were analysed, but notwhen each population was analysed alone. No linkage wasdetected among 122 paired loci comparisons (P < 0.05;
genepop version 3.4; Raymond & Rousset 1995). blast
searches of the barn swallow sequences indicate partialhomology with two loci from other birds [110 bp of Hir 6
matched partially to accession AY366081 (Garrulax canorus)and 205 bp of Hir19 matched partially to accession
XM416761 (Gallus gallus)].
Overall, the high numbers of alleles per locus, high PIC
values and heterozygosities demonstrate the potentialof the developed microsatellite primers for a variety ofpurposes, including detection of genetic effects of radiationin H. rustica populations from contaminated environments.

Acknowledgements
This research was supported by the Environmental Remediation
Sciences Division of the Office of Biological and Environmental
Research, US Department of Energy, through Financial AssistanceAward no. DE-FC09-96-SR18546 to the University of Georgia© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd

Research Foundation. The financial support also came from grants
to T.A.M. and A.P.M. from the National Geographic Society,
the Civilian Research Development Foundation, and the SamuelFreeman Charitable Trust.

References
Brohede J, Primmer C, Møller A, Ellegren H (2002) Heterogeneity
in the rate and pattern of germline mutation at individual
microsatellite loci. Nucleic Acids Research, 30, 1997–2003.
DeWoody AJ, Schupp J, Kenefic L et al. (2004) Universal method
for producing ROX-labeled size standards suitable for automated genotyping. BioTechniques, 37, 348–350.
Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991)‘Touchdown’ PCR to circumvent spurious priming during gene
amplification. Nucleic Acids Research, 19, 4008.
Ellegren H, Lindgren G, Primmer CR, Møller AP (1997) Fitness
loss and germline mutations in barn swallows breeding in
Chernobyl. Nature, 389, 593–596.
Glenn TC, Schable NA (2005) Isolating microsatellite DNA loci.
Methods in Enzymology, 395, 202–222.
Marshall TC, Slate J, Kruuk L, Pemberton JM (1998) Statisticalconfidence for likelihood-based paternity inference in naturalpopulations. Molecular Ecology, 7, 639–655.
Primmer CR, Møller AP, Ellegren H (1996) A wide-ranging surveyof cross-species microsatellite amplification in birds. MolecularEcology, 5, 365–378.
Raymond M, Rousset F (1995) genepop (version 1.2): population
genetics software for exact tests and ecumenicism. Journal ofHeredity, 86, 248–249.