TECHNICAL NOTE

Characterization of 17 microsatellite loci for use in populationgenetic and mating system studies of the endangered NorthAmerican passerine, loggerhead shrike (Lanius ludovicianus)

Angela Coxon • Amy A. Chabot • Stephen C. Lougheed •

Jose Antonio Davila • Bradley White

Received: 18 November 2011 / Accepted: 1 December 2011 / Published online: 25 December 2011

� Springer Science+Business Media B.V. 2011

Abstract Seventeen polymorphic microsatellite DNA

markers were isolated from the loggerhead shrike (Lanius

ludovicianus) using individuals from two geographically

distinct populations (Ontario and mainland California). The

number of alleles per locus ranged from 6 to 20 and expected

heterozygosity varied from 0.639 to 0.902. All loci were

heterozygous and private alleles were found for most.

Combined non-exclusion probability of identity ranged from

0.047 to 0.122 in the Ontario population and 0.011–0.060

in the California population. Our results indicate that these

markers will be useful for studies of genetic population

structure, parentage and pedigree analysis.

Keywords Laniidae � Passerine � Microsatellites �Conservation genetics � Pedigree analysis � Parentage �Population genetic structure

Introduction

The loggerhead shrike (Lanius ludovicianus) is one of 30

‘true’ shrikes in the broadly distributed passerine family

Laniidae (Monroe and Sibley 1993), and the only species

that occurs exclusively in North America. It is one of the

most persistently declining species in the Breeding Bird

Survey (BBS), decreasing at an average annual rate of

3.7% in North America during the period 1966–1998

(Pruitt 2000). Miller (1931) recognized 11 subspecies in

the most comprehensive assessment of the species. How-

ever, some designations have been questioned (Rand 1957;

Phillips 1986). In the United States, only the subspecies L.l.

mearnsi, which occurs exclusively on San Clemente Island

off the coast of California, is listed federally as endangered,

but the species is listed as endangered or threatened in 14

states (Pruitt 2000). In Canada, the species is listed as

‘threatened’ (L.l. excubitorides) in the west and ‘endan-

gered’ in the east (L.l. migrans). A modern biosystematic

survey is urgently needed to aid conservation efforts (Pruitt

2000).

Highly variable DNA markers that have sufficient res-

olution for detecting intra-population genetic differences

are required to address questions of conservation concern

in shrikes. To this end, we developed primer pairs for 17

microsatellite loci to quantify genetic population structure

within and among populations of the loggerhead shrike.

Together with the 4 microsatellite markers previously

developed for this species (Mundy and Woodruff 1996),

researchers can now generate robust data sets to address

questions that can assist in recovery efforts for the log-

gerhead shrike.

Primer pairs Llu011, Llu20, Llu40, Llu45, Llu89 and

Llu133 were developed using cloning methods described in

Hamilton et al. (1999a). Genomic DNA was extracted from

Electronic supplementary material The online version of thisarticle (doi:10.1007/s12686-011-9585-2) contains supplementarymaterial, which is available to authorized users.

A. Coxon � B. White

Natural Resources DNA Profiling and Forensic Centre, Trent

University, 1600 West Bank Drive, Peterborough, ON K9J 7B8,

Canada

A. Coxon

Department of Environment, Government of Nunavut,

PO Box 209, Igloolik, Nunavut X0A 0L0, Canada

A. A. Chabot (&) � S. C. Lougheed

Department of Biology, Queen’s University, 116 Barrie Street,

Kingston, ON K7L 3N6, Canada

e-mail: amy@chabotcuddy.ca

J. A. Davila

Instituto de Investagacion en Recursos Cinegeticos, IREC

(C.S.I.C.-U.C.L.M.-J.C.C.M.), Ronda de Toledo s/n,

13005 Ciudad Real, Spain

123

Conservation Genet Resour (2012) 4:503–506

DOI 10.1007/s12686-011-9585-2

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504 Conservation Genet Resour (2012) 4:503–506

123

a blood sample obtained from a female loggerhead shrike

(L.l. migrans) using standard phenol–chloroform methods

(Sambrook et al. 1989) and digested with restriction

enzymes AluI, HaeIII, NheI and RsaI. Fragments ranging

from 300 to 900 base pairs electrophoresed in a 1.5%

agarose gel and visualized with ethidium bromide were

excised, cleaned using QIAquick Gel Extraction Kit�

(Qiagen), blunt ended, dephosphorylated, and ligated to

SNX linkers. Linked fragments were then hybridized to 30

biotinylated TG13, AG13, GATA8 and AAAG8 probes for

3 h at 63�C. Enriched DNA was separated using strepta-

vidin beads (DynaBeads� M-280 Spretavidin). Microsat-

ellite enriched DNA was ligated into the XbaI site of

pBluescript� II SK(?) (Stratagene) and transformed into

Electron-Blue� Escherichia coli competent cells (Strata-

gene) using electroporation. Cells were grown on ampi-

cillin-treated agar plates and colonies were screened using

a DIG fluorescent kit (Roche Diagnostics). Positive clones

were sequenced on a Beckman Coulter capillary machine

(CEQ 8000). Primer development was done using PRIMER

3 (Rozen and Skaletsky 2000).

Primer pairs Llu15, Llu39, Llu55, Llu82, Llu85, Llu90,

Llu95, Llu102, Llu112, Llu157, Llu176 were developed

using cloning methods adapted from Hamilton et al.

(1999b) and Refseth et al. (1997). Whole genomic DNA

was isolated from a male and female loggerhead shrike

(L.l. mearnsi) using standard phenol–chloroform proce-

dures (Sambrook et al. 1989). Pooled genomic DNA from

the two individuals was digested with the restriction

enzyme HaeIII, and SNX linkers were ligated to the

digested fragments. Enrichment was carried out using

GATA8 biotin labeled probe and streptavidin coated beads

(Dynal Biotech). 150 ng of DNA were hybridized to

100 lmol of probe in a 50 lL volume, the complex was

added to 50 lL of washed beads and the two were mixed

for 15 min at room temperature. The unbound DNA was

washed away using three washes each of 100 lL of 29 and

19 SSC. Single-stranded DNA, once released from the

probe, was amplified to double stranded form. The result-

ing product was ligated into vector and transformed into

cells using pCR�2.1-TOPO� vector, and TOP10 chemi-

cally competent cells (Invitrogen). Colonies positive for an

insert were amplified using M13 forward and reverse

primers, the resulting product sequenced using the DYE-

namicTM ET terminator cycle sequencing kit, and run on a

MegaBASE 1000 (Amersham-Pharmacia).

The number of alleles, and observed and expected het-

erozygosities were calculated using GenAlEx 6 (Peakall

and Smouse 2006). The number of alleles present ranged

from 6 to 20 (Table 1). Observed heterozygosity ranged

from 0.367 to 0.953 (Table 1). Expected heterozygosity

ranged from 0.639 to 0.902 (Table 1). We checked for

the presence of null alleles using Micro-checker (VanTa

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Conservation Genet Resour (2012) 4:503–506 505

123

Oosterhout et al. 2004) and calculated null allele frequen-

cies using the Brookfield-1 equation (Brookfield 1996).

Null alleles were found at one locus (Table 1). Loci were

tested for departures from Hardy–Weinberg Equilibrium

using GenePop 4.0 (Rousset 2008)—two showed devia-

tions from Hardy–Weinberg equilibrium after Bonferroni

correction (Rice 1989) (Table 1). While there was no

indication of large allele dropout (Table 1), the departure

from Hardy–Weinberg Equilibrium at locus Llu102 may be

due to null alleles. PCR conditions, assessment of sex

linkage and comparative tests of variation in shrikes from

Ontario versus San Clemente Island are provided as sup-

plemental information.

Acknowledgments This study was funded by the United States

Navy Region Southwest (grant to BW) and Environment Canada

(Canadian Wildlife Service—Ontario Region) (grant to SCL) and the

Natural Sciences and Research Council of Canada (grant to SCL). We

express our sincere thanks to Gary Santolo of CH2MHill, the San

Diego Zoo Center for Reproduction of Endangered Species, Catalina

Island Conservancy, UCLA, Santa Barbara Museum of Natural His-

tory, Los Angeles County Museum of Natural History, the San Diego

Museum of Natural History, the Toronto Zoo, McGill University’s

Avian Science Centre and the Canadian Eastern Loggerhead Shrike

Recovery Team for supplying samples. We thank Nadine Sharpe and

Zhengxin Sun, Queen’s University, for assistance in the laboratory.

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