MICROSATELLITE LETTERS

Isolation of 13 tetranucleotide microsatellite loci in the RockBunting (Emberiza cia)

Gernot Segelbacher • Ingolf Schuphan

Received: 8 January 2014 / Accepted: 13 January 2014

� Springer Science+Business Media Dordrecht 2014

Abstract We isolated 13 polymorphic microsatellite DNA

loci from Rock Bunting (Emberiza cia) to investigate pop-

ulation fragmentation in a species adapted to different

environments. The loci were screened for polymorphism

using 30 individuals from a population in Germany and

primers amplified loci with high numbers of alleles ranging

from 5 to 9 alleles per locus.

Keywords Tetranucleotide microsatellites �Emberiza cia � Primer

Introduction

The Rock Bunting (Emberiza cia) is distributed within

Europe mainly in the Mediterranean and steppe areas in

Eastern Europe. In Central Europe it occurs in mountain

regions of the Alps and reaches its most northern distri-

bution in climatic favoured areas in Central Germany,

which are often rocky, south-exposed and dry cliffs or

steep terrace vine cultivation habitats along the rivers Ahr,

Mosel, Middle-Rhine, Nahe and Main (Schuphan 2011).

Thus the rock bunting covers a range of different climatic

habitats from a mild climate of a Mediterranean type to

more harsh conditions in the montane zones of the Vosges,

Black Forest and in the Alps, where they breed up till

2,300 m altitude. The species is distributed patchily within

Germany and connectivity among local breeding sites and

potential subpopulations is unknown. The plasticity of

habitat choice and the distinct distribution pattern thus

makes the rock bunting an ideal species for studying

potential adaptions to climate change in a fragmented

landscape.

Genomic DNA was extracted from blood samples using

the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Ger-

many) and microsatellites were isolated using magnetic

bead capture enrichment (Glenn and Schable 2005). A

genomic library was made after double enrichments for the

motifs (AACT)8, (AAGT)8, (ACAT)8, (AGAT)8. Total

DNA was digested with Rsa I (New England Biolabs), and

fragments were ligated to double stranded SuperSNX24

linkers. Fragments were hybridized to biotinylated oligo-

nucleotides and captured with magnetic streptavidin beads

(Invitrogen). Enriched DNA was amplified using the

polymerase chain reaction (PCR) primer forward Super-

SNX24. Cloning was conducted using TOPO-TA Cloning

Kit (Invitrogen). Clones with inserts between 300 and

700 bp length were purified using QIAquick PCR Purifi-

cation Kit (Qiagen, Hilden) and sequenced. Sequences

from both strands were assembled and microsatellites

located using Tandem Repeats finder (http://tandem.bu.

edu/trf/trf.html) and confirmed by eye.

Primers were designed from the flanking sequences of

the tandem repeats using Primer 3 software http://frodo.wi.

mit.edu/primer3/input.htm and were tested for amplifica-

tion on 1.2 % agarose gels.

PCR amplifications were performed in a 10-ll volume

consisting of 1 9 QIAGEN PCR buffer, 0.025 mM of each

primer, 3 mM MgCl2, 0.40 mM of each dNTP and 0.5 U

Taq DNA Polymerase (Qiagen) and 1 ll template using an

Eppendorf Mastercycler Gradient. A Touchdown thermal

G. Segelbacher (&)

Wildlife Ecology and Management, University Freiburg,

Tennenbacher Str. 4, 79106 Freiburg, Germany

e-mail: gernot.segelbacher@wildlife.uni-freiburg.de

I. Schuphan

Institute for Plant Physiology (Bio III), Aachen University

(RWTH), Worringerweg 1, 52054 Aachen, Germany

123

Conservation Genet Resour

DOI 10.1007/s12686-014-0149-0

cycling program encompassing a 10� C span of annealing

temperatures ranging between 60 and 50 �C was used for

the amplification. Following an initial denaturation step of

95 �C for 3 min, cycling parameters were 20 cycles at

95 �C for 30 s, 60� annealing temperature (decreased

0.5 �C per cycle) for 30 s and 72 �C for 40 s and 15 cycles

of 95 �C for 30 s, 50 �C for 30 s and 72 �C for 40 s and a

final extension step of 72 �C for 5 min. PCR products were

run on Elchrom Spreadex EL 400 gels in an Elchrom SEA

2000 apparatus and sized with M3 size standard (Elchrom,

Switzerland).

Each locus was tested for polymorphism and heterozy-

gosity using 30 individuals. Characteristics of the 13

working primer pairs are given in Table 1. We estimated the

number of alleles per locus (k), polymorphic information

content (PIC), observed and expected heterozygosity (Ho

and He), and frequency of null alleles and tested for devia-

tions from Hardy–Weinberg-equilibrium (HWE) using

CERVUS version 3.0 (Marshall et al. 1998). Loci Embc13

deviated significantly from HWE for our sample dataset and

no significant deviations for a gametic disequilibrium was

detected among all paired loci comparisons (p \ 0.05);

GENEPOP Version 3.4, Raymond and Rousset 1995).

Overall the high numbers of alleles per locus and het-

erozygosity and paternity exclusion probabilities of 0.99

demonstrate the potential of these novel species specific

rock bunting microsatellite primers for a variety of ques-

tions like kinship analysis and population differentiation.

Acknowledgments Katja Fleckenstein helped developing the prim-

ers. IS was supported by a grant of the Deutsche Ornithologen-

Gesellschaft (DO-G).

Table 1 Microsatellite loci in Emberiza cia including GenBank accession number, primer sequence, repeat motif, size of cloned allele in bp,

number of alleles (k); PIC, HE, expected heterozygosity; HO, observed heterozygosity and frequency of null alleles

Locus

Accession

number

Primer sequence (50–30) Repeat motif Size

(bp)

K PIC He Ho Null allele

frequency

embc4 F: TGAAGTGGAAAATACCAGTCAGG (GATA)9 122 5 0.69 0.74 0.73 0.008

KF969222 R: CAGTGACACTTCAGACAGCTCA

embc5 F: TCCTGGTTGTATTATTCTCCAAA (GATA)13 170 9 0.75 0.80 0.75 0.031

KF969223 R: CCTGCCTGAAACTAAGACAAATC

embc8 F: TGTTTTAAAATTGCTTTTTCAGTG (TCTA)14 176 6 0.58 0.63 0.76 -0.132

KF969230 R: TTTTCAGATCAAGTGTCTGCCTA

embc11 F: TCCCTACAGACACACACACACA (GATA)11 123 5 0.49 0.45 0.53 0.098

KF969224 R: TGTGCAAGAAAACATCTGAGG

embc13 F: AAAAACAGCCTATGGAAAATAACTT (GATA)5 (GACA)4 (GATA)7 157 6 0.56 0.64 0.81 -0.151

KF969225 R: AAACAGATGGGTGTGAAGACA

embc14 F: GGTGCAGGCCTGTATTTTTAAC (TATC)8 170 5 0.71 0.77 0.54 0.163

KF784805 R: CCCAAAATAAAACTAGAACAAGAACT

embc16 F: TGTGGGGAAGTTCACAAAGA (GATA)15 116 6 0.70 0.76 0.72 0.015

KF784805 R: AAACCTGCAAACACAAGGAAA

embc19 F: GAAGCTACAGCCCAAACTCC (CTCA)13 155 4 0.63 0.69 0.74 -0.041

KF784805 R: TTCATTTTCTTGTTCCACTCTACG

embc21 F: GGACTATTTAATTTCTTCAAATTGGT (GATA)11 (GACA)3 170 5 0.64 0.68 0.48 0.169

KF969226 R: CAATGAGTAGTTTTATTTGCAGCAG

embc22 F: TGTCCCATGGAGGGTTCTAC (GATA)13 149 7 0.77 0.80 0.68 0.078

KF969227 R: TGAGATATAATGTTTGTTTCATCCAA

embc28 F: GTCTGCAGAAGGGCAGATTC (TG)17 109 7 0.81 0.85 0.76 -0.097

KF969228 R: TGTGATTGGACACAATCCCTTA

embc29 F: TGAAATGATGCTGAATGACCA (CCAT)4(CCAA)4(CCAT)10 132 6 0.72 0.75 0.65 0.064

KF969229 R: TGTCATGTATAGGTGAATAGATGATGA

embc30 F: CATCCATTCATCATCTATTCACCT (GATA)11 114 5 0.70 0.76 0.65 0.060

KF969231 R: CGATTGCAACCAAATTGCTC

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