Molecular Ecology Notes (2006)

6

, 104–106 doi: 10.1111/j.1471-8286.2005.01154.x

© 2006 Blackwell Publishing Ltd

Blackwell Publishing, Ltd.

PRIMER NOTE

Isolation and characterization of highly polymorphic microsatellite loci for the garden tiger moth

Arctia caja

(Lepidoptera: Arctiidae)

SARAH J . ANDERSON,*† DEBORAH A. DAWSON† and JOANNA R. FREELAND**

Department of Biological Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK,

†

Sheffield Molecular Genetics Facility, Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK

Abstract

A set of microsatellite markers was isolated from

Arctia caja

, a species that has been decliningin UK for several decades. As has been found with other Lepidoptera species, this proved tobe a difficult task. Eight enriched libraries identified 103 positive clones, and these yielded onlyseven polymorphic microsatellites. Allelic diversity ranged from 10 to 23 alleles per locus ina population of 30 individuals. Significant heterozygosity deficits were shown by three ofthese loci, presumably due to null alleles. The remaining four loci were in Hardy–Weinbergequilibrium and will be used to investigate the population genetic structure of

A. caja

.

Keywords

:

Arctia caja

, Arctiidae, Lepidoptera, microsatellite, moth

Received 6 July 2005; revision accepted 25 July 2005

A long-term data set from the Rothamsted Insect Surveyhas shown that the number of garden tiger moths in UKfell by nearly 30% in the mid-1980s, a decline from whichthey have not yet recovered and one that is expected tocontinue (Conrad

et al

. 2002). Over a similar period, theirdistribution has been moving northwards; populationshave almost completely disappeared from the southeast ofEngland, while previously small or nonexistent popula-tions in Scotland are flourishing. These well-documentedchanges make

Arctia caja

an ideal species in which to studythe effects that regional declines, in conjunction with localextinctions and recolonizations, can have on genetic diversityand gene flow. Microsatellite markers for

A. caja

weretherefore developed with the aim of linking patterns ofpopulation genetics with long-term population dynamics.

Larvae were collected from the Sands of Forvie NatureReserve near Aberdeen, Scotland, and stored in 100% eth-anol. Genomic DNA was obtained separately from threeindividuals of unknown sex using a salt extraction method(Bruford

et al

. 1998) and then pooled. The characterizationof microsatellite loci in the Lepidoptera is known to beproblematical, and in the past there have been varyinglevels of success (reviewed in Zhang 2004). In order to

maximize the success of microsatellite isolation, eightdifferent libraries enriched for eight different motifs werecreated. The libraries were prepared following the methodsof Armour

et al

. (1994) with modifications by Dawson

et al

.(2003). Genomic DNA was digested with

Mbo

I, and 300–1000 bp fragments were separately enriched for (CA)

15

,(GA)

25

, (GC)

25

, (GAA)

18

, (TTAG)

14

, (GATA)

14

, (TTTC)

14

and (GTAA)

14

(Amersham Pharmacia Biotech). Enrichedfragments were ligated into pUC18-

Bam

HI/BAP (Qbiogene)and transformed into XL1-Blue Competent Cells (Strata-gene). In total, 4992 clones were transferred to nylon mem-branes (Hybond-N) and screened by hybridization ateither 55

°

C (tri- and tetranucleotide motifs) or 65

°

C(dinucleotide motifs) using the same stocks of dinucle-otides, trinucleotides and tetranucleotides as used for theenrichment, but this time radiolabelled with [

α

32

P]-dCTP.All 103 positively hybridizing clones were sequencedusing BigDye terminators (Applied Biosystems) GenBank[Accession nos AJ809344–AJ809406, AJ829727–AJ829729and AJ869349–AJ867385]. Fifty of these sequences werediscarded because their flanking sequences were either tooshort to allow primer design, or they were duplicates ofother sequenced loci (identified using

blastn

version 2.2.4software, Altschul

et al

. 1997).Primers were designed for the remaining 53 unique loci

using

primer

3 (Rozen & Skaletsky 2000). Primers were

Correspondence: Sarah Anderson, Fax: +44 1908 654 167; E-mail:s.j.e.anderson@open.ac.uk

P R I M E R N O T E

105

© 2006 Blackwell Publishing Ltd,

Molecular Ecology Notes

, 6, 104–106

initially tested in polymerase chain reactions (PCRs) usingfive individuals from each of three populations. Each 10

µ

LPCR contained 10 ng DNA, 5 pmol of each primer, 0.15 m

m

of each dNTP, 1.5 m

m

MgCl

2

and 1 U

Taq

polymerase (Bio-line) in the manufacturer’s buffer [160 m

m

(NH

4

)

2

SO

4

,670 m

m

Tris-HCl (pH 8.8 at 25

°

C), 0.1% Tween 20]. PCRamplification was carried out using a Tetrad thermal cycler(MJ Research) with a PCR profile of 2 min at 94

°

C for onecycle followed by 35 cycles of 1 min at 94

°

C, 1 min at theannealing temperature (Table 1) and 1 min at 72

°

C. Visu-alization of the PCR products on a 1% agarose gel stainedwith ethidium bromide indicated that 17 primer sets failedto amplify any product, and eight primer sets amplifiednonspecific products with multiple bands or smears. Of the24 primer sets that amplified a specific product, the forwardprimer from each of 14 primer pairs was fluorescentlylabelled with HEX or FAM (Sigma Genosys). Using the samePCR conditions, labelled primers were used to amplify thealleles from 30 presumably unrelated individuals that hadbeen collected from Belford, Northumberland. Genotypeswere scored on an ABI 3730 DNA sequencer with the ROXfilter set D and analysed using

genemapper

3.5 software(Applied Biosystems).

Of the 14 genotyped loci, five failed to amplify despiteattempts at re-optimization, one locus was fixed for a singleallele and one locus showed nonspecific amplification. Forthe remaining seven loci, observed and expected hetero-zygosities and null allele frequencies were calculated using

cervus

version 2.0 (Marshall

et al

. 1998). Tests for departurefrom Hardy–Weinberg equilibrium and linkage disequi-librium were conducted using a Markov chain method in

genepop

on the web (an update of Raymond & Rousset 1995).

Some males and females were heterozygous at eachlocus, and therefore no loci were sex-linked. No loci werefound to be in linkage disequilibrium. Three of the loci hadhigh-predicted null allele frequencies and showed significantdeparture from Hardy–Weinberg equilibrium (Acaja02,Acaja25 and Acaja55; Table 1) and may not be suitable forfuture population genetic studies. The remaining four loci,however, appear to be reliable codominant markers with ahigh level of polymorphism that should provide a fine-scaleresolution for analysing the population genetic structure of

A. caja

. This work has further emphasized the difficultiesthat typically accompany the development of microsatellitemarkers in Lepidoptera, since a combination of approxi-mately 4992 clones and eight different enrichment motifswas required before four markers suitable for populationgenetic analysis could be identified.

Acknowledgements

The microsatellite library was created at the Sheffield MolecularGenetics Facility supported by the Natural Environment ResearchCouncil, UK. We wish to thank Rothamsted Insect Survey andSands of Forvie Nature Reserve for supplying samples, and Jag-roop Pandhal and Andrew Pemberton for assistance with librarydevelopment and genotyping.

References

Altschul SF, Madden TL, Schaffer AA

et al.

(1997) Gapped

blast

and

psi-blast

: a new generation of protein database search pro-grams.

Nucleic Acids Research

,

25

, 3389–3402.Armour JAL, Neumann R, Gobert S, Jeffreys AJ (1994) Isolation of

human simple repeat loci by hybridization selection.

HumanMolecular Genetics

,

3

, 599–605.

Table 1 Characteristics of garden tiger moth Arctia caja microsatellite loci based on a population sample of 30 presumably unrelatedindividuals

Locus

EMBL Accession no. Repeat motif Primer sequence (5′−3′)

Ta(°C)

MgCl2(mm) A

Allele size range (bp) HE HO HW

Acaja02 AJ809345 (CA)18 HEX-TGCAATAGAACTTTCAACCCCTTTGAGGGGCGACTTACCTAAACAAGG

59 1.5 21 181–245 0.80 0.23 P < 0.01

Acaja10 AJ809353 (CT)21 HEX-GTCCTTCTCATGAACGACTG TTCTAACGCAATAGAATGGTG

56 1.5 22 189–239 0.89 0.92 0.94

Acaja25 AJ809368 (CT)3TTC(CT)3CC FAM-CGTTGGCAGATACATCAGAATCG 61 1.5 10 175–196 0.69 0.47 P < 0.01(CT)5TT(CT)13 TCAGTGAGAATATGGCATTGGTAGG

Acaja28 AJ809371 (TC)9(TA)1(TC)16 FAM-GCGCGTGAGGTGTGTCAAG CGAAACGAAAATAACTGGCTTGC

62 1.5 23 190–248 0.82 0.89 0.70

Acaja41 AJ809384 (TTAC)5 FAM-GCGGTTTCAGTACGCACTTCC TGTCAATAGGTTATGGACCTGCAAC

62 1.5 10 160–181 0.75 0.60 0.35

Acaja42 AJ809385 (TACT)2(ACT)3 HEX-ACAACCCGGACACGACACTG 62 1.5 10 186–216 0.30 0.27 0.12(TACT)2(ACT)1(TACT)1 CATGGTTCCAAAGGGGCTTG

Acaja55 AJ809398 (CA)16 FAM-CCTTGTAATAATCAAAATGACCAATCTGAAACTGTTTGATAACCATTTGAG

63 1.5 12 132–170 0.84 0.61 0.01

Ta, annealing temperature; A, number of alleles when scored on an ABI 3730 (Applied Biosystems); HO, observed heterozygosity; HE, expected heterozygosity; HW, probability of the population being in Hardy–Weinberg equilibrium.

106 P R I M E R N O T E

© 2006 Blackwell Publishing Ltd, Molecular Ecology Notes, 6, 104–106

Bruford MW, Hanotte O, Brookfield JFY, Burke T (1998) Multi-locus and single-locus DNA fingerprinting. In: Molecular geneticanalysis of populations (ed. Hoelzel AR), pp. 287–336. Oxford Uni-versity Press, New York.

Conrad KF, Woiwod IP, Perry JN (2002) Long-term decline inabundance and distribution of the garden tiger moth (Arctia caja)in Great Britain. Biological Conservation, 106, 329–337.

Dawson D, Bretman A, Tregenza T (2003) Microsatellite loci forthe field cricket, Gryllus bimaculatus and their cross-utility inother species of Orthoptera. Molecular Ecology Notes, 3, 191–195.

Marshall TC, Slate J, Kruuk LEB, Pemberton JM (1998) Statistical

confidence for likelihood-based paternity inference in naturalpopulations. Molecular Ecology, 7, 639–655.

Raymond M, Rousset F (1995) genepop (version 1.2): populationgenetics software for exact tests and ecumenicism. Journal ofHeredity, 86, 248–249.

Rozen S, Skaletsky HJ (2000) primer 3 on the WWW for generalusers and for biologist programmers. In: Bioinformatics Methodsand Protocols: Methods in Molecular Biology (eds Krawetz S, MisenerS), pp. 365–386. Humana Press, Totowa, NJ.

Zhang DX (2004) Lepidopteran microsatellite DNA: redundant butpromising. Trends in Ecology & Evolution, 19, 507–509.