Microsatellite loci for the green and golden bell frog (litoria aurea)

Emma Burns* & Gianfrancesco FerrariSchool of Biological, Earth and Environmental Sciences, University of New South Wales, Sydney 2052,Australia (*Author correspondence: fax: +61-2-9385-1558; e-mail: e.burns@austmus.gov.au)

Received 5 May 2003; accepted 10 June 2003

Key words: dinucleotide, Litoria aurea, microsatellite

The Green and Golden Bell Frog (Litoria aurea) isa large and distinctive tree frog species currentlyprotected under Australian legislation due todramatic declines and range contractions (Pykeand White 2001). Little is known about the pop-ulation structure of persisting populations, al-though a small-scale allozyme study suggested alow level of structuring for an amphibian (Colgan1996). Microsatellites are increasingly accepted asthe marker of choice for conservation genetics andrecently have been used to investigate amphibianpopulation structure (Newman and Squire 2001).

Dinucleotide microsatellite markers were gen-erated using the enrichment technique of Gardneret al. (1999). In brief, genomic DNA (10 lg) wasdigested with restriction enzyme Sau 3A andligated to the S62/S61 adaptor (0.9 mmol) (S62:5¢-GATCCGAAGCTTGGGGTCTCTGGCC-3¢;(S61: 5¢-GGCCAGAGACCCCAAGCTTCG-3¢)(Gardner et al. 1999). Fragments between 400 and1200 bp were excised from a 1% agarose gel andpurified using the freeze-squeeze technique (Tautzand Renz 1983).

Streptavidin MagneShpere� ParaMagneticbeads (100 ll) (Promega) and 200 pmol of the5¢-(CA12)GCTC[Biotin]A-3¢oligonucleotide wereincubated for 15 min at room temperature andwashed (Gardner et al. 1999). The DNA/adaptorsolution (50 ll in 1 · hybridisation solution)(Gardner et al. 1999) was heat denatured at 95 �Cfor 5 min in the presence of 20 pmol of S61, cooledto 55 �C, added to the magnetic bead/biotin-oli-gonucleotide, incubated for 20 min at 55 �C andthen washed to remove unbound DNA fragments.

Captured CA-enriched DNA fragments wereeluted, purified using a QIAquick column (QIA-

GEN) and used as template in a PCR reaction(Gardner et al. 1999). The PCR product was thenpurified using a QIAquick column, eluted in 30 ll10 mM Tris (pH 8.5), cloned into a pGEM Tvector (Promega) and used to transform compe-tent E. coli (JM109)(Promega) according to themanufacturers instructions. Colonies were trans-ferred onto Hybond N+ membranes and screenedusing a synthetic copolymer poly(dA/dC)/poly(dG/dT) probe (Pharmacia), labelled byincorporation of [a32P]-dATP using a ‘nick’ trans-lation kit (Amersham). A total of 65 positiveclones, from 400 screened, were sequenced usingM13 primers to characterise each locus. Primerswere designed for 26 loci using PRIMER3 (Rozenand Skaletsky 1996). For a number of loci multipleprimer combinations were trailed, in total 108different primers were designed and tested. Onlyfour loci amplified consistently and were poly-morphic. Each of these forward primers was syn-thesised with a fluorchrome label (TET or FAM).

PCR reactions (10 ll) for loci Laurea4-49 andLaurea5M consisted of DNA (50–100 ng),1.5 pmol of each primer, 1.25 mM MgCl2, 67 mMTris–HCl, 16.6 mM (NH4)2SO4, 0.45% Triton X-100, 0.2 mg/ml gelatin, 2 mM dNTPs and 0.5 UTth+ (Biotech Australia). The following MJ Re-search PTC-100 thermal cycling profile was em-ployed: (i) initial 1 min denaturation at 94 �C; (ii)35 cycles of denaturation (20 s at 94 �C), anneal-ing (1 min at the selected temperatures in Table 1),and extension (45 s at 72 �C); 3 min at 72 �C. PCR(10 ll) for loci Laurea2A and Laurea4-10 wereperformed using a FailSafe� PCR PreMix Selec-tion Kit (EPICENTRE). Reactions consisted ofDNA (50–100 ng), 1.5 pmol of each primer,

Conservation Genetics 5: 421–423, 2004.� 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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Table

1.Characteristics

ofLitoriaaureamicrosatelliteloci

Locusname

Repeatmotif

Primer

sequence

Allelesize

range(bp)

Ta�C

No.of

alleles

HO

HE

H–W

exact

P

GenBank

No.

Laurea4-49

(CA) 9GA(C

A) 19

F-G

CTGCCTATGGACTCAAGGA

213–229

55–50TD*

60.421

0.734

0.006*

AY273937

R-TTCAGCCTTTGGCAGACAG

Laurea5M

(CA) 4AA

(CA) 9

F-TTCACCCAGTGCTTGATTCA

116–136

58

60.684

0.759

0.216

AY273938

R-C

AGGGTTGTCAGTTGTCCCT

Laurea2A

(CA) 7AA(C

A) 13

F-C

CATAGCTTTTGAAACAGTGTTTA-

ACCCTTTGAC

196–216

66–62TD**

50.895

0.745

0.930

AY273939

R-G

ATTGCCGCATTTGACCTAGTGGGTTT

Laurea4-10

(CA) 6CT(C

A) 6

F-A

CTCCAAATCCAGACCTCCATGGG

212–227

66–62TD**

30.474

0.465

0.422

AY273940

R-A

GGATCAGGGCGCACTCATCTCTAA

Ta=

optimalannealingtemperature;TD*=

PCR

program

ofdecreasinginitialannealingtemperatures1�C

/cyclefor5cycles.

TD**=

PCR

program

ofdecreasingannealingtemperatureswith10cycles

@66�C

,10cycles

@64�C

,15cycles

@62�C

.

HO=

observed

heterozygosity;H

E=

expectedheterozygosity;H–W

Exact

P=

Probabilityvaluefrom

Hardy-W

einbergtest

forheterozygote

deficiency.

*Significant(P

<0.05).

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0.25 U FailSafe PCR Enzyme Mix and 5 ll Fail-Safe PCR 2X PreMix (100 mM Tris–HCl,100 mM KCl, 400 lM dNTPs, MgCl2 3–7 mMand FailSafe PCR Enhancer 0–8 X) A and PreMixC respectively. FailSafe PCR 2X PreMix A andPreMix C differ in MgCl2 and FailSafe PCR En-hancer concentrations, not disclosed by the man-ufacturer. The thermal cycling profile employedwas: (i) initial 2 min denaturation at 95 �C; (ii) 35cycles of denaturation (20 s at 93 �C), annealing(1 min at the selected temperatures in Table 1),and extension (45 s at 72 �C); 5 min at 72 �C. Thefluorchrome-labelled microsatellites were elec-trophoresed using 4.25% acrylamide gels on anABI PRISM� 377 DNA sequencer. Bandingpatterns were analysed using GENESCAN� 3.1and GENOTYPER� 1.1.1 (Applied Biosystems).

Using the species-specific loci reported here,genetic diversity was assessed in 19 captive indi-viduals from Taronga Zoo, Sydney Australia. Allloci were polymorphic with between three and sixalleles per locus (Table 1). Three of the locishowed an expected dinucleotide allele distributionwhilst locus Laurea4-10 exhibited alleles of bothodd and even base pair sizes, suggesting thatmutation may not be restricted to repeat units(Table 1). Observed and expected heterozygositieswere estimated using the software package GENE-POP (Raymond and Rousset 1995) version 3.2.Observed heterozygosity (HO) ranged from 0.421to 0.895 (Table 1). Deviations from Hardy–Weinberg Equilibrium and linkage disequilibriumwere tested using Marchov chain approximation(Gou and Thompson 1992) in GENEPOP (Ray-

mond and Rousset 1995). There was no evidenceof linkage disequilibrium, however a heterozygotedeficiency (P ¼ 0.006) at locus Laurea4-49 (Ta-ble 1) may indicate null allele(s).

Acknowledgements

This work was funded by the ZPB of NSW, RTAand a ARC small grant. We thank Candice Webb,Dion Hobcroft and Alaxandra Schulmeister.

References

Colgan D (1996) Electrophoretic variation in the Green andgolden bell frog Litoria aurea. Aust. Zool., 30, 170–176.

Gardner MG, Cooper SJB, Bull CM, Grant WN (1999) Iso-lation of microsatellite loci from a social lizard, Egerniastokesii, using a modified enrichment procedure. J. Hered.,90, 301–304.

Gou SW, Thompson EA (1992) Performing the exact test ofHardy–Weinberg proportion for multiple alleles. Biometrics,48, 361–372.

Newman RA, Squire T (2001) Microsatellite variation and fine-scale population structure in the wood frong (Rana sylavat-ica). Mol. Ecol., 10, 1087–1100.

Pyke GH, White AR (2001) A review of the biology of the greenand golden bell frog Litoria aurea. Aust. Zool., 31, 563–598.

Raymond M, Rousset F (1995b) GENEPOP (Version 1.2):Population genetic software for exact tests and ecumen-icisms. J. Hered, 86, 248–249.

Rozen S, Skaletsky HJ (1996) Primer 3. Code available at:http//www.genome.wi.mt.edu/genome_software/other/pri-mer3.html.

Tautz D, Renz M (1983) An optimised freeze squeeze methodfor the recovery of DNA fragments from agarsoe gels. Anal.Biochem., 132, 14–19.

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