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Microsatellite Loci Isolated from the Mediterranean Species Cicada Barbara (Stål) and C

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Microsatellite Loci Isolated from the Mediterranean Species Cicada Barbara (Stål) and C Molecular Ecology Notes (2002) 2, 173–175 PRIMERBlackwell Science, Ltd NOTE Microsatellite loci isolated from the Mediterranean species Cicada barbara (Stål) and C. orni L. (Hemiptera, Cicadoidea) S. G. SEABRA,*† H. R. WILCOCK,* J. A. QUARTAU† and M. W. BRUFORD* *School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff, CF10 3TL, UK, †Centro de Biologia Ambiental e Departamento de Zoologia e Antropologia, Bloco C2, 3° Piso, Faculdade de Ciências de Lisboa, Campo Grande 1700 Lisboa, Portugal Abstract We describe the isolation of six polymorphic microsatellites for Cicada barbara (Stål), four of which are also polymorphic for the closely related Cicada orni L. Cicadas from several sites in the Iberian Peninsula, North Africa (C. barbara) and Greece (C. orni) were genotyped at these loci. Polymorphism is higher than that previously obtained with allozymes for these species. One locus allows species diagnosis (nonoverlapping allele size ranges) between C. barbara and C. orni and the others have some exclusive alleles for each species. Keywords: cicadas, Cicada barbara, Cicada orni, insects, microsatellites Received 6 November 2001; revision received 21 December 2001; accepted 21 December 2001 The genus Cicada L. (Hemiptera, Cicadoidea) includes agarose gel by electroelution into dialysis tubing and some sibling species that are morphologically very similar purified using Centricon Microconcentrators (Amicon). but distinguishable by the specific calling songs produced The fragments were enriched for CA and GA repeats using by males for mate recognition. Cicada barbara and C. orni are biotin-labelled probes and then ligated into a pUC18 vector two of these species that exist in sympatry in some areas (Amersham Pharmacia Biotech) (Hammond et al. 1998). in the southern Iberian Peninsula. C. barbara is widely Ligation products were cloned into One Shot® competent distributed in North Africa while C. orni occurs mostly in cells TOP 10 (Invitrogen). Colonies were screened follow- European Mediterranean countries. Previous studies ing the polymerase chain reaction (PCR) -based screening using allozymes showed low levels of genetic variation method of Lunt et al. (1999). Of the 432 colonies screened, within and between these species (Quartau et al. 2001). 71 were positive and were sequenced directly from the With the aim of studying the genetic structure of popu- PCR products using a Big Dye™ Terminator Cycle lations, dispersal and potential interspecific hybridiza- Sequencing Ready Reaction Kit (PE Biosystems), following tion in these species, microsatellites were isolated and the cycle sequencing conditions: 25 cycles of 10 s at 96 °C, described. 5 s at 50 °C and 4 min at 60 °C. The fragments were Two separate partial genomic libraries were constructed, separated on an ABI 377 Sequencer. Fifty-nine colonies one for each species, using the protocol of Hammond et al. had repeat sequences (14% enrichment). Ten primer pairs (1998). Total DNA was extracted from the muscle tissue of were designed using the software programme omiga™ 2.0 the thorax of five specimens from different populations of (Oxford Molecular Ltd). each species using the QIAamp DNA Mini Kit (QIAGEN). Amplification of microsatellite loci was performed on a The DNA was digested with MboI (Promega) and selected Gene Amp 9700 Perkin Elmer thermal cycler under the fragments (300–900 base pairs) were isolated from a 1.5% following conditions: 2 min at 94 °C, then 30 cycles of 30 s at 94 °C, 40 s at 58–62 °C (Table 1) and 1 min at 72 °C, followed by an extension period of 72 °C for 10–30 min. Correspondence: Sofia Gonçalves Seabra. Fax: +44(0)2920874305; Optimized PCR reaction mixes contained approximately E-mail: [email protected] 100 ng of template DNA, 1.5 or 2 mm MgCl2 (Table 1), © 2002 Blackwell Science Ltd 174 PRIMER NOTE PRIMER Table 1 Characterization of Cicada L. microsatellite loci. The motif, primer sequences, annealing temperatures (Ta) and MgCl2 concentrations are given for each locus. Allele size ranges, number of alleles and heterozygosity (observed – HO and expected – HE) were determined from genotyping 31 specimens of C. barbara (Cb) from seven sites in the Iberian Peninsula (Foz Côa, Crato, Monforte, Arrábida, Alcalar, Toledo and Cordoba) and one site in North Africa (Ceuta), and 32 specimens from C. orni (Co) from six sites in the Iberian Peninsula (Lisboa, Monte da Caparica, Sesimbra, Arrábida, Crato and Monforte) and one site in Greece (Athens) Allele size range (bp) No. alleles HO (HE) Repeat in sequenced Primer sequence GenBank MgCl2 ′ ′ ° Locus allele (5 to 3 ) accession no. Ta ( C) (mm) Cb Co Cb Co Cb Co Cib01 (GA)18 F: GATAAAATCAGTGGAGTGC AF437631 58 2 164–194 162–208 10 6 0.759 0.120 R: AGTCGATACAATCGAACC (0.828) (0.607) © 2002BlackwellScienceLtd, Cib03 (GT)2T(GT)13 F: ATATCTGATGGACCCTCG AF437632 62 2 257–267 235–253 5 7 0.333 0.607 R: AGGTATCATGCCTTATTGC (0.625) (0.759) Cib06 (GT)2TT(GT)11TGT F: CGCAGCAGACGATTTTATCC AF437633 60 1.5 256–294 — 8 — 0.793 — R: GCTCAATAACTGCCCATAACG (0.849) Cib07 (GT)15 F: TTGGAATTCAGAGTGTCG AF437634 60 1.5 133–165 129–197 7 11 0.160 0.484 R: GTGTCCTGTGTATCCTACG (0.706) (0.740) Cio08 (CA)7TA(CA)5 F: CGATGGTTGTAATTTAGTGG AF437636 60 1.5 201–217 181–213 8 10 0.667 0.241 R: TCAATAGCATATCTTGCTCC (0.811) (0.735) Cib10 (CA)16 F: AGAAGAAGGTGGACAACC AF437635 60 1.5 141–165 — 5 — 0.160 — R: TGACGACTTGAAAACAGC (0.675) Molecular Ecology Notes , 2,173–175 PRIMER NOTE 175 0.2 mm dNTPs, 0.2 mm of each primer (forward primer 5′ high levels of localized inbreeding. These are currently end-labelled with a fluorescent dye), 0.25 U of Taq poly- under investigation. merase (GIBCO Life Technologies) and the manufacturer’s buffer (20 mm Tris–HCl pH 8.4, 50 mm KCl) in a final reac- Acknowledgements tion volume of 10 µL. Amplified microsatellites were run on polyacrylamide gels (Gene-PAGE, Amresco) on an ABI This work was supported by Fundação para a Ciência e a Tecno- 377 Automated Sequencer using an internal lane standard logia, Portugal (PhD grant SFRH/BD/1027/2000). We thank Genage André, Gabriela Pinto, Mónica Ribeiro, Paula Simões and (genescan-350 tamra from PE Applied Biosystems). Teresa Fernandes for their help in the field. Six out of the 10 loci tested were polymorphic in C. bar- bara and four of these were also polymorphic in C. orni (Table 1). All loci were obtained from the C. barbara library References ± except Cio08. The mean number of alleles was 7.2 1.94 Hammond RL, Saccheri IJ, Ciofi C et al. (1998) Isolation of micro- (SD) for C. barbara and 8.5 ± 2.38 (SD) for C. orni. One of the satellite markers in animals. In: Molecular Tools for Screening loci (Cib03) had nonoverlapping allele size ranges between Biodiversity (eds Karp A, Isaac PG, Ingram DS), pp. 279–287. the species. In other loci (Cib01, Cib07 and Cio08) there Chapman & Hall, London. were some exclusive alleles for each species. Observed Lunt DH, Hutchinson WF, Carvalho GR (1999) An efficient heterozygosity was significantly lower than expected for method for PCR-based isolation of microsatellite arrays (PIMA). Molecular Ecology, 8, 891–894. all except one species–locus combination (that of C. barbara Quartau JA, Ribeiro M, Simões PC, Coelho MM (2001) Genetic for Cib06). Since the DNA was of high quality and geno- divergence among populations of two closely related species of types were repeated up to three times, allelic dropout is Cicada Linnaeus (Hemiptera: Cicadoidea) in Portugal. Insect unlikely, but there remains the possibility of null alleles or Systematics and Evolution, 32, 99–106. © 2002 Blackwell Science Ltd, Molecular Ecology Notes, 2, 173–175.
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