Molecular Ecology (2000) 9, 1171–1193 © 2000 Blackwell Science Ltd Blackwell Science, Ltd PRIMER NOTES 1171 1173 Graphicraft Limited, Hong Kong Identification of microsatellite loci in olive (Olea europaea) and their characterization in Italian and Iberian olive trees K. M. SEFC,*‡ M. S. LOPES,* D. MENDONÇA,* M. RODRIGUES DOS SANTOS,* M. LAIMER DA CÂMARA MACHADO† and A. DA CÂMARA MACHADO* *Universidade dos Açores, Departamento de Ciências Agrárias, Terra Chã, 9700 Angra do Heroismo, Açores, Portugal, †Institut für Angewandte Mikrobiologie, Universität für Bodenkultur Wien, Nussdorfer Lände 11, 1190 Vienna, Austria Keywords: Olea europaea, olive, microsatellite Received 17 November 1999; revision received 31 January 2000; accepted 5 February 2000 Correspondence: Artur da Câmara Machado. Fax: + 351 295332605; E-mail: amachado@angra.uac.pt ‡Present address: Zentrum für Angewandte Genetik, Universität für Bodenkultur Wien, Muthgasse 18, 1190 Vienna, Austria. Olive (Olea europaea L. ssp. europaea) is one of the most ancient cultivated fruit trees and an important oil-producing crop in the Mediterranean basin. Feral olives and wild oleaster forms add to the intraspecific variability of O. europaea and the number of species within the genus Olea is high and con- troversial. Genetic studies have already been used to address and clarify Olea systematics (Angiolillo et al. 1999). The identifi- cation of olive cultivars is a further application of genetic markers in this crop (Fabbri et al. 1995). In this paper, we report the development of microsatellite primers from O. europaea and their polymorphism in olive trees from Italy and the Iberian Peninsula. For the construction of the genomic library, young leaves and flower buds were harvested from an olive tree in Porto Martins, Terceira, Açores. The DNA extraction protocol described by Fabbri et al. (1995) was modified as follows: 5 g plant material were ground in liquid nitrogen and incubated in 20 mL CTAB buffer (100 mm Tris-HCl pH 8.0, 1.4 m NaCl, 20 mm EDTA, 2% CTAB, 1% PVP, 0.2% β-mercaptoethanol) for one hour at 65 °C. After centrifugation, the supernatant was extracted twice with chloroform–isoamyl alcohol (24 : 1), and DNA was precipitated from the recovered aqueous phase with 0.6 vol. isopropanol. The pellet was resuspended in 4 mL H 2 O and proteins were precipitated with 0.5 vol. 7.5 m ammonium acetate. After ethanol precipitation of the DNA, resuspension of the pellet in 4 mL H 2 O, RNase digest and second protein precipitation, the supernatant was extracted with phenol– chloroform–isoamyl alcohol (25 : 24 : 1). DNA was precipitated from the aqueous phase with ethanol, washed and resuspended in 1 mL H 2 O. A size-selected genomic library of O. europaea was screened for (GA) n and (CA) n sequences as described previously (Sefc et al. 1999). After two rounds of screening, plasmids were isolated from positive Escherichia coli clones (Flexi- Prep Kit, Amersham Pharmacia Biotech) and inserts were sequenced using the dRhodamine Terminater Cycle Sequencing Kit (PE Applied Biosystems) and an automated sequencer (ABI Prism 310 Genetic Analyser, PE Applied Biosystems). For polymerase chain reaction (PCR), DNA was extracted from leaves as described in Fabbri et al. (1995). PCR reactions were carried out in a vol. of 20 μL, containing 50 ng DNA, 50 mm KCl, 10 mm Tris-HCl pH 9.0, 0.1% Triton X 100, 1.5 mm MgCl 2 , 100 μm of each dNTP, 1 μm of each primer and 0.5 U Taq DNA polymerase (Promega, Mannheim). The following temperature regime was applied: 5 min denaturation at 95 °C, 35 cycles of 20 s denaturation at 95 °C, 30 s at the appropri- ate annealing temperature (Table 1), 30 s elongation at 72 °C. Electrophoresis was carried out using an automated sequencer (ABI Prism 310 Genetic Analyser, PE Applied Biosystems) and fragment lengths were determined with the help of internal size standards (Genescan 350 TAMRA Size Standard, PE Applied Biosystems). Simultaneous screening with (GA) n and (CA) n probes revealed 20 (GA) n , 4 (CA) n and 5 (CA) n -(GA) n com- pound repeats. Primers could be designed for 28 loci, and specific amplification was achieved at 15 microsatellite loci (Table 1). At locus ssrOeUA-DCA11, the size of the cloned allele was 179 bp, while amplification from the individual used for library construction yielded a 125-bp band. Sequencing of the 125 bp fragment revealed a microsatellite locus with flanking sequences identical to those of the originally cloned allele and a (GA) 7 microsatellite. A 139-bp fragment ampli- fied from another tree again showed the identical flanking sequences and a (GA) 12 (GGGA) 1 microsatellite. The repeat motif in the originally cloned fragment is (GA) 26 (GGGA) 4 . However, two individuals showed alleles in the expected size range of 177 and 179 bp. Possibly, the primers designed for the cloned allele preferentially amplify a duplicated locus with lower repeat numbers whenever this locus is present. These amplification results were reproducible at various annealing temperatures. At locus ssrOeUA-DCA17, the amplified fragments ranged from 101 bp to 183 bp, with 109 bp and 111 bp being the most frequent fragment sizes. A comparison of the sequences of the 111 bp and 183 bp fragments showed identical flanking sequences, whereas the repeat sequence of the short allele dis- played a deletion, which reduced the (GT) 9 (AT) 7 AGATA(GA) 38 motif of the cloned 183 bp allele to (GT) 7 (GA) 13 in the 111 bp allele. The markers were characterized in olive trees from the Iberian Peninsula (n = 38) and from Italy (n = 9; Table 2). All loci were polymorphic with 4 –15 alleles per locus.