Isolation of polymorphic microsatellite markers for the Alpine Lady Fern, Athyrium distentifolium Tausch ex Opiz, from an enriched genomic library J. Squirrell 1 *, M. Woodhead 2 , P.M. Hollingsworth 1 , J. Russell 2 , M. Gibby 1 & W. Powell 2 1 Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh, Scotland EH3 5LR; 2 Scottish Crop Research Institute, Invergowrie, Dundee, Scotland DD2 5DA (*Author for correspondence: Fax: +44-131- 2482901; e-mail: J.Squirrell@rbge.org.uk) Received 12 August 2003; accepted 11 September 2003 Key words: fern, microsatellites, pteridophyte, SSR, Woodsiaceae Athyrium distentifolium Tausch ex Opiz, the Al- pine Lady fern, is a perennial diploid fern of the Woodsiaceae and has a scattered northern cir- cumboreal distribution. Its scarcity in Britain, and its progenitor role in the evolution of the British endemic A. flexile (Newman) Druce has resulted in conservation interest (Stewart et al. 1994; McHaffie 1999). A set of co-dominant microsat- ellite markers from an enriched library has been developed to investigate population diversity and differentiation in this species and to provide a complementary set of microsatellite markers to those already developed from a cDNA library (Woodhead et al. 2003). Total genomic DNA was isolated from frozen frond material of Athyrium distentifolium (Bridge of Orchy, Scotland) using a DNeasy Plant Maxi kit (Qiagen). DNA (2.5 lg) was enriched for the dinucleotide microsatellites CA and GA using a membrane enrichment method (Hale et al. 2001) with modifications detailed in Hughes et al. (2002) and Brennan et al. (2002). The enriched DNA was ligated into a pGEMT-Easy vector (Promega) and transformed into ElectroMAX DH10B cells (Invitrogen) according to the manufacturer’s instructions. Transformed colonies were identified, using blue/white screening, and plasmids from individual colonies isolated. Inserts were se- quenced (Brennan et al. 2002) and analysed on either an ABI Prism 377 or 3700. Primers were designed to flank suitable microsatellites (those considered were ‡ 16 bp and either perfect or compound in sequence) using the program PRI- MER-3 (Rozen and Skaletsky 1998). The reverse primer for each primer pair was modified accord- ing to Magnuson et al. (1996) by the addition of a 5¢ dGTP. This increases the likelihood that all PCR products will be of the +A type which can facilitate microsatellite profile interpretation. In total 576 plasmids were isolated from the library. Of the 321 inserts that were successfully sequenced, 231 contained a microsatellite motif. Of these 19 were duplicates or partial sequence duplicates, 51 had repeat motifs less than 16 bp long, 57 were too close to the cloning site and 13 had unsuitable flanking sequence to allow suc- cessful primer design. The remaining 91 loci were potentially suitable for primer design. Of these, 44 were chosen and primers synthesised (MWG-Bio- tech). To evaluate levels of polymorphism and interpretability of these loci the primer pairs were assessed initially against 8 individuals that encompassed the wide geographical distribution of this species. The microsatellite loci were amplified in 10 ll reactions [1· PCR buffer: 16 mM (NH 4 ) 2 SO 4 , 67 mM Tris–HCl (pH 8.8), 0.01% Tween-20; 2 mM MgCl 2 , 200 nM of each primer, 100 lM of each dNTP, 0.25 units Taq polymerase (Bioline), 1.0 lM F-dCTP and approximately 10 ng DNA] using the following PCR cycling conditions: 95 °C for 5 min followed by 30 cycles of 94 °C for 15 s, T an (°C) for 15 s, 72 °C for 30 s and a final cycle of 72 °C for 30 min. All reactions were performed in a GeneAmp Ò PCR System 2700 (Applied Biosys- tems). The microsatellites were then detected using an ABI 377 Prism genetic analyser by use of the incorporated fluorescent dCTPs (either [R6G] Conservation Genetics 5: 283–286, 2004. Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands. 283