Isolation and characterization of 12 microsatellite loci in soapbark, Quillaja saponaria (Quillajaceae)1

Ap Applicati tions ons in in Pl Plant t Scien Sciences ces Quillaja saponaria Molina is an endemic Chilean tree, known as soapbark, soap bark tree, or quillay (as it is known in Chile). It is an evergreen tree of the family Quillajaceae (Kubitzki, 2007), found from the Coquimbo Region to Arauco Province in the Bío-Bío Region, approximately between 31 ° and 38 ° south (García and Ormazabal, 2008). It grows from sea level to 1600 m a.s.l., preferably in dry areas that are poor in nutrients. The family is monotypic with a single genus and two species from warm-temperate South America (Chile, Brazil, and northern Argentina). Quillaja saponaria is considered one of the most important and representative species of the sclero-phyllous forest from central Chile, and these communities are part of a biodiversity hotspot called the Chilean Winter Rain-Our purpose is to evaluate the effects of anthropogenic fragmentation on the patterns of genetic variation and connectivity in populations of Q. saponaria. For this reason, we isolated and characterized 12 nuclear microsatellite loci that are being successfully applied to describe spatial patterns of genetic structure. These are the fi rst microsatellite markers developed for a Quillaja Molina species. METHODS AND RESULTS Microsatellite isolation was performed by the simple sequence repeat (SSR) development company Genetic Marker Services (Brighton, United Kingdom; www.geneticmarkerservices.com). Genomic DNA was extracted from a single Q. saponaria (Qsa) individual collected in the locality of Coya, near the city of Rancagua, O'Higgins Region, Chile (locality 5 in Appendix 1), using a modifi ed cetyltrimethylammonium bromide (CTAB) protocol described by Doyle and Doyle (1990) , and used to develop an enriched library to isolate microsatellite (SSR)–containing loci. Enrichment involved incubat-ing adapter-ligated restricted DNA with fi lter-bonded synthetic repeat motifs:positive Escherichia coli JM109 clones were detected and sequenced, of which 23 contained exploitable repeat motifs with suffi cient fl anking regions to design forward/reverse primer pairs. The online primer design software Primer3 (Rozen and Skaletsky, 1999) was used to develop primer pairs amplifying fragments ranging in size from 100 to 250 bp, to help minimize later multiloading overlap ambiguities during sequencer genotyping. The primers were then tested for successful amplifi cation on one individual from each of seven populations, chosen to represent the whole latitudinal range of the spe-cies' distribution Appendix 1), using a touchdown PCR protocol. PCR amplifi cations were performed in a 25-μ L fi nal volume containing 7 …