Content area
Full text
The Smilacaceae is grouped within the Monocotyledoneae of the Liliales and has only two genera: Smilax L., with 300 species, and Heterosmilax Kunth, with 15 species (Angiosperm Phylogeny Group III, 2009). The family is distributed worldwide and is composed mainly of herbaceous vines and shrubs, and rarely of subshrubs and dioecious species. In Brazil, Smilax comprises 31 species, 14 of which are exclusively Brazilian (Andreata, 1997). Smilax species, which are popularly known as sarsaparilla, are used in folk medicine as tonics, antirheumatics, and antisyphilitics and are sold in Brazilian pharmacies without any quality control over their origin and effectiveness (Andreata, 1997). The quality control of herbal drugs should be more stringent, and molecular markers may be useful tools for the identification of species sold in pharmacies. Thus, the aim of the current study was to isolate and characterize microsatellite markers to identify Smilax species.
METHODS AND RESULTS
Genomic DNA was extracted from fresh leaves of S. brasiliensis Spreng., S. campestris Griseb., S. cissoides Mart. ex Griseb., S. fluminensis Steud., S. goyazana A. DC., S. polyantha Griseb., S. quinquenervia Vell., S. rufescens Griseb., S. subsessiliflora Duhamel, and S. syphilitica Humb. & Bompl. ex Willd. using the cetyltrimethylammonium bromide (CTAB) protocol described by Doyle and Doyle (1990) with modifications. The plant samples were registered (Appendix 1) and added to the plant collection of the Herbarium of the Escola Superior de Agricultura “Luiz de Queiroz” (ESA) of the Universidade de São Paulo, Brazil, and the Herbarium “Coleção de Plantas Medicinais e Aromáticas” (CPMA) of the Universidade Estadual de Campinas, Brazil.
A microsatellite‐enriched library was obtained using protocols adapted from Billotte et al. (1999). Genomic DNA from one individual of S. brasiliensis (Campina Verde, Minas Gerais) was digested with AfaI (Invitrogen, Carlsbad, California, USA) and enriched in microsatellite fragments using (CT)8 and (GT)8 motifs. Microsatellite‐enriched DNA fragments were ligated into pGEM‐T Easy Vectors (Promega Corporation, Madison, Wisconsin, USA), which were used to transform Epicurian Coli XL1‐Blue Escherichia coli competent cells (Promega Corporation). Positive clones were selected using the β‐galactosidase gene and grown overnight with ampicillin. The sequencing reactions (10 μL) contained 200 ng of plasmid DNA, 0.5 pmol SP6 primer, 0.4 μL of BigDye Terminator mix...