A combined approach using RAPD, ISSR and volatile analysis for the characterization of Thymus caespititius from Flores, Corvo and Graciosa islands (Azores, Portugal) Helena Trindade * , Monya M. Costa, Sofia B. Lima, Luis G. Pedro, Ana C. Figueiredo, Jose ´ G. Barroso Universidade de Lisboa, Faculdade de Cieˆncias de Lisboa, DBV, IBB, Centro de Biotecnologia Vegetal, C2, Campo Grande,1749-016 Lisbon, Portugal article info Article history: Received 27 May 2009 Accepted 10 October 2009 Keywords: Thymus caespititius Lamiaceae Aromatic plants Molecular analysis RAPD ISSR Essential oils Volatiles abstract RAPDs and ISSRs were used as molecular markers and combined with the chemical analysis of the volatiles, aiming at characterizing Thymus caespititius from the islands of Corvo, Flores and Graciosa (Azores). Thirty-one individuals were analyzed using 17 RAPD and 11 ISSRs primers, which generated respectively 199 and 127 polymorphic scorable bands. Volatiles were isolated by distillation–extraction and analyzed by GC and GC–MS. Molecular data obtained from both markers discriminated assessments from Corvo and Flores which clustered separately from plants collected in Graciosa. Volatile data grouped plants in a similar way, giving rise to two groups, depending on their oil type; plants collected on Corvo and Flores yielded carvacrol-rich volatiles while those from Graciosa yielded both carvacrol and a-terpineol-rich oils. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction The genus Thymus (Labiatae/Lamiaceae) is comprised of important aromatic species, one of which is Thymus caespititius. This species is endemic of the NW Iberian Peninsula, occurring also on the islands of the Azores and Madeira archipelagos. The molecular and volatile characterization of T. caespititius from Azores has been performed previously on plants from Pico, Sa ˜o Jorge and Terceira islands using RAPD (Random Amplified Polymorphic DNA) as molecular markers and no straight corre- lation could be found between molecular markers and chemotypes in those assessments (Trindade et al., 2008). T. caespititius plants found in these islands belong to very distinctive chemotypes and it is not clear the reason for this chemical variability. In continuation of this characterization we collected plants from Corvo, Flores and Graciosa, which are locations where plants, based on previous studies on populations, were reported to be respectively carvacrol and a-terpineol chemotypes (Graciosa) or carvacrol-rich (Corvo and Flores) (Pereira et al., 2003; Santos et al., 2005). The study we present here was performed on individual plants and not in populations and uses as molecular markers both RAPD and ISSR (Inter Simple Sequence Repeat), in a joint chemical–volatile analysis. ISSR (Gupta et al., 1994) and RAPD (Williams et al., 1990) share in common some advantages, such as the high poly- morphism they generate and the fact they don’t require previous knowledge of the genome. Primers for ISSR are micro- satellite sequences which generate multilocus markers following a polymerase chain reaction (Reddy et al., 2002), while * Corresponding author. Tel.: þ351 217500199; fax: þ351 217500048. E-mail address: htrindade@fc.ul.pt (H. Trindade). Contents lists available at ScienceDirect Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco 0305-1978/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2009.10.006 Biochemical Systematics and Ecology 37 (2009) 670–677