PCR Techniques and Automated Sequencing in Lichens

Abstract

The Polymerase Chain Reaction (PCR) is a fairly simple but powerful technique that allows the amplification of small amounts of genetic material in vitro (Saiki et al. 1985). The versatility of this technique has made it a routine method in any molecular work and its application in evolutionary biology has led to important progress in understanding the phylogeny of organisms. Several studies have focused on the phylogeny of lichenized and non-lichenized ascomycetes or bacidiomycetes inferred from data of the nuclear small subunit ribosomal DNA (SSU rDNA; e.g., Gargas and Taylor 1992a, Gargas et al. 1995a, Lutzoni and Vilgalys 1995, Wedin et al. 1998). Studies on the SSU rDNA have also revealed that insertions are apparently more common in lichenized fungi than in many other organisms (e.g., Gargas et al. 1995b). Most studies on lichen-forming fungi were facilitated by the design of specific primers that do not amplify algal DNA (e.g., Gargas and Taylor 1992b). During the last few years, studies on the internal transcribed spacer regions (ITS1 and ITS2) and the large subunit (LSU) rDNA have become more common. The ITS regions and some parts of the LSU are generally more variable than the SSU, and sequences from these regions have been used to study phylogenies within and between genera (e.g., Arup and Grube 1998, Crespo and Cubero 1998, Groner and LaGreca 1997) and at the population level (DePriest 1994).