Polymerase chain reaction restriction fragment length polymorphism identification of trebouxia lichen photosymbionts

Lichens are defined as symbiotic associations composed of a fungal partner, the mycobiont, and one or more photosynthetic partners, the photobiont (1). A currently employed method for the identification of photobionts is the culture of photobiont from the lichen, but this method employs a labor intensive and long cultivation period, thus identification has been neglected. Out of the approximatelyl4,000 lichen described, only about 4% oflichen photobionts have been identified to species (1). In this study we investigated the feasibility of developing a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) identification system for rapidly identifying lichen algae of the genus Trebouxia (In this study we consider Pseudo-Trebouxia part of the genus Trebouxia). DNA was isolated and purified from cultures of each Trebouxia species. A 1300 hp fragment of the 5\u27 region of the nuclear-encoded large subunit (26S) ribosomal RNA genes was amplified by PCR (2). This 5\u27region of the 26S region is considered to be a byper-variable region because it differs amongst Trebouxia (2) making it a good candidate for RFLP. The sequences were then analyzed with restriction analysis software to determine restriction maps and individual virtual RFLP patterns. Patterns were constructed using the program SPR Opt (SNP and PCR-RFLP Optimization) allowing each Trebouxia species to be identified by a distinctive restriction pattern. We were unable to validate the key due to contamination of materials, which lead to inconclusive data. Future experiments aim to validate the key by comparing the virtual RFLP patterns to the actual patterns obtained for each type culture of each species