Clone Based Sequence for the Identification and Phylogenetic Study of Lichenized Fungi: A Case Study from Usnea

Abstract

Background: Usnea is a large genus in the family Parmeliaceae, with more than 350 species widely distributed in polar, temperate and tropical regions. Aim: The Usnea genus is recognized based on the fruticose thallus, branches with a cartilaginous central axis and usnic acid in the cortex. Materials and Methods: The phylogenetic relationships and the morphological variation among Usnea species have studied. The morphological characters traditionally used for species recognition of several European Usnea species analysed regarding their reliability. The evolution and distribution of the morphological characters looked to a phylogeny based on sequence data. It is easiest to obtain sequences from fresh Usnea material (not older than five years). DNA from the central axis has extracted to minimize the risk of contamination with lichen parasites. Since lichens are a combination of multiple organisms (fungi and algae), obtaining a single organism's DNA is difficult. These lichenized organisms cannot quickly be grown in axenic culture or manually teased apart from their associated microbial communities. It is a common phenomenon to observe multiple DNA bands following PCR caused by DNA from various organisms. Results: There is a greater chance of failure in the sequencing process. The cloning approach is the best one to check the sequence. To do the cloning, consider purifying at least five bands separately from the gel electrophoresis. Thus, get five clones to get an accurate picture of which sequences contained in the DNA. That way, even if the sequencing fails, you will still have the cloned products as a backup. Thus, clone-based sequencing is more efficient than that traditional sequencing methods. Conclusion: In the present study, we use this method for the phylogenetic interpretation of the genus Usnea and the results compared with the global data sets.