Epsilon Archive for Student Projects

Abstract

The fungal kingdom is a large group of eukaryotic organisms consisting of more than 100 000 known and an expected number of more than 1 million species with a wide variety of lifestyles. Among these lifestyles is a symbiotic lifestyle that has been of importance for lichen forming fungi. The lichens are a polyphyletic group consisting of around 18000 different species. These fungi belong to both the Ascomycota and Basidiomycota, with the majority of the lichen forming fungi being Ascomycota. Though lichens have been known of and studied since the 19th century, many of the basic biological features are unknown due to their cryptic nature. With new and modern methods, such as next generation sequencing (NGS) and barcoding, it has become possible and more available to study lichens in different substrates.
Different barcoding regions are used in the three major eukaryotic groups, animals, plants and fungi. The internal transcribed spacer (ITS) region is the adopted default barcoding region in fungi and has proven to be efficient in most taxa. In this study, the fungal ITS2 region is amplified with the primers gITS7, ITS4A and ITS4 from samples collected from naturally decaying Norway spruce logs. The aim of this study is to: (1) provide practical experience, (2) examine where the highest fungal DNA concentration in the logs is and (3) examine the similarity between the technical replicates.
In this experiment, 32 wood discs collected from 8 logs of Norway spruce in two forests in Arvidsjaur, Sweden by my supervisor Veera Tuovinen were examined. From 13 different places on the wood discs, drill-samples were taken and extracted for DNA. Two replicates of each drill- sample were taken. In addition, from each wood disc four biofilm samples were taken and DNA extracted. The samples were run in PCR with tagged primers for preparations to Illumina- sequencing. After the PCR, the samples were cleaned and the DNA-concentrations measured.
The concentration of fungal DNA in the log decreased from the edge to the center of the logs, being twice as high close to the edge compared to the center. The similarity between the two technical replicates was between 39-45% at the different sampling points. The highest similarity was at the outmost and the fourth outmost samples which were 45% similar and the lowest similarity was at the center samples with a similarity of 39%. The PCR-cycles used were also similar for all sampling points with 29 ± 2 cycles.
The distribution of the DNA concentration in the wooden disc could possibly be due to where the fungi get in contact and colonize the log. Mycelial growth into the heartwood will take more time and is likely to explain the lower DNA concentration there. Some fungi may through assistance by bark beetles or by colonizing highly degraded parts enter deeper part of the wood than what they normally tend to colonize.