1. Fungus Among Us at Brookside County Park
Julia Kaczmarek and Emily Manning assisted by Mrs. Maria Brown
Abstract
Materials & Methods
There are 5.1 million species of fungi in the world. We collected 18 different fungi samples from Brookside County Park which were all thought to be different species based on morphology. We used the Cold Spring Harbor Barcode Long Island Footlocker Method for DNA extraction and the Fungi ITSIF/ITS4 primers for PCR . We did not get successful results of our fungi with this method and could not send samples to the sequencing lab. In the future we would use the Qiagen™ Dneasy Blood & Tissue Kit for DNA extraction as we observed that the silica seems to dry out very quickly and is likely impacting our DNA extraction process. Also in the future we would experiment with a different primer for our fungi samples which will be determined after a comprehensive literature review.
Primers: Fungi cocktail (ITSIF/ITS4)
Thermal Profile for PCR (35 cycles):
Initial step: 94 degrees Celsius, 1 minute
Denaturing step: 94 degrees Celsius
Annealing step: 55 degrees Celsius
Extending step: 72 degrees Celsius
Introduction
It is difficult to accurately identify fungi using standard taxonomic keys to species level, but experienced mycologists can do a fairly good job using a good microscope to carefully examine structures associated with the carposphore (Pacione, 1981).
It therefore, makes sense that barcoding could improve identification to species level since barcoding is an approach to rapidly identify species using short, standard genetic markers. Dentinger, Didukh, & Moncalvo (2011) demonstrated through 167 partial cytochrome oxidase I (COI) gene sequences, that COI may not be the best choice for a marker for fungi.
The ITS regions the most frequently sequenced genetic marker for fungi and it is routinely used to identify species at or below the species level (Begerow, Nilsson, & Maier, 2010).
Figure 2: Methods schematic showing basic steps in the experimental design with an overview of the Footlocker Method Protocols for Barcode Long Island, the DNA Subway pathway for the Blue Line and selected photos of fungus collected in the field.
Discussion
Our samples were not successful . We think that the Footlocker Method may have had some issues as the silica dried out quickly after pickup from DNALC in our refrigerater at the high school.
Salt spray on the fungi we collected as a result of our proximity to the bay may have interfered with the process, but we have not been able to find literature to support that theory.
Future Work
Figure 1: Green’s Creek watershed showing the area at Brookside County Park where fungi samples were collected within the palustrine forested wetlands (circled).
Collect new samples and wash them completely to remove salts prior to DNA extraction.
Use Qiagen™ Dneasy Blood & Tissue Kits for DNA extraction.
Goals
References
Ainsworth, A.M., Cannon, P.F., & Dentinger, T.M. (2013). DNA barcoding and morphological studies reveal two new species of waxcap mushrooms (Hygrophoraceae) in Britain. MycoKeys 7, pp
Begerow, D., Nilsson, R.H., Unterseher, M. & Maier, W. (2010). Current state and perspectives of fungal DNA barcoding and rapid identification procedures. Appl Microbiol Bitechnol 3 (34), Springer.
Biosphere. (2007). New York: Facts On File. Retrieved November 15, 2015, from of fungi.
Blackwell, M. (2011). The Fungi: 1,2,3….5.1 million species? Am. J. Bot. 98 (3);
Chase, M.W. & Fay, M.F. (2009). Barcoding of plants and fungi. Ecology 33, pp
Cold Spring Harbor Laboratory (2014). Using DNA Barcodes to Identify and Classify Living Things. pp1-41.
Dentinger, B.T., Didukh, M.Y., & Moncalvo, J.M. (2011). Comparing COI and ITS as DNA barcode markers for mushrooms and allies (Agaricomycotina). PLoS One 6 (9).
Pacione, G. (1981). Guide to mushrooms. Simon & Schuster. New York, New York.
Use barcoding techniques to confirm mushroom species that cannot be identified using standard taxonomic keys.
Identify closely matched barcodes in Genbank using the DNA Subway Blue Line and bioinformatics techniques identified in Using DNA Barcodes to Identify and Classify Living Things.
Acknowledgements
We would like to thank our parents for supporting us throughout this year. We would also like to thank Mrs. Brown for helping and supporting us all year long and making sure we do our best. Thank you to the staff at DNALC for providing this opportunity for us!