Course Goals: An introduction to microscopic and molecular methods used in the identification of mushrooms  

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Entry Level : Biological Sciences 101 or equivalent and Plant Pathology 135 or Plant Pathology 148
   
Texts: Hills, D. M., Moritz, C., and Mable, B. K., eds. 1996. Molecular Systematics, 2nd Edition. Sinauer Assoc., Inc. and Largent, D. L., Johnson, D., and Watling, R. 1987. How to Identify Mushrooms to Genus III: Microscopic Features. Mad River Press
   
Course Format:

One-day mandatory field trip and 3-hour discussion/laboratory per week. Students will collect wild mushrooms on the field trip and will be responsible for their identity with provided keys. Each student will provide thorough written and sketched descriptions of macroscopic and microscopic characteristics of two species. The students will also use molecular methods to further characterize their specimens. Procedures will include DNA extraction, PCR-amplification of nuclear ribosomal DNA, purification and quantification of the amplified product, digestion of the product with restriction enzymes, and DNA sequencing. The students will gain experience using SEQWeb (web-based sequence analysis) as a tool for mushroom identification by comparing sequences of their two specimens with those available in GenBank. Finally, a cursory phylogenetic analysis will be conducted with PAUP* (Phylogenetic Analysis Using Parsimony). Grades will be determined by completion of laboratory exercises and two exams.

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Topical Outline: 1.     Resources available for mushroom identification
2.   Species concepts in mycology
3.     Laboratory techniques used in the study of microscopic features
4.     Pelis, trama, cystidia, etc. characteristics
5.    Using the ITS regions of nuclear ribosomal DNA in mushroom systematics
6.     DNA extraction methods
7.     PCR amplification of ITS regions; product purification and quantification
8.     Restriction enzyme digestions
9.     Gene sequencing; analysis of sequences using SEQWeb and  sequence databases
10.  Generating phylogenetic trees with PAUP
   


Exercises:
(each submitted as a lab report, 10 points each)

  1. Photography and identification of two species of mushrooms
  2. Written description of macroscopic features and hand-drawn figures of spores (including ornamentation), pileipellis, cystidia, and gill trama, all to scale.
  3. Extraction of DNA, PCR amplification of a portion of the nuclear rDNA and electrophoresis of PCR product; photograph of gel will be submitted. 
  4. Purification of PCR products and their quantification by two methods: fluorometry and visual comparison of specific volumes of unknowns with standard concentrations of DNA.
  5. Restriction enzyme digestions of PCR products with Hae III and Nla III; photograph of gel will be submitted.
  6. Sequencing of PCR product. Printed sequences will be submitted. ITS regions will be highlighted on report.
  7. Exercises using SEQWEB:  Sequences from collected specimens will be compared to others from a BLAST search (the first five similar sequences will be submitted in the report); the location of restriction sites will be identified using MAP; and sequences of specimens will be compared with sequences of other mushrooms with PILEUP.
  8. A phylogeny of the mushroom specimens will be constructed with PAUP (other sequences will be provided).






   
Grading: Each exercise, 10 points; a midterm and final, each 10 points; total 100 points.