Molecular Biology of Fungi

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Molecular biology of Fungi – Syllabus

Course Information – Spring 2019

Postgraduate program in Biological Sciences, UNAM

100 hours

Program (in Spanish)

Instructors
Camille Truong, UNAM

Roberto Garibay Orijel, UNAM

Andres Argüelles Moyao, UNAM

Rodolfo Ángeles Argáiz, UNAM

Location & Time
Instituto de Biología UNAM, intensive course 19 March to 12 April 2019

Objectives

Students will develop theoretical and technical abilities in molecular biology related to the systematics and evolution of fungi. Specifically they will acquire:

Fundamentals of DNA extraction, PCR amplification and sequencing
Laboratory skills to achieve efficient sample processing
Critical thinking in the creation and troubleshooting of laboratory protocols
Basic knowledge of DNA sequence analysis

Prerequisites

Having basic knowledge of molecular biology and a thesis project that includes a mycological aspect. Each student will bring at least 5 fungal samples (sporocarp, fungal culture or root tip) to be sequenced and analyzed during the course.

Topics covered
UNIT 1 – DNA extraction, molecular markers for fungi

1.1 Theory and laboratory practice

1.2 DNA extraction using phenol-chloroform methods

1.3 DNA extraction using commercial kits (columns)

1.4 Rapid DNA extraction methods

1.5 Primers: characteristics and design

1.6 Regions and primers for fungi

1.7 Barecodes and MOTUs in fungi

UNIT 2 – Polymerase chain reaction

2.1 Theory and laboratory practice

2.2 Gradient and touchdown PCR

2.3 Real-time PCR

2.4 Reverse transcription PCR

2.5 PCR troubleshooting

UNIT 3 – DNA sequencing

3.1 Theory and laboratory practice

3.2 DNA electrophoresis

3.3 Cleaning of PCR products

3.4 Sanger sequencing by capillary electrophoresis

3.5 Basics of NGS methods on Ion Torrent and Illumina platforms

UNIT 4 – Sequence data analysis

4.1 Sequence editing

4.2 BLAST

4.3 Uses of the NCBI, UNITE and BOLD databases

4.4 Sequence alignment

4.5 Basics of maximum likelihood and bayesian phylogenetic methods

4.6 Basics of genomic and meta-genomic pipelines

Evaluation

Participation in class and in the laboratory – 50%

Final report (theoretical basis, protocols, results and discussion) based on the samples that were analyzed during the course – 50%

Bibliography

Blaxter M, Mann J, Chapman T, Thomas F, Whitton C, et al. (2005) Defining operational taxonomic units using DNA barcode data. Phil. Trans. R. Soc. London B Biol. Sci. 360:1935-43.

Cubero OF, Crespo A, Fatehi J, Bridge PD (1999). DNA extraction and PCR amplification method suitable for fresh, herbarium-stored, lichenized, and other fungi. Pl. Syst. Evol. 216: 243-249.

Kleparnik K, Bocek P (2007) DNA diagnostics by capillary electrophoresis. Chem. Rev. 107: 5279-5317.

Lindahl BD, Nilsson RH, Tedersoo L, Abarenkov K, Carlsen T, et al. (2013) Fungal community analysis by high-throughput sequencing of amplified markers – a user’s guide. New Phytol. 199: 288–99.

Nilsson RH, Taylor AFS, Bates ST, Thomas D, Bengtsson-Palme, et al. (2013) Towards a unified paradigm for sequence-based identification of fungi. Mol Ecol. 22: 5271–77.

Nguyen NH, Smith D, Peay K, Kennedy P. (2015) Parsing ecological signal from noise in next-generation amplicon sequencing. New Phytol. 205: 1389–93.

Peay KG, Kennedy PG, Bruns TD (2008) Fungal community ecology: A hybrid beast with a molecular master. BioScience 58: 799-810.

Schoch CI et al., Fungal Barcoding Consortium (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. PNAS 109: 6241–6246.