Abstract Detail

Mycology

Olds, Gary [1], Berta-Thompson, Jessie W. [2], Loucks, Justin [2], Levy, Rick [2], Wilson, Andrew [3].

Applying a Modified Metabarcoding Approach for the Sequencing of Macrofungal Specimens.

Natural history collections, such as fungaria, contain thousands of specimens that can be used to explore patterns and processes in diversity. This is especially relevant to fungaria because the taxonomic study of fungi is complicated due to the cryptic nature of fungal species and the need to work with specimens to describe new taxa. However, the potential of fungaria remains largely untapped due to the need and expense of producing DNA sequence data. Sanger sequencing is the traditional method for generating DNA barcode sequence data in fungi. However, this method can be costly both financially and in effort, and does not scale well with increased numbers. A cost-effective method is needed for efficient sequencing of specimens on a large scale. Metabarcoding methods are currently able to multiplex samples and produce high-quality nrITS2 sequence data (part of the fungal DNA barcode) using Illumina MiSeq. This study applies a modified metabarcoding sequencing approach for fungi to produce a labor- and cost-effective solution for sequencing macrofungal collections. A two-step metabarcoding approach was used with nested barcodes. PCR1 uses eight unique barcodes to pool specimens for PCR2, which adds Illumina paired indexes. All PCR2 product is then pooled, sequenced, and demultiplexed. Over 768 specimens were selected to examine efficacy across broad taxonomic scales in Basidiomycota and Ascomycota, and detect interspecific diversity via operational taxonomic units. Demultiplexing and sequence quality is evaluated. Sequence data is compared to BLAST and Sanger produced sequences to demonstrate efficacy of the protocol. The results show these methods hold promise for generating OTU sequence data to study taxonomic diversity across large numbers of macrofungal specimens in fungaria.

1 - Denver Botanic Gardens, Research & Conservation, 1007 York St, Denver, CO, 80206, United States
2 - Denver Botanic Gardens, 1007 York St, Denver, CO, 80206, United States
3 - Denver Botanic Gardens, Research And Conservation, 909 York St, Denver, CO, 80206, United States

Keywords:
fungi
Genetics
Fungal diversity
Fungarium
high-throughput Illumina MiSeq
Systematics
Sanger sequencing
Mycology.

Presentation Type: Poster
Session: MYP1, Mycology Posters I
Location: /
Date: Monday, July 19th, 2021
Time: 5:00 PM(EDT)
Number: MYP1013
Abstract ID:863
Candidate for Awards:MSA Best Poster Presentation Award by a Graduate Student