Simultaneous Metabarcoding and Quantification of Neocallimastigomycetes from Environmental Samples: Insights into...

Area of Science:

• Microbiology
• Mycology
• Molecular Biology

Background:

• Anaerobic fungi (Neocallimastigomycetes) are key lignocellulose degraders in herbivore guts with biotechnological potential.
• Current molecular detection methods lack specificity, hindering evolutionary and ecological research on these fungi.

Purpose of the Study:

• To develop and validate a Neocallimastigomycetes-specific PCR primer pair for accurate molecular detection.
• To enable improved screening, quantification, sequencing, and phylogenetic analysis of anaerobic gut fungi.

Main Methods:

• Designed and tested a PCR primer pair targeting the D2 region of the ribosomal large subunit.
• Evaluated primer specificity and performance in silico, in vitro (pure cultures), and on environmental samples.
• Utilized short-read high-throughput sequencing and qPCR assays.

Main Results:

• The primer pair demonstrated high specificity across known Neocallimastigomycetes genera and species.
• Successfully amplified and sequenced anaerobic gut fungi from diverse herbivore fecal samples.
• Identified new Neocallimastigomycetes clades, including potential novel lineages from rodent microbiomes.

Conclusions:

• The developed primer pair is a valuable tool for Neocallimastigomycetes research, facilitating ecological and evolutionary studies.
• The method allows for robust phylogenetic differentiation and discovery of novel anaerobic gut fungal diversity.