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Protein Coding Low-Copy rpb2 and ef1-α Regions Are Viable Fungal Metabarcoding DNA Markers Which Can Supplement ITS for Better Accuracy

Summary

Researchers tested different DNA markers for identifying fungal species using DNA sequencing technology. They compared the standard fungal marker (ITS) with two alternative protein-coding markers (rpb2 and ef1-α) on closely related mushroom species. The results showed that using multiple markers together provides better and more reliable identification of fungal species compared to using just one marker, which is especially useful for environmental monitoring and species identification studies.

Background

The internal transcribed spacer (ITS) region is the universal fungal barcode marker, but it often lacks a significant barcoding gap between closely related species and has limitations including unequal copy numbers and intragenomic variability. Alternative protein-coding low-copy markers such as rpb2 and ef1-α have shown promise for fungal species identification in phylogenetic studies.

Objective

This study compared the performance of protein-coding low-copy genes (rpb2 and ef1-α) with the standard ITS1 and ITS2 regions for fungal metabarcoding in three mock communities of closely related agaric species (Dermoloma, Hodophilus, and Russula). The goal was to evaluate species recovery, data quality, filtering complexity, threshold consistency, and reference data applicability.

Results

ITS regions produced higher sequencing depth but required more sophisticated filtering due to higher chimera rates (2.5-3.7%) compared to protein-coding markers (0.5-0.7%). All four markers recovered similar species overall, but best-fitted clustering thresholds varied among genera except for ef1-α which showed consistent 99% threshold performance. Low-copy regions occasionally recovered species missed by ITS and confirmed OTU reliability when combined with ITS data.

Conclusion

Protein-coding markers rpb2 and ef1-α are viable alternatives for fungal metabarcoding that can supplement ITS for improved accuracy and species detection. A combined marker approach is recommended, especially for group-specific studies, as it improves taxonomic resolution and confirms clustering reliability while building reference databases for understudied fungal groups.
  • Published in:Ecology and Evolution,
  • Study Type:Experimental Comparison Study,
  • Source: 10.1002/ece3.71352, PMID: 40260151
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