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Experimental quantification of pollen with DNA metabarcoding using ITS1 and trnL.

Sandra Baksay1, André Pornon2, Monique Burrus2

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Summary

Metabarcoding reliably quantifies pollen abundance when using light microscopy and the trnL marker. Optimizing PCR cycles to 30 improves accuracy in DNA-based pollen analysis.

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Area of Science:

  • Ecology
  • Molecular Biology
  • Bioinformatics

Background:

  • Metabarcoding is widely used for identifying taxa in DNA mixtures.
  • Its accuracy in quantifying taxon abundance, particularly for pollen, remains debated.
  • Factors influencing quantification reliability need further investigation.

Purpose of the Study:

  • To assess the relationship between pollen grain counts and high-throughput sequencing reads.
  • To evaluate how different methodologies affect pollen quantification accuracy.
  • To provide recommendations for improving pollen quantification using metabarcoding.

Main Methods:

  • Created mock solutions with varying amounts of pollen grains.
  • Compared pollen counting methods: light microscopy and flow cytometry.
  • Tested different polymerase chain reaction (PCR) cycle numbers (25, 30, 35).
  • Evaluated two DNA markers: chloroplast trnL and ribosomal ITS1.
  • Analyzed data considering different plant species' pollen characteristics.

Main Results:

  • A significant positive correlation was found between DNA sequence abundance and pollen grain number.
  • Light microscopy yielded better quantification accuracy than flow cytometry.
  • The chloroplast trnL marker showed a stronger relationship than the ribosomal ITS1 marker.
  • 30 PCR cycles resulted in more accurate quantification compared to 25 or 35 cycles.

Conclusions:

  • Metabarcoding can reliably quantify pollen abundance under optimized conditions.
  • Methodological choices (counting technique, marker, PCR cycles) significantly impact results.
  • Recommendations are provided to enhance the precision of pollen quantification in ecological studies.