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Experimental quantification of pollen with DNA metabarcoding using ITS1 and trnL.
Sandra Baksay1, André Pornon2, Monique Burrus2
1Laboratoire Evolution and Diversité Biologique EDB, CNRS, UMR 5174, Université Toulouse III Paul Sabatier, F-31062, Toulouse, France. sandra.baksay@gmail.com.
Scientific Reports
|March 8, 2020
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.
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.
