Experimental quantification of pollen with DNA metabarcoding using ITS1 and trnL

Sandra Baksay,Nathalie Escaravage,André Pornon,Christophe Andalo,Monique Burrus,Jérôme Mariette

doi:10.1038/s41598-020-61198-6

Abstract

Although the use of metabarcoding to identify taxa in DNA mixtures is widely approved, its reliability in quantifying taxon abundance is still the subject of debate. In this study we investigated the relationships between the amount of pollen grains in mock solutions and the abundance of high-throughput sequence reads and how the relationship was affected by the pollen counting methodology, the number of PCR cycles, the type of markers and plant species whose pollen grains have different characteristics. We found a significant positive relationship between the number of DNA sequences and the number of pollen grains in the mock solutions. However, better relationships were obtained with light microscopy as a pollen grain counting method compared with flow cytometry, with the chloroplastic trnL marker compared with ribosomal ITS1 and with 30 when compared with 25 or 35 PCR cycles. We provide a list of recommendations to improve pollen quantification.

Highlights

The use of metabarcoding to identify taxa in DNA mixtures is widely approved, its reliability in quantifying taxon abundance is still the subject of debate
Our main objectives were to investigate, for pollen quantities in the range of insect pollen loads involved in plant pollination: (1) whether using flow cytometry on extracted solutions provides more accurate estimates of pollen grain abundance than light microscopy, and a stronger relationship between the number of pollen grains and sequence abundance; (2) how the relationship is affected by the number of PCR cycles, the type of markers and plant species with different pollen characteristics
56.68% were assigned to Hippeastrum sp. (HIP) and 43.32% to Chrysanthemum sp. (CHR)

Summary

Introduction

The use of metabarcoding to identify taxa in DNA mixtures is widely approved, its reliability in quantifying taxon abundance is still the subject of debate. Many studies which investigated artificial[26,30,31] or natural pollen mixtures[17] used samples with huge pollen concentrations (from 30,000 to potentially more than 1,000,000 pollen grains) stored in a single bee corbicula[30,31] or several grams of pollen[26,27] Such large quantities of pollen could release large amounts of DNA polymerase inhibitors[32] and cause PCR dysfunction. Our main objectives were to investigate, for pollen quantities in the range of insect pollen loads involved in plant pollination: (1) whether using flow cytometry on extracted solutions provides more accurate estimates of pollen grain abundance than light microscopy, and a stronger relationship between the number of pollen grains and sequence abundance; (2) how the relationship is affected by the number of PCR cycles, the type of markers and plant species with different pollen characteristics

Methods

Results

Conclusion