Looking for Hidden Enemies of Metabarcoding: Species Composition, Habitat and Management Can Strongly Influence DNA Extraction while Examining Grassland Communities.

Anna Rucińska,Arkadiusz Nowak,Marcin Nobis,Grzegorz Kusza,Szymon Zubek,Marcin Olszak,Sebastian Świerszcz,Maja Boczkowska

doi:10.3390/biom11020318

Abstract

Despite the raising preoccupation, the critical question of how the plant community is composed belowground still remains unresolved, particularly for the conservation priority types of vegetation. The usefulness of metabarcoding analysis of the belowground parts of the plant community is subjected to a considerable bias, that often impedes detection of all species in a sample due to insufficient DNA quality or quantity. In the presented study we have attempted to find environmental factors that determine the amount and quality of DNA extracted from total plant tissue from above- and belowground samples (1000 and 10,000 cm2). We analyzed the influence of land use intensity, soil properties, species composition, and season on DNA extraction. The most important factors for DNA quality were vegetation type, soil conductometry (EC), and soil pH for the belowground samples. The species that significantly decreased the DNA quality were Calamagrostis epigejos, Coronilla varia, and Holcus lanatus. For the aboveground part of the vegetation, the season, management intensity, and certain species—with the most prominent being Centaurea rhenana and Cirsium canum—have the highest influence. Additionally, we found that sample size, soil granulation, MgO, organic C, K2O, and total soil N content are important for DNA extraction effectiveness. Both low EC and pH reduce significantly the yield and quality of DNA. Identifying the potential inhibitors of DNA isolation and predicting difficulties of sampling the vegetation plots for metabarcoding analysis will help to optimize the universal, low-cost multi-stage DNA extraction procedure in molecular ecology studies.

Highlights

Model Performance highest relative influence was recorded for vegetation type (55.7%), electrical conductivity
The model revealed that DNA with the lowest contamiA230, both for below- and aboveground biomass (85.8 and 69.2%, respectively), quality nation of carbohydrates was in the belowground plant material taken from the dry and wet
The lowest values of deFor the ratio A260–A280, the most parsimonious model contains four predictors, with viance explained were observed for DNA quality A260–A230 for belowground biomass the largest relative influence recorded for soil group predictors, obtaining 90.5% in total, and

Summary

Introduction

Biomolecules 2021, 11, 318 differentiation, biotic interactions, environmental filtering, species coexistence, functional diversity or typology of plant communities, as well as temporal and spatial changes in vegetation, or the influence of environmental variables on vegetation, considers only the aboveground components of the plant communities as a representative diversity measure e.g., [1,2,3,4,5]. In many ecosystems, in stressful habitats, the absolute plant richness and majority of biomass (e.g., 50–90% or even more) is located belowground as roots, bulbs, rhizomes, and shoot bases [6]. This is supposed to be due to persistent belowground meristems, enabling dormancy in the soil without producing aboveground shoots [7,8]. Plants can spread roots farther than shoots [9], via stolons or rhizomes [10], resulting in overlapping root systems and increased species coexistence belowground

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