msGBS: A new high‐throughput approach to quantify the relative species abundance in root samples of multispecies plant communities

Abstract

Plant interactions are as important belowground as aboveground. Belowground plant interactions are however inherently difficult to quantify, as roots of different species are difficult to disentangle. Although for a couple of decades molecular techniques have been successfully applied to quantify root abundance, root identification and quantification in multispecies plant communities remains particularly challenging. Here we present a novel methodology, multispecies genotyping by sequencing (msGBS), as a next step to tackle this challenge. First, a multispecies meta‐reference database containing thousands of gDNA clusters per species is created from GBS derived High Throughput Sequencing (HTS) reads. Second, GBS derived HTS reads from multispecies root samples are mapped to this meta‐reference which, after a filter procedure to increase the taxonomic resolution, allows the parallel quantification of multiple species. The msGBS signal of 111 mock‐mixture root samples, with up to 8 plant species per sample, was used to calculate the within‐species abundance. Optional subsequent calibration yielded the across‐species abundance. The within‐ and across‐species abundances highly correlated (R2 range 0.72–0.94 and 0.85–0.98, respectively) to the biomass‐based species abundance. Compared to a qPCR based method which was previously used to analyse the same set of samples, msGBS provided similar results. Additional data on 11 congener species groups within 105 natural field root samples showed high taxonomic resolution of the method. msGBS is highly scalable in terms of sensitivity and species numbers within samples, which is a major advantage compared to the qPCR method and advances our tools to reveal hidden belowground interactions.

Assembly history modulates vertical root distribution in a grassland experiment

“…Studying the extent to which the behaviour of the roots of species inhabiting plant communities changes as a function of the order of arrival of plants, as well as studying how these plastic root responses would be reflected at the community level, requires information on the distribution of roots at the species level, which unfortunately was not available in our study. Given the above, as well the inherent limitations of any rhizobox experiment, we see two main avenues for future research aimed at better understanding the roles played by priority effects in root dynamics and their consequences for species coexistence: (1) non-destructively monitoring root development at different soil depths in the field using minirhizotrons (Rewald and Ephrath, 2013;Freschet et al, 2020), and (2) quantifying species relative abundance in root samples taken from plant communities at different soil depths using state-of-the-art molecular techniques (Wagemaker et al, 2021). Only by going underground can we improve our mechanistic understanding of priority effects in plant communities and their implications for species coexistence.…”

Optimisation of root traits to provide enhanced ecosystem services in agricultural systems: A focus on cover crops

“…However, both methods have a relatively low throughput and require a labour‐intensive calibration that prevents their use in species‐rich mixtures (Rewald et al, 2012 ). Recently, a new high‐throughput next generation sequencing‐based method was developed to quantify species proportions in mixed root samples (Wagemaker et al, 2021 ). This method, referred to as multispecies genotyping by sequencing (msGBS), proved to be as accurate as the PCR‐based method of (Mommer et al, 2008 ).…”

“…It is also more sensitive and less labour‐intensive. In addition, it does not require the development of species‐specific DNA primers, which makes it possible to analyse root samples collected from species‐rich polycultures and measure traits such as rooting depth for each species individually (in‘t Zandt et al, 2020 ; Wagemaker et al, 2021 ). Root phenotyping in polycultures can also be facilitated using crop lines that were genetically modified to express a green or red fluorescent protein (Faget, Nagel, et al, 2013 ).…”

A snapshot of the root phenotyping landscape in 2021

“…This is particularly true for approaches relying on root observation windows (rhizoboxes) and minirhizotrons, 3D imaging, plant modelling, or (un)targeted metabolomics. Results from our survey also highlighted the popularity of next generation sequencing-based methods in root phenotyping, which have become methods of choice to characterise the plant and soil-associated microbiota (Schöler et al ., 2017) as well as to quantify the relative abundance of plant species in mixed root samples (Wagemaker et al ., 2021).…”

“…They include image-based approaches using scanners, cameras and microscopes, as well as chemical abundance measurements based on infrared gas analysis, chromatography and mass spectrometry (van Dam & Bouwmeester, 2016; Atkinson et al ., 2019; Wasson et al ., 2020). Next-generation sequencing-based methods have also become very popular for characterising root-associated microbiota (Hannula et al ., 2021) and quantifying species proportions in mixed root samples (Wagemaker et al ., 2021). The diversity of approaches used in root phenotyping is illustrated in Fig.…”