msGBS: A new high-throughput approach to quantify relative species
abundance in root samples of multi-species plant communities
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 multi-species plant communities remains particularly
challenging. Here we present a novel methodology, multi-species
Genotyping By Sequencing (msGBS), as a next step to tackle this
challenge. First, a multi-species 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
multi-species 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 analyze 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 the hidden belowground interactions.