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A high-throughput belowground plant diversity assay using next-generation sequencing of the trnL intron

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Abstract

Aims

We refine and test a next-generation sequencing assay for the molecular identification and quantification of plant roots from mixed-species samples.

Methods

We modified primers targeting the trnL intron to provide greater taxonomic resolution and developed an improved bioinformatics pipeline that can identify roots based on global, site-, and plot-specific taxon lists. We tested our primers and pipeline on mock plant communities of known composition and mixed-species samples of roots collected from temperate grassland and high Arctic tundra communities.

Results

We retrieved a high correlation (0.72) between observed and expected community dissimilarities. We found positive linear relationships between aboveground and belowground species richness in the grassland community, with higher correlations for plot-specific reference databases (R = 0.70) than a global reference database (R = 0.48). This highlights the importance of local reference databases within the bioinformatics pipeline. Lower root than aboveground richness suggests that typical root lateral spread in this grassland is less than 25 cm. We observed lateral rooting extents of up to 40 cm and rooting depths of up to 30 cm for six high Arctic species.

Conclusions

Testing on mock community, grassland, and tundra samples demonstrated that next-generation sequencing using our improved trnL primers and pipeline can successfully recover plant root community structure.

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Acknowledgments

This work was funded by NSERC Discovery grants to SDS and EGL. Drs. Ben Raymond and John McKinlay (Australian Antarctic Division) and Dr. Shaun Neilsen (University of New South Wales, Sydney Australia) provided expertise in development of R code for the pipeline automation stages. The manuscript was substantially improved based on comments from Katherine Stewart and several anonymous reviewers. The authors declare no conflict of interest with this work.

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Authors and Affiliations

  1. Department of Plant Sciences, University of Saskatchewan, 51 Campus Dr., Saskatoon, SK, S7N 5A8, Canada

    E. G. Lamb, C. L. Piper & S. A. Freidrich

  2. Australian Antarctic Division, Department of the Environment, Kingston, Tasmania, 7050, Australia

    T. Winsley

  3. Department of Soil Science, University of Saskatchewan, 51 Campus Dr., Saskatoon, SK, S7N 5A8, Canada

    T. Winsley & S. D. Siciliano

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  1. E. G. Lamb
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  2. T. Winsley
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  3. C. L. Piper
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  4. S. A. Freidrich
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  5. S. D. Siciliano
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Correspondence to E. G. Lamb.

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Responsible Editor: Peter J. Gregory.

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Lamb, E.G., Winsley, T., Piper, C.L. et al. A high-throughput belowground plant diversity assay using next-generation sequencing of the trnL intron. Plant Soil 404, 361–372 (2016). https://doi.org/10.1007/s11104-016-2852-y

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