Finding our roots: A PCR-based method for species identification in mixed root samples of grassland species

Species identification is critical for most ecological studies, and is typically achieved through the use of morphological keys. Unfortunately in some biological systems morphological keys are not easily applied. For example, fine roots of naturally co-occurring plant species are densely mixed and visually indistinguishable. However, in many plant systems, more than 60% of plant biomass occurs belowground, and as a result ecological interactions occur predominantly in the soil. A detailed understanding of how species interact belowground is therefore critical to our understanding of plant communities. DNA barcoding is applicable to situations where morphological differences are absent and offers a solution to this problem. However, DNA barcoding is not typically applied to mixed species samples, and methods for doing so are lacking. We developed a PCR based method that uses species-specific primers designed from databased plant barcode sequences to identify mixed roots of 10 co-occurring grassland plant species. We tested this method in two ways; 1) We identified species present in mixtures of DNA containing random combinations of 4, 6, 8 and 10 species. 2) We grew random combinations of 4 species in pots, and identified the species present in DNA extractions from mixed root subsamples. Using this method we were able to make reproducible positive species identifications 100% of the time. Application of this method will allow us to shed light on how belowground plant communities are structured, and how plant roots interact among species.