Molecular Identification of Boreal Forest Roots: An Expansion of Techniques and Investigation of Limitations and Biases

Plant identification is a fundamental ecological tool. While identifying flowers and leaves is relatively straightforward, identifying roots can be difficult. Here, I expand the use of fluorescent amplified fragment length polymorphisms (FAFLPs) as a tool to identify roots. Using this molecular tool, I examine possible limitations of identifying a large set of boreal plant species and compare the utility of FAFLPs to DNA barcoding. In addition, I address some challenges specific to belowground detection of roots, namely, the influence of species and root size class. To identify roots, fragment lengths of three non-coding cpDNA regions, the trnT-trnL intergenic spacer, trnL intron, and trnL-trnF intergenic spacer, were resolved using capillary electrophoresis for 194 plant species common to the Canadian boreal forest. To determine whether DNA sequencing increases successful identification of closely related species, Sanger sequencing of the trnL intron of a subset of 24 species across nine genera was compared to FAFLPs. FAFLPs produced unique size profiles for 74% of species using all three cpDNA regions. In contrast, only 27 species (14%) could be identified using the relatively conserved trnL intron alone. DNA sequencing did not increase detection success: eight (33%) species could be distinguished by sequences of the trnL region, nine (38%) by fragment lengths of the same region. Fifteen (63%) congeneric species could be distinguished by FAFLPs of all three regions. Fine roots yielded higher DNA concentrations as well as higher DNA purity than larger root classes. Fine roots of the grass species Poa pratensis, produced the highest yield and quality of DNA. This suggests that false-positives in belowground assays of roots may most likely to occur from fine roots of specific species. Overall, I found that molecular tools can be effective in identifying roots, but FAFLPs and DNA sequencing have strengths and limitations, and more assumptions of the methods presented here need to be tested before accurate multiplexing of roots from large species pools can occur.

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