Biases underlying species detection using fluorescent amplified-fragment length polymorphisms yielded from roots

Background: Roots of different plant species are typically morphologically indistinguishable. Of the DNA-based techniques, fluorescent amplified-fragment length polymorphisms (FAFLPs) are considered reliable, high throughput, inexpensive methods to identify roots from mixed species samples. False-negatives, however, are not uncommon and their underlying causes are poorly understood. We investigated several sources of potential biases originating in DNA extraction and amplification. Specifically, we examined the effects of sample storage, tissue, and species on DNA yield and purity, and the effects of DNA concentration and fragment size on amplification of three non-coding chloroplast regions (trnT-trnL intergenic spacer, trnL intron, and trnL-trnF intergenic spacer). Results: We found that sample condition, tissue and species all affected DNA yield. A single freeze-thaw reduces DNA yield, DNA yield is less for roots than shoots, and species vary in the amount of DNA yielded from extractions. The effects of template DNA concentration, species identity, and their interaction on amplicon yield differed across the three chloroplast regions tested. We found that the effect of species identity on amplicon production was generally more pronounced than that of DNA concentration. Though these factors influenced DNA yield, they likely do not have a pronounced effect on detection success of fragments and only underscore the restriction on the use of FAFLPs for measuring species presence rather than their abundance. However, for two of the regions tested-the trnT-trnL intergenic spacer and the trnL intron-size-based fragment competition occurred and the likelihood of detection was higher for smaller than larger fragments. This result reveals a methodological bias when using FAFLPs. Conclusions: To avoid potential bias with the use of FAFLPs, we recommend users check for the disproportionate absence of species detected belowground versus aboveground as a function of fragment size, and explore other regions, aside from the trnT-trnL intergenic spacer and trnL intron, for amplification.