Expanding and testing fluorescent amplified fragment length polymorphisms for identifying roots of boreal forest plant species

Abstract

Premise of the Study Identifying roots to species is challenging, but is a common problem in ecology. Fluorescent amplified fragment length polymorphisms ( FAFLP s) can distinguish species within a mixed sample, are high throughput, and are inexpensive. To broaden the use of this tool across ecosystems, unique size profiles must be established for species, and its limits identified. Methods Fragments of three noncoding cp DNA regions were used to create size profiles for 193 species common to the western Canadian boreal forest. We compared detection success among congeners using FAFLP s and Sanger sequencing of the trnL intron. We also simulated and experimentally created communities to test the influence of species richness, cp DNA regions used, and extraction/amplification biases on detection success. Results Of the 193 species, 54% had unique size profiles. This value decreased when fewer cp DNA regions were used. In simulated communities, ambiguous species identifications were positively related to the species richness of the community. In mock communities, some species evaded detection owing to poor extraction or amplification. Sequencing did not increase detection success compared to FAFLP s for a subset of 24 species across nine genera. Discussion We recommend FAFLPs are best suited to confirm rather than discover species occurring belowground.

Shifts in ectomycorrhizal exploration types parallel leaf and fine root area with forest age

“…The trn T‐ trn L intergenic spacer, the trn L intron and the trn L‐ trn F intergenic spacer were targeted and amplified with primers (A2: FAM, C: VIC, E: NED) developed by Taberlet, Gielly, Pautou, and Bouvet (1991) and modified by Cronn, Small, Haselkorn, and Wendel (2002). DNA fragment lengths were obtained following Metzler, La Flèche, and Karst (2019). Resolved fragment lengths were compared to those developed by Metzler et al.…”

“…DNA fragment lengths were obtained followingMetzler, La Flèche, and Karst (2019). Resolved fragment lengths were compared to those developed byMetzler et al (2019) to assign root identities to species. To confirm the identity of roots classified as 'pine,' DNA was extracted from 30 root tips using Extract-N-Amp solution (Sigma-Aldrich, Inc.).…”

“…Again, the trnT-trnL, trnL intron and trnL-trnF regions were targeted with primers specified above. DNA was amplified, amplicon fragment lengths were obtained and species were identified followingMetzler et al (2019; Supporting Information, S2.1).…”

Botanical microbiomes on the cheap: Inexpensive molecular fingerprinting methods to study plant‐associated communities of bacteria and fungi

“…Although LH‐PCR products can be separated using polyacrylamide gel electrophoresis (as in SSCP or DGGE), the resolution, reproducibility, and analysis time is much improved by instead using fluorophore‐tagged primers to create fluorescent amplicons that are analyzed using capillary electrophoresis and laser detection in DNA sequencing machines. This technique has also been called fluorescent amplified fragment length polymorphism, and is used for identifying plant roots in soil samples (Metzler et al., ). The most common variant of LH‐PCR for studying microbiomes is ARISA.…”

Methods in belowground botany

“…Fluorescent amplified fragment length polymorphisms (FAFLPs) are a low-cost, high-throughput method for identifying multiple species in a single sample. Here, Metzler et al (2019) test the efficacy of FAFLPs for identifying mixed samples of roots and provide new size profiles for 193 species, effectively doubling the previous number of available reference species. They find that the FAFLP method can be as effective or more effective in detecting species than traditional Sanger sequencing, although ambiguous species identifications in mock communities increase with species richness.…”