title: Molecular identification of roots from a grassland community using size differences in fluorescently labelled PCR amplicons of three cpDNA regions.
abstract: Elucidating patterns of root growth is essential for a better understanding of the functioning of plant-dominated ecosystems. To this end, reliable and inexpensive methods are required to determine species compositions of root samples containing multiple species. Previous studies use a range of PCR-based approaches, but none have examined a species pool greater than 10 or 30 when evaluating mixed and single species samples, respectively. We present a method that evaluates size differences in fluorescently labelled PCR amplicons (fluorescent fragment length polymorphism) of the trnL intron and the trnT-trnL and trnL-trnF intergenic spacers. Amplification success of the trnT-trnL spacer was limited, but variation in the trnL intron and the trnL-trnF spacer was sufficient to distinguish over 80% of the 95 species (97% of the 77 genera) evaluated from a diverse fescue grassland community. Moreover, we identified species known to be present in mixed samples of 4, 8, 12, and 16 species on average 82% of the time. However, this approach is sensitive to detecting species known to be absent (false positives) when using our key of 95 species. Comparing unknowns to a limited species pool ameliorates this problem, comparable to a researcher using prior knowledge of what species could be found in a sample to constrain the identification of species. Comparisons to other methods and future improvements are discussed. This method is efficient, cost- effective and broadly applicable to many ecosystems.
authors.affiliation: Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9.
authors.forename: John M
authors.initials: JM
authors.lastname: Taggart
authors.forename: James F
authors.initials: JF
authors.lastname: Cahill
authors.forename: Gordon G
authors.initials: GG
authors.lastname: McNickle
authors.forename: Jocelyn C
authors.initials: JC
authors.lastname: Hall
mesh_terms: D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D018742:DNA, Chloroplast / Q000235:genetics*; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics; D018517:Plant Roots / Q000145:classification / Q000235:genetics; D006109:Poaceae / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length
chemical_list: D017931:DNA Primers; D018742:DNA, Chloroplast; D005456:Fluorescent Dyes; D010940:Plant Proteins