A DNA barcode for land plants

Abstract

DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF-atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK-psbI spacer, and trnH-psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL؉matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.matK ͉ rbcL ͉ species identification L arge-scale standardized sequencing of the mitochondrial gene CO1 has made DNA barcoding an efficient species identification tool in many animal groups (1). In plants, however, low substitution rates of mitochondrial DNA have led to the search for alternative barcoding regions. From initial investigations of plastid regions (2-4), 7 leading candidates have emerged (5, 6). Four are portions of coding genes (matK, rbcL, rpoB, and rpoC1), and 3 are noncoding spacers (atpF-atpH, trnH-psbA, and psbK-psbI). Different research groups have proposed various combinations of these loci as their preferred plant barcodes, but no consensus has emerged (5-12). This lack of an agreed standard has impeded progress in plant barcoding.Our aim here is to identify a standard DNA barcode for land plants. To achieve this goal, we have pooled data across laboratories including sequence data from 907 samples, representing 445 angiosperm, 38 gymnosperm, and 67 cryptogam species. Using various subsets of these data, we evaluated the 7 candidate loci using criteria in the Consortium for the Barcode of Life's (CBOL) data standards and guidelines for locus selection (http:// www.barcoding.si.edu/protocols.html). Universality: Which loci can be routinely sequenced across the land plants? Sequence quality and coverage: Which loci are most amenable to the production of bidirectional sequences with few or no ambiguous base calls? Discrimination: Which loci enable most species to be distinguished? ResultsUniversality. Direct universality assessments using a single primer pair for each locus in angiosperms resulted in 90%-98% PCR and sequencing success for 6/7 regions. Success for the seventh region, psbK-psbI, was 77% (Fig. 1A). Greater problems were encountered in other land plant groups, with rpoB, matK, atpF-atpH, and psbK-psbI all showing Ͻ50% success in gymnosperms and/or cryptogams based on data compiled from several laboratories (Fig. 1 A).Sequence Quality. Evaluation of sequence quality and coverage from the candidate loci demonstrated that high quality bidirectional sequences were routinely obtained from rbcL, rpoC1, and rpoB (Fig. 1B, x axis). The remaining 4 loci required more manual editing and produced f...

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Utility of DNA barcoding to identify rare endemic vascular plant species in Trinidad

“…Three markers were assessed for species identification: the recommended two‐locus cpDNA barcode ( matK  +  rbcL ; CBOL Plant Working Group 2009), and one cpDNA regions ( rpoC1 ; Chase et al., 2005; Kress, Wurdack, Zimmer, Weigt, & Janzen, 2005; Table 2). PCR amplification reagents and thermal conditions were used according to the CBOL laboratory manual guidelines (http://www.barcoding.si.edu/ plant_working_group.html).…”

“…Selection of a plant DNA barcode must meet a number of criteria which have already been described elsewhere (Ford et al., 2009; Hollingsworth et al., 2009; Kress & Erickson, 2008; Li et al., 2015). The chloroplast ribulose‐1, 5‐bisphosphate carboxylase/oxygenase large subunit gene ( rbcL ) and maturase K gene ( matK ) are the approved barcodes for land plants (CBOL Plant Working Group 2009). However, plant‐plastid barcodes typically have lower resolving power to separate closely related plant species compared to the animal barcode, and in several cases, conspecifics or recently diverged species do not form highly supported, distinct sequence clusters that allow species discrimination (Hollingsworth et al., 2016; van Velzen, Weitschek, Felici, & Bakker, 2012; Zhang et al., 2012).…”

Phylogenetic congruence between subtropical trees and their associated fungi

“…For each of the 26 species, we searched GenBank (Benson et al., 2013) for four gene sequences: matK , rbcL , atpB, and 5.8S , which are the standardized DNA barcodes for land plants (CBOL Plant Working Group 2009) and are commonly used in published angiosperm phylogenies (e.g., Cadotte, Cardinale, & Oakley, 2008; Wojciechowski, Lavin, & Sanderson, 2004). Of the 26 species, 24 had at least one gene represented in GenBank.…”