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...
Ford, C. S., Ayres, K. L., Toomey, N., Haider, N., Stahl, J. V., Kelly, L. J., Wikstrom, N., Hollingsworth, P. M., Duff, R. J., Hoot, S. B., Cowan, R. S., Chase, M. W., Wilkinson, M. J. (2009). Selection of candidate coding DNA barcoding regions for use on land plants. Botanical Journal of the Linnean Society, 159, (1), 1-11. Sponsorship: Alfred P. Sloan Foundation Gordon and Betty Moore Foundation IMPF: 00.98 RONO: 00An in silico screen of 41 of the 81 coding regions of the Nicotiana plastid genome generated a shortlist of 12 candidates as DNA barcoding loci for land plants. These loci were evaluated for amplification and sequence variation against a reference set of 98 land plant taxa. The deployment of multiple primers and a modified multiplexed tandem polymerase chain reaction yielded 85?94% amplification across taxa, and mean sequence differences between sister taxa of 6.1 from 156 bases of accD to 22 from 493 bases of matK. We conclude that loci should be combined for effective diagnosis, and recommend further investigation of the following six loci: matK, rpoB, rpoC1, ndhJ, ycf5 and accD.Peer reviewe
Fitness of hybrids between genetically modified (GM) crops and wild relatives influences the likelihood of ecological harm. We measured fitness components in spontaneous (non-GM) rapeseed x Brassica rapa hybrids in natural populations. The F1 hybrids yielded 46.9% seed output of B. rapa, were 16.9% as effective as males on B. rapa and exhibited increased self-pollination. Assuming 100% GM rapeseed cultivation, we conservatively predict < 7000 second-generation transgenic hybrids annually in the United Kingdom (i.e. approximately 20% of F1 hybrids). Conversely, whilst reduced hybrid fitness improves feasibility of bio-containment, stage projection matrices suggests broad scope for some transgenes to offset this effect by enhancing fitness.
We estimate the global BOLD Systems database holds core DNA barcodes (rbcL + matK) for about 15% of land plant species and that comprehensive species coverage is still many decades away. Interim performance of the resource is compromised by variable sequence overlap and modest information content within each barcode. Our model predicts that the proportion of species-unique barcodes reduces as the database grows and that ‘false’ species-unique barcodes remain >5% until the database is almost complete. We conclude the current rbcL + matK barcode is unfit for purpose. Genome skimming and supplementary barcodes could improve diagnostic power but would slow new barcode acquisition. We therefore present two novel Next Generation Sequencing protocols (with freeware) capable of accurate, massively parallel de novo assembly of high quality DNA barcodes of >1400 bp. We explore how these capabilities could enhance species diagnosis in the coming decades.
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