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PLoS One. 2015 Sep 25;10(9):e0138888. doi: 10.1371/journal.pone.0138888. eCollection 2015.

Refining DNA Barcoding Coupled High Resolution Melting for Discrimination of 12 Closely Related Croton Species.

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Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.
State Key Laboratory of Genetic Resources and Evolution and Yunnan Laboratory of Molecular Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China; Science and Technology Research Institute, Chiang Mai University, Chiang Mai, 50200, Thailand.
Institute of Applied Biosciences, Centre for Research & Technology Hellas, Thessaloniki, Greece.
Department of Organismal Biology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, SE-75236 Uppsala, Sweden; The Natural History Museum, University of Oslo, P.O. Box 1172, NO-0318 Oslo, Norway.


DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Croton (Euphorbiaceae), one of the largest genera of plants with over 1,200 species. Seven primer pairs were evaluated (matK, rbcL1, rbcL2, rbcL3, rpoC, trnL and ITS1) from four plastid regions, matK, rbcL, rpoC, and trnL, and the nuclear ribosomal marker ITS1. The primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL1 primer pair gave the lowest resolution. It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Our Bar-HRM results here also provide further support for the hypothesis that both sequence and base composition affect DNA duplex stability.

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