Evaluation of Potential DNA Barcodes for Identifying Thunbergia spp.
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Abstract
Introduction: A number of Thunbergia species are widely used as medicinal plants. Identifi -
cation by traditional morphological methods is diffi cult especially when specimens are commercial
herbal products, which morphology is unattainable. To facilitate the identifi cation process, this study
searched for potential DNA barcodes by evaluating three DNA loci, the chloroplast psbA-trnH and
trnL-trnF intergenic spacers and the second nuclear internal transcribed spacer (ITS2), among eight
species of Thunbergia. Methods: Plant samples were collected and fresh leaves were used for
genomic DNA extractions. Polymerase chain reaction amplifi cations of the psbA-trnH, trnL-trnF and
ITS2 regions were performed. The PCR products were sequenced and verifi ed by BioEdit v7.0.8 and
Clustal W programs. Kimura 2-Parameter (K2P) were computed via MEGA 6.06 to reveal the genetic
divergence. Results: Three candidate regions of all Thunbergia DNA samples were successfully
amplifi ed with the rate of 100 %. These amplifi ed products were also successfully sequenced (100%)
in both directions. The average interspecifi c divergence values calculated from K2P for psbA-trnH,
trnL-trnF, and ITS2 were 0.088, 0.054 and 0.121, respectively, implying that each Thunbergia spp.
could be discriminated from one another. Conclusion: This study confi rmed the universality of the
primers used for psbA-trnH, trnL-trnF and ITS2 PCR amplifi cation and sequencing. The K2P results
showed that each locus could individually differentiate between Thunbergia spp. under studied while
the most variable region that can make a distinction effectively was ITS2. The combination of ITS2
with psbA-trnH and/or trnL-trnF sequences was proposed as a potential barcode for identifying
Thunbergia species.
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