Abstract
Members of Aquilaria Lam. (Thymelaeaceae) are evergreen trees that are widely distributed in the Indomalesia region. Aquilaria is highly prized for its unique scented resin, agarwood, which is often the subject of unlawful trade activities. Survival of the tree is heavily threatened by destructive harvesting and agarwood poaching, leading to its protection under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Unfortunately, an efficient species identification method, which is crucial to aid in the conservation efforts of Aquilaria is lacking. Here, we described our search for a suitable specific DNA barcode for Aquilaria species using eight complete plastome sequences. We identified five highly variable regions (HVR) (matK-rps16, ndhF-rpl32, psbJ-petA, trnD, and trnT-trnL) in the plastomes. These regions were further analyzed using the neighbor-joining (NJ) method to assess their ability at discriminating the eight species. Coupled with in silico primer design, two potential barcoding regions, psbJ-petA and trnT-trnL, were identified. Their strengths in species delimitation were evaluated individually and in combination, via DNA barcoding analysis. Our findings showed that the combined dataset, psbJ-petA + trnT-trnL, effectively resolved members of the genus Aquilaria by clustering all species into their respective clades. In addition, we demonstrated that the newly proposed DNA barcode was capable at identifying the species of origin of six commercial agarwood samples that were included as unknown samples. Such achievement offers a new technical advancement, useful in the combat against illicit agarwood trades and in assisting the conservation of these valuable species in natural populations.
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Data Availability
The DNA sequences generated and analysed in this paper are available in GenBank (https://www.ncbi.nlm.nih.gov/genbank/, accession numbers listed in Table 1). The DNA sequences were submitted on March 8, 2021 (Accession number: MW712538–MW712585).
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Acknowledgements
Muhammad Syahmi Hishamuddin received Graduate Research Fellowship from Universiti Putra Malaysia, UPM Serdang, Malaysia. This manuscript is part of the requirement for his Degree of Doctor of Philosophy.
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This work was supported by Universiti Putra Malaysia under the Research University Grant Scheme—PUTRA Grant [Project No. UPM/800-3/3/1/9630100] from the Ministry of Education of Malaysia.
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RM and SYL designed the study, collected, identified plant materials, and edited the manuscript. MSH conducted the experiments, drafted, and revised the manuscript. MSH performed data analysis guided by RM and SYL. SAS assisted in primer design and figure formatting. SIR, DUL and RM supervised. RM acquired funding. All authors reviewed and approved the final manuscript.
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Hishamuddin, M.S., Lee, S.Y., Syazwan, S.A. et al. Highly divergent regions in the complete plastome sequences of Aquilaria are suitable for DNA barcoding applications including identifying species origin of agarwood products. 3 Biotech 13, 78 (2023). https://doi.org/10.1007/s13205-023-03479-1
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DOI: https://doi.org/10.1007/s13205-023-03479-1