DNA Barcoding of the Endangered Aquilaria (Thymelaeaceae) and Its Application in Species Authentication of Agarwood Products Traded in the Market

Abstract

The identification of Aquilaria species from their resinous non-wood product, the agarwood, is challenging as conventional techniques alone are unable to ascertain the species origin. Aquilaria is a highly protected species due to the excessive exploitation of its precious agarwood. Here, we applied the DNA barcoding technique to generate barcode sequences for Aquilaria species and later applied the barcodes to identify the source species of agarwood found in the market. We developed a reference DNA barcode library using eight candidate barcode loci (matK, rbcL, rpoB, rpoC1, psbA-trnH, trnL-trnF, ITS, and ITS2) amplified from 24 leaf accessions of seven Aquilaria species obtained from living trees. Our results indicated that all single barcodes can be easily amplified and sequenced with the selected primers. The combination of trnL-trnF+ITS and trnL-trnF+ITS2 yielded the greatest species resolution using the least number of loci combination, while matK+trnL-trnF+ITS showed potential in detecting the geographical origins of Aquilaria species. We propose trnL-trnF+ITS2 as the best candidate barcode for Aquilaria as ITS2 has a shorter sequence length compared to ITS, which eases PCR amplification especially when using degraded DNA samples such as those extracted from processed agarwood products. A blind test conducted on eight agarwood samples in different forms using the proposed barcode combination proved successful in their identification up to the species level. Such potential of DNA barcoding in identifying the source species of agarwood will contribute to the international timber trade control, by providing an effective method for species identification and product authentication.

DNA barcoding of vouchered xylarium wood specimens of nine endangered Dalbergia species

“…Our results have clearly demonstrated that DNA barcoding using only a single DNA locus was insufficient to provide resolution for Dalbergia species. These results are similar to previous finding for Aquilaria species (Lee et al 2016).…”

“…In recent years, DNA methods to verify species identity and origin of internationally traded woods have attracted increasing interest to combat illegal logging (Lowe and Cross 2011;Lowe et al 2016). Several studies have shown the ability of barcoding to effectively discriminate important timber species, such as agarwood (Aquilaria) (Jiao et al 2014;Lee et al 2016), oak (Quercus) (Deguilloux and Petit 2004), and mahogany (Swietenia) (Degen et al 2013). More recent studies have included Dalbergia species, focusing on species identification in Southeast Asia and Madagascar (Hartvig et al 2015;Hassold et al 2016;Yu et al 2016).…”

Optimizing Genomic DNA Isolation and PCR Amplification For Pasak Bumi (Eurycoma longifolia)

“…The successfulness of amplification using trnL-trnF have been reported in previous research for Aquilaria, Styrax sumatrana, Shorea, Taxus sumatrana, and Dipterocarpus [33][34][35][36][37]. Furthermore, Fig.…”

Characterization of the complete chloroplast genome of Aquilaria sinensis, an endangered agarwood-producing tree

“…Agarwood is widely used as traditional drug, interior decoration and valuable perfume for thousands of years in Asia (Hashim et al 2016). The genus Aquilaria is well known as the main material of the agarwood (Lee et al 2016). Unfortunately, due to the overharvesting of wild resources for high demand of agarwood products, all Aquilaria species are currently endangered and regulated under the CITES (http://checklist.cites.org).…”