DNA Barcoding

Seed Germination | Micropropagation | Sexual Characteristics | DNA Barcoding | Nursery Management

DNA barcoding is an essential scientific tool to verify the species identity of agarwood, especially when dealing with CITES-listed species like Aquilaria malaccensis. It provides genetic proof that the material you’re cultivating, harvesting, or trading is the correct and legally recognized species.

Species identified: Aquilaria malaccensis
Sequence match: 100.00% identity
Accession Number: MH265672.1
GenBank Reference: Aquilaria malaccensis voucher A3-LI-NP (confirmed)
Confidence level: High

DNA samples were extracted and chloroplast barcoding genes (rbcL and matK) were optimized and sequenced. DNA sequencing was completed for the six samples. We used the Basic Local Alignment Search Tool (BLAST) implemented in the NCBI database to compare our samples with known deposited sequences.

Ac, Ac/p, and Af from Putz Agarwood Farm did not have sequence variation among them in terms of the rbcL gene
However, the sequence deposited in the NCBI for A. cumingiana was highly different with A. cumingiana,
A.cumingiana/parvifolia or A. filaria as identified by the Farm at three distinct positions (Table 2). To confirm A.
cumingiana, we will be sequencing samples collected from the natural populations. For A. malaccensis our sequences were confirmed 100% similar with A. malaccensis deposited in the NCBI (BLAST results not shown).

In terms of the matK gene, Ac, Ac/p, and Af did not also have sequence variation among them (Table 3). However, the
sequence deposited in the NCBI for A. malaccensis was different by two base points from the A. malaccensis from the
Putz Agarwood Farm (Table 3). To confirm A. malaccensis, more samples from the natural populations are needed to be sequenced and compare it with the current sequences of A. malaccensis.

Aquilaria malaccensis that we have in our nursery matched 100% with reference database when the sequences ran. Simply means, what we propagated, distributed, cultivated and planted in our farm is the correct and certified species of Aquilaria malaccensis (BARI).

Specimens sample used has been kept in our arboretum.

DNA sequencing of Aquilaria species plays a vital role in:

  • Species identification (especially because many Aquilaria species look similar morphologically),
  • Conservation efforts (monitoring wild populations),
  • Traceability and legal trade compliance (e.g., CITES),
  • Breeding and propagation programs (selecting high resin-yielding genotypes).

DNA Sequencing Techniques for Aquilaria

  1. DNA Barcoding (Standard for Species ID)
    • Common markers used:
      • ITS (Internal Transcribed Spacer) – nuclear DNA
      • matK and rbcL – chloroplast DNA
      • trnL-trnF, psbA-trnH – intergenic spacers in chloroplasts
    • Application:
      • Differentiating species such as Aquilaria malaccensis, A. crassna, A. sinensis, etc.
  2. Next-Generation Sequencing (NGS)
    • Whole genome sequencing or transcriptome sequencing
    • Use: Genetic diversity, gene discovery (e.g., genes involved in resin biosynthesis), phylogenetic analysis
  3. Microsatellite (SSR) Analysis
    • Useful for studying population structure and genetic variation
    • Developed from genome or transcriptome data
  4. RAPD, ISSR, AFLP (older methods, less specific)
    • Still used in some developing country labs for genetic diversity analysis
    • RAPD (Random Amplified Polymorphic DNA)
    • ISSR (Inter Simple Sequence Repeats)

Why you need DNA Barcoding For Agarwood Trade?

Use CaseWhy It’s Needed
CITES Export PermitVerifies the species as legally tradable under Appendix II
DENR-BMB Registration (WCuP, CWR)Confirms your plantation’s species identity
Quality assurance for buyersEspecially for Middle East and Japanese markets
Inoculation researchEnsures proper matching of strain-to-species compatibility
Plantation investment & certificationAdds credibility to provenance claims and traceability

Notable Research Findings

  • Studies in Thailand, India, Malaysia, and the Philippines have developed genetic fingerprints for key Aquilaria species.
  • Research has identified candidate genes involved in agarwood resin production (e.g., sesquiterpene synthases, defense response genes).
  • DNA barcoding is being used in CITES enforcement to detect illegal or mislabeled trade samples.
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