Characterization of selected aquilaria species through DNA barcodes and identification of wound-response proteins of aquilaria malaccensis Lam.

Over-exploitation in search of its valuable non-wood fragrance product, the agarwood, has put pressure on the survival of the endangered Aquilaria trees in the wild. The lack of genetic information on these species rendered conservation efforts a tough task.Agarwood species identification is challenging as conventional techniques alone are unable to ascertain the species origin. In this work, the genetic variation within Aquilaria species in Peninsular Malaysia, and their relationship to several Aquilaria species of foreign origins were studied using molecular approaches. The internal transcribed spacer (ITS) of the nuclear region of 19 wild Aquilaria populations from different states in Peninsular Malaysia were sequenced and compared to the same species residing outside of Malaysia using fresh leaf samples. Single nucleotide polymorphisms (SNPs) were identified when comparisons were made between A. malaccensis from different countries, suggesting geographical segregation is a contributing factor toward genetic variation in A. malaccensis. The phylogenetic analysis conducted on the nuclear ribosomal ITS and the intergenic spacer region trnLtrnF regions of selected agarwood-producing species further revealed that both sequences were able to separate two important genera of agarwood tree species (Aquilaria and Gyrinops) into two clades, indicating they are paraphyletic. In addition,during this study, a critically endangered species, Aquilaria rostrata, endemic to Peninsular Malaysia, was rediscovered and compared to other Aquilaria species using DNA sequence and taxonomic treatments. For the identification of agarwood species of origin, a reference DNA barcode library was developed 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. 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. However the trnL-trnF+ITS2 was proposed as the best candidate barcode for Aquilaria because the ITS2 is shorter in sequence length compared to ITS. This eases PCR amplification especially when using degraded DNA samples. In an attempt to identify processed agarwood products, real-time PCR technique coupled with species-specific primers derived from SNP positions in the matK and trnL-trnF sequences successfully targeted three commercial Aquilaria species: A. crassna, A. malaccensis, and A. sinensis,demonstrating their specificity for the purpose of DNA tracing. For identification of wound-response related proteins from A. malaccensis tree stem, 16 protein spots were identified reproducible between biological replicates under 2D-PAGE, with only two protein spots showing regulation in expression after wounding treatment. The two proteins were predicted as malate synthase and NADPH quinone oxidoreductase subunit 2B. Both proteins were reported to be directly and indirectly related to wounding treatments in plants, and thus may be involved in agarwood formation mechanism. In conclusion, the molecular information obtained from this study will serve as a useful reference in designing in-situ programs to conserve this threatened species, contributes to the international timber trade control by providing an effective method for species identification and agarwood product authentication, and contributes in preliminary information on protein expressions related to agarwood formation in Aquilaria.

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