Comparison of DNA extraction methods on CITES-listed timber species and application in species authentication of commercial products using DNA barcoding

Sources of samples

In this study, 12 authentic wood samples of Aquilaria, Dalbergia and Pterocarpus were from the collection of Shiu-Ying Hu Herbarium and donations of Agriculture, Fisheries and Conservation Department of the Hong Kong Special Administrative Region Government (AFCD of HKSAR Government). For commercial wood products, four samples were donated by AFCD of HKSAR Government and six samples were purchased from local retail stores, either ordered online or bought from physical stores. Leaf and twig samples were also collected as positive controls during amplification and for generating reliable reference barcode sequences. These samples were collected from Hong Kong by the staff members of Shiu-Ying Hu Herbarium of The Chinese University of Hong Kong with deposited voucher specimens. The collections are permitted under the Permission to Make Field Collection for Research Purpose issued by AFCD of HKSAR Government. Two samples, namely KFBG_7521 and KFBG_9203, were sampled from the living individuals cultivated in Kadoorie Farm & Botanic Garden in Hong Kong on 25^th September, 2019. All collections are permitted and legal in Hong Kong. Details of collected authentic wood samples, commercial wood products and reference leaf and twig samples are listed in Tables 1, 2 and Supplementary Table S1, respectively. Figure S1 shows the photos of the authentic wood samples, with different sampled parts annotated. Photos of the commercial wood products are shown in Fig. S2.

Genomic DNA extraction

For leaf and twig samples used as reference, DNA of these samples were extracted using BioMed Plant Genome DNA Extraction Kit (Biomed, Beijing, China) following manufacturer’s instructions. Small wood pieces were first sampled from both authentic and commercial wood products using electric drill or scalpel blades and further disrupted into fine powder using Precellys Evolution Tissue Homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). For authentic wood samples T5196, T4966, T5155, T5152 and T5197, small wood pieces were taken from three parts namely sapwood, outer heartwood and inner heartwood following the practice of Rachmayanti²⁷ and Jiao¹³. Inner heartwood was sampled about 2 cm diameter from pith, while outer heartwood was defined as the transition zone between sapwood and inner heartwood. For authentic wood samples T4964 and T4965, since sapwood is absent, only the outer and inner heartwood were sampled. Because authentic wood sample T4961 is an incomplete wood block lacking the inner heartwood, small wood pieces of sapwood and heartwood were taken, without further differentiating the heartwood into inner and outer part.

The quality, quantity and amplifiability of DNA extracted using three different genomic DNA extraction methods, including SDS-based method, QIAGEN method and another silica-column-based method using guanidine thiocyanate (GuSCN)-containing binding buffer developed by The Canadian Centre for DNA Barcoding (CCDB method), were compared. For fair comparison, the amount of wood powder and the amount of water/elution buffer used to elute DNA were kept the same. The same silica columns, QIAquick Spin Columns (Qiagen, Hilden, Germany), were used in all three methods. After evaluating the results of DNA extracted from the three DNA extraction methods, DNA of commercial wood products were extracted using the CCDB method.

SDS-based method

The extraction protocol was modified from Little²⁹. A total of 0.3 g wood powder were mixed with 0.1 g of polyvinylpolypyrrolidone (PVPP) and incubated with 2 mL of extraction buffer (8 mM NaCl, 16 mM sucrose, 5.8 mM EDTA, 0.5% (w/v) sodium dodecyl sulphate, 12.4 mM Tris (pH 7.4)) and 20 µL of proteinase K (20 mg/mL) for 18 h at 42 °C. After incubation, 400 µL of 3 M potassium acetate (pH 4.7) was added followed by incubation at 0 °C for 10 min. After incubation, mixtures were centrifuged at 15,000 rpm for 5 min. Clear supernatant was collected and mixed with 1.5 × volume of 2 M guanidine hydrochloride in 95% (v/v) ethanol. Mixtures were applied to QIAquick Spin Columns followed by centrifugation at 5000 rpm for 5 min. After all mixtures were applied, sample was washed twice with 500 µL of washing buffer (50% (v/v) ethanol, 10 mM Tris (pH 7.4), 0.5 M EDTA, 50 mM NaCl) with centrifugation at 5000 rpm at 5 min. Ethanol residue was removed by centrifugation at 15,000 rpm for 5 min. Genomic DNA were eluted with 50 µL of pre-warmed molecular biology grade water with centrifugation at 15,000 rpm for 5 min after incubation at room temperature for 2–3 min. Time consumed in this method was approximately within 20 h for 24 samples and each sample costed USD 6.4.

QIAGEN method

DNeasy Plant Pro Kit (Qiagen, Hilden, Germany) was used according to manufacturer’s instructions with modification. A total of 0.3 g wood powder was first mixed with 0.1 g of PVPP and incubated with 1.8 mL of CD1 solution and 200 µL of PS solution for 1 h at 65 °C. After incubation, mixtures were centrifuged at 15,000 rpm for 20 min. Clear supernatant was collected and mixed with same volume of solution CD2 followed by centrifugation at 15,000 rpm for 20 min. Clear supernatant was collected and mixed with 1000 µL of APP solution. Mixtures were applied to QIAquick Spin Columns followed by centrifugation at 5000 rpm for 5 min. After all mixtures were applied, column was washed with 650 µL of AW1 solution followed by centrifugation at 5000 rpm at 5 min and 650 µL of AW2 solution followed by centrifugation at 5000 rpm at 5 min. Ethanol residue was removed by centrifugation at 15,000 rpm for 5 min. Genomic DNA were eluted with 50 µL of pre-warmed EB solution with centrifugation at 15,000 rpm for 5 min. Time consumed in this method was approximately within 3.5 h for 24 samples and each sample costed USD 17.

CCDB method

Genomic DNA was extracted using extraction protocol modified from Ivanova³⁰. A total of 0.3 g sample powder was mixed with 0.1 g of PVPP and mixed with 2 mL of lysis buffer (700 mM GuSCN, 30 mM EDTA (pH 8.0), 30 mM Tris–HCl (pH 8.0), 0.5% Triton X-100, 5% Tween-20), 200 µL of proteinase K (20 mg/mL) and 50 µL of α-amylase. The mixtures were incubated at 56 °C for 30 min followed by an incubation at 65 °C for 1 h. After incubation, mixtures were centrifuged at 15,000 rpm for 10 min and clear supernatant was collected and mixed with equal volume of extraction binding buffer (6 M GuSCN, 20 mM EDTA (pH 8.0), 10 mM Tris–HCl (pH 6.4), 4% Triton X-100). Mixtures were applied to QIAquick Spin Columns followed by centrifugation at 5000 rpm for 5 min. After all mixtures were applied, sample was washed with 180 µL of extraction wash buffer (60% ethanol, 50 mM NaCl, 10 mM Tris–HCl (pH 7.4), 50 mM EDTA (pH 8.0)) with centrifugation at 5000 rpm for 5 min. After that, sample was washed twice with 650 µL of extraction wash buffer. Ethanol residue was removed by centrifugation at 15,000 rpm for 5 min. Genomic DNA were eluted with 50 µL of pre-warmed water with centrifugation at 15,000 rpm for 5 min after incubation at room temperature for 2–3 min. Time consumed in this method was approximately within 4 h for 24 samples and each sample costed USD 13.5.

DNA quantity and quality analysis

For comparison of DNA extracted using the three genomic DNA extraction methods, the quality and quantity of the DNA extracted were evaluated by a NanoDrop Lite spectrophotometer (Thermo Scientific, Massachusetts, USA).

PCR amplification, agarose gel electrophoresis and DNA sequencing

For each genus, different DNA mini-barcode loci were selected for amplification and sequencing based on their species discrimination ability evaluated by previous research. For Aquilaria, DNA barcode loci ITS2, matK and trnL–trnF were selected²⁰. For Dalberiga, DNA barcode loci ITS2, matK, trnH–psbA and trnL were selected¹⁸ and for Pterocarpus, DNA barcode loci ITS2, matK, rbcL and ndhF–rpl32 were selected¹⁹. DNA extracted were amplified using the GoTaq G2 Flexi DNA Polymerase (Promega, Wisconsin, USA). Each 15-μL PCR contained 10 ng template DNA, 3 μL 5X Colorless GoTaq Flexi Buffer, 2.4 μL MgCl₂ (25 mmol/L), 0.3 μL dNTP mixture (10 mmol/L each), 0.75 μL forward primer (10 μmol/L), 0.75 μL reverse primer (10 μmol/L) and 0.125 μL GoTaq polymerase (5 U/μL). Sequences of primers and the corresponding reaction conditions are listed in Supplementary Table S2. PCR products were mixed with 6X loading dye in a ratio of 5:2 and visualized in 2% agarose gel and sizes of fragments were compared with GeneRuler 100 bp DNA Ladder. After evaluating the result of DNA extracted from the three DNA extraction methods, PCR products with successful amplification of DNA extracted with the CCDB method were purified with Biomed gel extraction kit (Biomed, Beijing, China). Sanger sequencing was performed by Tech Dragon Ltd., Hong Kong.

Additional sequences

In addition to sequences generated from reference leaf and twig samples, sequences of Aquilaria (ITS2, matK and trnL–trnF), Dalberiga (ITS2, matK, trnH–psbA and trnL) and Pterocarpus (ITS2, matK, rbcL and ndhF–rpl32) were downloaded from GenBank for data analysis (Supplementary Tables S3–S5).

Data analysis

Raw sequences generated from reference leaf and twig samples in this study were assembled and aligned followed by a manual adjustment using BioEdit³¹, saved in FASTA format and deposited to GenBank. Reference DNA barcode libraries for each genus (Aquilaria, Dalbergia and Pterocarpus) were developed from sequences obtained from leaf and twig samples and sequences obtained from GenBank for species identification of authentic wood samples and commercial products based on single DNA barcodes and multi-loci combinations through phylogenetic tree-based analysis. Phylogenetic trees were constructed based on the aligned sequences from single loci or concatenated loci using neighbour-joining (NJ) method using the Kimura 2-Parameter (K2P) model³² with bootstrap re-sampling (n = 1000) and pairwise deletion using MEGA X³³.

Plant collection statement

All plant samples in this study were collected in Hong Kong and the experiments were performed locally. The authors carefully checked that proper sample collection permits were in place at the time of collection. The authors declare that all the experimental research and field sampling of plant material comply with institutional, local, national and international guidelines and legislation.