DNA barcoding for the discrimination of Uncaria gambir and its closely related species using internal transcribed spacer genes

DNA extraction, PCR amplification and sequencing

Six fresh leaves of four Uncaria gambir variants were collected from Siguntur, West Sumatera Province, Indonesia (1°05'36.8"S 100°28'27.0"E -1.093562, 100.474153). Morphology identification was conducted in the Herbarium of Andalas University. Harvested fresh leaves were frozen immediately in liquid nitrogen and was stored at -80°C for 24 hours prior to DNA isolation.

DNA isolation was performed using the CTAB method (Allen et al., 2006). In total 300 mg of frozen leaf sample was ground and put into a 2 ml Eppendorf tube. 1 ml of 2x CTAB buffer and merchaptoethanol was added and vortexed until homogenized. The solution was incubated at 65°C for 30 minutes and inverted every 10 minutes. 500 µL of phenol: chloroform: isoamylalcohol mixture (25:24:1) was added and vortexed for 1 minute then centrifuged at 12,000 rpm for 10 minutes. The supernatant was transferred to a new 2 ml Eppendorf tube. 500 µL of chloroform mixture: isoamylalcohol (24:1) was added and vortexed for 10 minutes then centrifuged at 12,000 rpm for 10 minutes. The supernatant was transferred to a new 1.5 mL Eppendorf tube. Sodium acetate was added as much as 1/10 times the volume of the supernatant, then 1 ml of cold ethanol 99% was added and swirled for 1 minute. The solution was centrifuged at 12,000 rpm for 5 minutes, supernatant was removed and 500 µL of 70% ethanol was added. The solution was centrifuged at 12,000 rpm for 5 minutes, the supernatant was discarded and the DNA was dried in the oven. Finally 100 µL of 1xTE buffer was added to dried DNA and stored at -20°C.

DNA amplification was performed using MyTaq^TM HS Red Mix Bioline kit. The final concentrations for a typical 25 μl reaction were as follows: 12.5 µL MyTaq^TM HS Red Mix Bioline, 1 µL reverse primer ITS-u4 (RGTTTCTTTTCCTCCGCTTA), 1 µL forward primer ITS-u1 (GGAAGKARAAGTCGTAACAAGG), 1 µL DNA template, 9.5 µL Nuclease-Free Water to obtain a 25 µL final volume. The thermal cycling conditions (BIO-RAD C1000^TM Thermal Cycler) were as follows: (a) Denaturation 96°C for 60 s, (b) Denaturation 96°C for 15 s, (c) Annealing 70°C for 45 s (-1°C decrement each cycle), (d) Extension 72°C for 60 s; (e) repeat step (b-d) for 25 cycles and followed by a final extension at 72 °C for 5 min.

The isolation and amplification results were visualized using electrophoresis with 1.5% agarose gel. The expected amplification product was sequenced by 1^st BASE DNA Sequencing Service (Apical Scientific Sdn Bhd) bidirectionally using the specific primer.

Sequence and distance analysis

A total of 132 Uncaria ITS sequences were downloaded from National Center for Biotechnology Information (NCBI) and were aligned with Uncaria gambir using MEGA X software version 10.2.6 (Tamura et al., 2013). The intraspecific and interspecific distances were computed with Kimura 2-Parameter (K2P) using MEGA X software (Kimura, 1980)(Meier et al., 2006).

Phylogenetic analysis

The sequences were analyzed by the maximum likelihood method for phylogenetic tree construction. The maximum likelihood analyses, including 1000 nonparametric bootstrap replicates, was performed using MEGA X software under the Kimura 2-parameter (K2P)+G model. Nauclea orientalis and Nauclea diderrichi sequences that are relative to the Uncaria genus were downloaded from NCBI and used as an outgroup species.

Sequences and distance analysis

The primer ITS-u1 and ITS-u4 showed 100% amplification efficiency to Uncaria gambir. Sequences were deposited in the NCBI GenBank database under the accession numbers MZ927014, MZ927015, MZ927016, and MZ926993 (Table 1). The region's length was obtained from 644 to 646 bp.

Phylogenetic analysis

The phylogenetic tree was constructed using 13 Uncaria species. Uncaria gambir were clustered from other species (Figure 1). The four variants of Uncaria gambir had a close relationship with Uncaria macrophylla MF033303.

Figure 1. Phylogenetic tree analysis of Uncaria gambir to another Uncaria species was computed using maximum likelihood method.

The phylogenetic tree showed Uncaria gambir has a close relationship with Uncaria yunnanensis but not Uncaria scandens. In contrast, the interspecific distance value showed Uncaria gambir had a close relationship with Uncaria yunnanensis and Uncaria scandens.

Underlying data

The ITS sequences from this research are available from NCBI:

GenBank: Uncaria gambir isolate Udang, Accession number MZ927014: https://www.ncbi.nlm.nih.gov/nuccore/MZ927014

GenBank: Uncaria gambir isolate Mancik, Accession number MZ927015: https://www.ncbi.nlm.nih.gov/nuccore/MZ927015

GenBank: Uncaria gambir isolate Riau, Accession number MZ927016: https://www.ncbi.nlm.nih.gov/nuccore/MZ927016

GenBank: Uncaria gambir isolate Cubadak, Accession number MZ926993: https://www.ncbi.nlm.nih.gov/nuccore/MZ926993

Extended data

Dryad: GenBank accession numbers of downloaded sequences used in this study https://doi.org/10.5061/dryad.dfn2z352w (Wardi et al., 2021a)

This project contains the following extended data:

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).