Zunera Shabbir PhD 1st semester Department of Botany PMAS AAUR DNA barcoding in medicinal plants: Testing the potential of a proposed barcoding marker for identification of Uncaria species from China

About 5 to 50 million plants and animals are living on earth, out of which less than 2 million have been identified. In recent years new ecological approach called DNA barcoding has been proposed to identify species and ecology research. DNA barcoding, a system for fast and accurate species identification which will make ecological system more accessible. DNA barcoding is a taxonomic method that uses a short genetic marker in an organism's DNA to identify it as belonging to a particular species.

History DNA barcoding first came to the attention of the scientific community in 2003 when Paul Hebert’s research group at the University of Guelph published a paper titled "Biological identifications through DNA barcodes". 

The short DNA sequence is taken from standard region of genome to generate DNA barcode. DNA barcode is short DNA sequence made of four nucleotide bases A (Adenine), T (Thymine), C (Cytosine) and G (Guanine). Each base is represented by a unique color in DNA barcode as shown in figure. Even non experts can identify species from small, damaged or industrially processed material.

There are an estimated 300,000 plant species in the world (IUCN, 2012) but relatively few of these can be identified based on traditional plant identification methods. Accurate classification and identification of this large number of species remains a significant challenge even for specialist taxonomists. The emergence of DNA barcoding has had a positive impact on biodiversity classification and identification (Gregory, 2005). DNA barcoding is a technique for characterizing species of organisms using a short DNA sequence from a standard and agreed-upon position in the genome. (http://barcoding.si.edu/DNABarCoding.htm).

Schematic timeline of plant barcoding history and possible developments. CO1, cytochrome c oxidase 1; cp, chloroplast; ITS, internal transcribed spacer.

The genus Uncaria, belongs to the family Rubiaceae, is mainly distributed in tropical Asia and Australia, with a distribution of 12 species in China. All 12 Uncaria species, known as “Gou-teng”, have long been used in traditional Chinese medicines (Yu et al., 1999). Consequently, the reserves of mainstream species have significantly decreased, and the market is becoming more and more irregular, facing issues such as adulteration, substitution and lower-quality products selling at top-quality prices (Yu et al., 1999). Uncaria rhynchophylla

Many Uncaria plants are morphologically similar, making identification of their origin species difficult. A reliable and quick method for the identification of Uncaria species needs to be developed for their further research and utilization. In this research paper, they have chose five DNA regions (rbcL, matK, trnH-psbA, ITS and ITS2) that have previously been proposed as DNA barcodes to evaluate and verify the feasibility of authenticating Uncaria medicinal plants and establishing a suitable DNA barcoding protocol for them.

Taxon sampling Ten of 12 (according to the Flora of China, 2013) species of Uncaria were collected from the field, individual cultivators and botanical gardens. Multiple samples of each species were collected to ensure that both the morphological and geographical ranges of each taxon were covered. The total number of samples was 54 and all corresponding voucher samples are in the Herbarium of the Yunnan branch of IMPLAD (Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences).

DNA extraction, PCR amplification and sequencing Total DNA was isolated from silica gel-dried leaves following the manufacturer's instructions for the Plant Genomic DNA Kit (DP305, Tiangen Biotech Co, China). Specific DNA fragments were amplified via standard polymerase chain reaction (PCR). The primers synthesized for PCR and sequencing were as described by Chen et al. (2010), and the reaction conditions are presented in Table. PCR amplification was performed in 20 mL reaction mixtures containing 10-30ng of genomic DNA template, 1 PCR buffer with 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 mM of each primer and 1.5 U Taq DNA Polymerase. The PCR products were purified using PCR Cleanup Kit (AXYGEN, USA) and sequenced in both directions with the primers used for PCR amplification on a 3730XL sequencer (Applied Biosystems, USA).

In this study, they initially tested five potential DNA barcode sequences, including psbA-trnH, ITS, ITS2, matk, and rbcL, but during the further analysis procedure, the matk was ignored because of the low PCR amplification efficiency.

DNA sequence data analysis To ensure sequencing accuracy, the raw sequencing results were corrected and assembled using CodonCode Aligner 3.0 (CodonCode Co., USA). Low-quality sequences (according to Chen et al., 2010) and primer sequences were deleted. For ITS2, they used Hidden Markov Models (HMMs) to remove possibly contaminated sequences from fungi (Keller et al., 2009). Candidate DNA barcodes were aligned by Clustal X V2.0. The output data were processed to calculate the Kimura-2-Parameter (K2P) distances for each region using MEGA v4.1 (Kumar et al., 2008). The neighbor-joining (NJ) method (Saitou and Nei,1987) was selected for the construction of phylogenetic trees.

A total of 1000 bootstrap replicates were calculated for the NJ tree construction (Saitou and Nei, 1987). To compare inter-specific divergence and intra-specific variation, average inter-specific distance and smallest inter-specific distance were calculated to characterize inter-specific divergence, average intra-specific distances and coalescent depth were calculated to determine intra-specific variation using a K2P distance matrix, and the software utilized was TaxonDNA 1.0 (Meier et al., 2006). The methods of species identification is Best Close Match (Meier et al., 2006). To increase the species identification accuracy of the potential barcodes, the sequence data deposited in the DNA database of GenBank was used.

Results 1. Efficiency of PCR amplification, sequencing and sequence character analysis The sequence lengths range from 219 to 719 bp. The shortest locus is ITS2, at 219-222 bp. The average GC content of each locus is also different, the highest one is ITS2 (reaching up to 66.2%), and the lowest is psbA-trnH (with a content of 27.0%). The efficiencies of PCR amplification and success rates of sequencing are important indicators for the evaluation of DNA barcodes. In this study, the efficiency of PCR amplification, from high to low, is 100.0% (ITS2), 70.4% (rbcL), 66.7% (ITS), and 64.8% (psbA-trnH). For the success rate of sequencing, the four loci are all 100.0%. Based on the efficiency of PCR amplification and the sequencing, the most suitable DNA barcode for Uncaria is ITS2.

2. Assessment of barcoding gap An ideal DNA barcode, the distributions of genetic variation between intra- and inter-specific should be as distinct and non-overlapping as possible. The results (data not shown) showed that: for three candidate DNA barcodes (ITS, rbcL, psbAtrnH), there was significant overlap in the distributions of intra- and inter-specific variation. Only ITS2 exhibits distinct gaps between the distributions of intra- and inter-specific variation that could be useful for the authentication of Uncaria plants.

Neighbor-joining trees produced from pairwise Kimura 2-parameter distances of ITS2 for analytical specimen. Numbers on branches represent NJ support values (%).

Conclusion An ideal DNA barcode should meet the following criteria: The inter-specific genetic variability and differentiation is obvious, and the intra-specific divergence is inconspicuous; The sequence is short enough and easy to be amplified and sequenced; The candidate DNA barcode should have conserved regions that are convenient for the design of universal primers. In the study we assessed the potential of five proposed DNA regions (ITS2, ITS, rbcL, matk and psbA-trnH) for differentiating Uncaria species, the results showed that the ITS2 region is most appropriate as a DNA barcode. For matk, difficulties in PCR amplification and sequencing are the most significant problems faced as a candidate DNA barcode. The results showed that matk had the lowest amplification (only 37.0%) and sequencing efficiency. Therefore, the matk region is not suitable for identifying Uncaria, even though it has been suggested as a core DNA barcode for plants (CBOL PlantWorking Group, 2009).

Among the other four candidate DNA barcodes that we tested, although three regions (ITS, rbcL, and psbA-trnH) had higher PCR amplification (66.7%, 70.4%, 64.8%, respectively) and sequencing efficiency (all were 100.0%), the authentication efficiencies were lower as a result of intra-specific divergences were similar to or exceeded the interspecific variation. The ITS2 region has been proposed as a universal DNA barcode for plant and animal identification in previous studies (Chen et al., 2010; Yao et al., 2010). In this study, ITS2 showed 100.0% amplification and sequencing efficiency, the interspecific and intra-specific variations were moderate and the inter-specific variation was higher than intra-specific divergence. In addition, among the five tested candidate DNA barcodes, ITS2 showed the highest ability of species identification on the basis of the Best Close Match (94.4%) and tree methods. The results also indicated that the higher authentication ability of ITS2 between the Uncaria species means that this region has a greater potential as a barcode than other candidate DNA barcodes.

Reference: Zhang, Z. L., Song, M. F., Guan, Y. H., Li, H. T., Niu, Y. F., Zhang, L. X., & Ma, X. J. (2015). DNA barcoding in medicinal plants: Testing the potential of a proposed barcoding marker for identification of Uncaria species from China. Biochemical Systematics and Ecology, 60, 8-14. Li, X., Yang, Y., Henry, R. J., Rossetto, M., Wang, Y., & Chen, S. (2015). Plant DNA barcoding: from gene to genome. Biological Reviews, 90(1), 157-166.