Identification and Phylogenetic Analysis of Selected Medicinal Plant Species from Pakistan: Dna Barcoding Approach

Pak. J. Bot., 50(2): 553-560, 2018. IDENTIFICATION AND PHYLOGENETIC ANALYSIS OF SELECTED MEDICINAL PLANT SPECIES FROM PAKISTAN: DNA BARCODING APPROACH ZABTA KHAN SHINWARI*1, 2, SOHAIL AHMAD JAN1, ALI TALHA KHALIL2, ADIL KHAN1, MUHAMMAD ALI1, M. QAISER4 AND NADIA BATOOL ZAHRA3 1Molecular Systematics and Applied Ethnobotany Lab, Quaid-i-Azam University, Islamabad, 45320-Pakistan 2Department of Eastern Medicine and Surgery, Qarshi University, Lahore, Pakistan 3Qarshi University, Lahore, Pakistan 4Centre of Plant Conservation, University of Karachi, Karachi *Corresponding author’s email: [email protected] Abstract Proper identification of medicinally important plant species is useful for their efficient utilization as food and medicine. Seventy eight species belonging to different plant families were characterized through chloroplast DNA gene sequences of rbcL, matK and intergenic spacer region psbA-trnH. These species were used to search possible relationships through phylogenetic analysis. The phylogenetic tree based on matK (~800 bp), rbcL (~1400 bp) and psbA-trnH (~500 bp) sequences showed 6, 8 and 4 diverged groups, respectively. Our analysis suggests that Ajuga bracteosa, Salvia aegyptiaca, Bupleurum falcatum and Acorus calamus are highly diverged from rest of seventy four different species. Based on the results of medicinal plants from Pakistan, we validate that matK and rbcL are more useful as barcode for studying large population of plant species. The present study is useful for authentic identification of new plant species and clearly shows high degree of intra-specific and inter-specific evolutionary relation among all tested species from different medicinally important plant families. Our findings suggest that proper identification of some of the plant species is still challenging and these unique species need further studies. The present study can serve as a model to identify and differentiate new medicinal plant species from Pakistan and neighboring countries through similar methods. Key words: DNA Barcode, Chloroplast DNA, Phylogenetics, Medicinal plants. Introduction to study both inter-specific and intra-specific variations. Initially DNA barcoding was used to identify some Identification of important medicinal plant species is metazoans species through cytochrome oxidase 1 (CO1) a difficult task and it needs expert taxonomists. At present gene sequences. Later barcode regions like rbcL, matK, ITS 391000 vascular plant species are known to science of and trnH-psbA were used to identify flowering species which about 94% are flowering plants (Anonymous., (Kress et al., 2005; Hollingsworth et al., 2011). The DNA 2016). However, large number of plant species are yet to barcode is an improved and efficient method to be identified. About 2000 species are being described differentiate among plant species (Zahra et al., 2016; Zahra every year. Therefore, limited number of species has been et al., 2014; Khan et al., 2015; Jamil et al., 2014; Shinwari identified through conventional identification and et al., 2014). Although, the selection of an ideal sequence classification methods (Chase & Fay, 2009; May, 1992). for DNA barcoding is a key to discover new plant species These medicinal plant species are mostly collected by the however, a single ideal barcode sequence is not discovered local untrained collectors based on the indigenous yet. Therefore two or multiple plastid sequences are used knowledge. During the collection process, sometimes the for discrimination/identification of plant species (Frezal & closely resembling undesirable species are also collected Leblois, 2008). Several different plant barcode sequences and sold with the same name. Therefore, it is necessary to were recommended by different researchers. These include collect proper species with the help of expert taxonomists rpoC1 + matK+ trnH-psbA or rpoC1 + rpoB + matK, from different regions of the country/state. In addition, rbcL+ trnH-psbA, matK + atpF/H + trnH-psbA and matK proper identification and classification would be useful to + atpF/H + psbK/I (Kress & Erickson, 2007). According to preserve the threatened or endangered plant species that CBOL (Consortium for the Barcode Life) the cp DNA seem decreasing day by day worldwide especially in regions rbcL and matK play vital role for rapid developing country like Pakistan (Hussain et al., 2009). identification of medicinally important plant species. DNA barcode is one of the advanced molecular The rbcL (1400 bp) and matK (1500 bp) plastid DNA marker-based methods that identify target plant species in sequences are considered important to study the similarity short duration. The purpose of DNA barcoding is and differences found among different plant species and nucleotide sequence based identification of multiple plant widely used to solve evolutionary and taxonomic issues species with accuracy and is one of the widely accepted (Janzen, 2009; Neuhaus & Link, 1987). These medicinal technology (Group et al., 2009). It is an efficient, quick, plants play key role in controling and treatment of many low-cost and standard method for evaluation and diseases (Khan et al., 2017; Habiba et al., 2016, Qasim et identification of different plant species (Khan et al., 2015). al., 2016; Hayat et al., 2016; Ullah et al., 2017; Jan et al., In addition, this method can efficiently identify unknown 2015). However, limited data is avalible to diffentiate species and/or species having complex morphometric medicinally important plant species from Pakistan. behaviors (Hebert et al., 2004). This technique is also used Therefore, comprehensive study was designed to test 554 Z.K. SHINWARI ET AL., rbcL, matK and trnH-psbA regions for authentication and Data analysis: The generated sequences were submitted phylogenetic investigation of various medicinal plant to GenBank under the accession numbers provided in species from different regions of Pakistan. Table 1. Sequence data from previous studies of same authors was also incorporated in the analysis. For Material and Methods analysis, target nucleotide sequences were aligned by using Mega 7.0 (Sudhir et al., 2015). The final alignments Plant material collection and storage: Selected native were analyzed separately by Neighbor Joining (NJ) plant species were collected from different regions of method using Mega 7.0 and final consensus phylogenetic Pakistan. However, two species of Mentha viz. M. trees were developed for rbcL, matK and psbA-trnH. aquatica and M. suaveolens, grown were collected from Bootstrap support was accessed by 1000 replicates. Qarshi Medicinal Plants garden, Hattar, Pakistan. These plants were collected during their flowering seasons. Results and Discussion Herbarium sheets were prepared and the voucher samples were deposited to the Molecular Systematics and Applied In the current study a total of 78 medicinally important Ethnobotany Lab (MoSAEL), Quaid -i-Azam University species collected from different regions of Pakistan were Islamabad, Pakistan. The plant material was kept in sealed assessed for identification and classification using DNA plastic bags added with the silica gel to absorb the barcoding data (Fig. 1). Although, the general protocol for moisture content. the isolation and amplification of DNA remained the same but in some cases the PCR conditions were optimized. DNA extraction, amplification, and sequencing: Rigorous optimizations for the isolation of DNA and the Total genomic DNA was extracted from specimens by PCR annealing temperature for family Rosaceae were grinding the tissue in liquid nitrogen and DNA was performed. Among the total selected species 54 matK, 72 isolated using 2% CTAB (Cetyltrimethyl ammonium rbcL and 25 psbA-trnH nucleotide sequences were found in bromide) method (Richards, 1997) with few good order to be further used for the phylogenetic analysis. modifications. The obtained pellet was washed with All these sequences were submitted to GenBank database 70% ethanol and allowed to dry at room temperature. with the accession numbers illustrated in Table 1. The DNA pellet was dissolved in TE buffer (10 The phylogenetic analysis based on maturase K mMTris–HCl, pH 8.0, 1 mM EDTA) to a final (matK) gene classified the target plant species into six concentration of 20–30 ng/μL. For the amplification of diverged groups (Fig. 2). The first group had 6 sub- the desired loci, PCR reaction was carried out in 0.5 ml groups. The first sub-group contained seven different PCR tubes. The desired loci were amplified using species. Among these species Mentha longifolia is KAPA3G Plant PCR Kit (Kapa Biosystems, Woburn, closely related to Mentha pulegium and Mentha x Massachusetts, USA) as described by Zahra et al., piperita as compared to Mentha spicata. The second sub- (2016). The cycling conditions used for rbcL were: group included three different species. Lallementia 95°C 10 min; 50 cycles: 95°C 20 s, 58°C 15 s, 72°C 90 royleana and Salvia aegyptiaca are closely related as s; and final elongation at 72 °C 90 s. The rbcL forward compared to Nepeta cataria. The third sub-group had primer 1F (Fay et al., 1998) and 1460R reverse four diverged species including Melissa officinalis, (Cuenoud et al., 2002) were used in this experiment. Salvia moorcroftiana, Rosmarinus officinalis and Salvia Same PCR parameters were used for matK 390F/1360R plebeia, respectively. The sub-group IV included four primers (Cuenoud et al., 2002) with 50°C annealing species from genus Ocimum and all accessions are in temperature for 40 cycles (Schori

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