ISSR-Derived Species-Specific SCAR Marker for Rapid and Accurate Authentication of Ocimum tenuiflorum L.

Abstract

Ocimum tenuiflorum has been widely used in traditional medicine and has high medicinal value. High volume trade of this potential medicinal plant species led to unscrupulous adulteration of both crude drugs as well as formulations. Morphology-based authentication is difficult in cases of incomplete or damaged samples and in dried herbal materials. In such cases, PCR-based molecular methods may aid in accurate identification. The present study aimed at developing species-specific DNA marker(s) for the authentication of O. tenuiflorum. A species-specific amplicon (279 bp) generated through an inter-simple sequence repeat marker (UBC 835) in all individuals of O. tenuiflorum was cloned, sequenced, and a primer pair was developed (designated as CIM-OT-835F/CIM-OT-835R). The newly developed sequence characterized amplified region marker was validated through PCR amplification in all available seven species of Ocimum, and its specificity for O. tenuiflorum was confirmed with the consistent generation of an amplicon of 177 bp. The developed marker can be used for accurate and rapid identification of the species for certification purposes and will be useful in quality control of medicinal preparations containing this important medicinal species.

Key words

adulteration - ISSR - Lamiaceae - Ocimum species - SCAR marker - authentication

• References

• 1 Shukla AK, Mall M, Rai SK, Singh S, Nair P, Parashar G, Shasany AK, Singh SC, Joshi VK, Khanuja SPS. A transcriptomic approach for exploring the molecular basis for dosha-balancing property-based classification of plants in Ayurveda. Mol Biol Rep 2013; 40: 3255-3262
• 2 Carvoic-Stanko K, Liber Z, Besendorfer V, Javornik B, Bohanec B, Kolak I, Satovic Z. Genetic relations among basil taxa (Ocimum L.) based on molecular markers, nuclear DNA content, and chromosome number. Plant Syst Evol 2010; 285: 13-22
• 3 Rastogi S, Kalra A, Gupta V, Khan F, Lal RK, Tripathi AK, Parameswaran S, Gopalakrishnan C, Ramaswamy G, Shasany AK. Unravelling the genome of Holy basil: an “incomparable” “elixir of life” of traditional Indian medicine. BMC Genomics 2015; 16: 413
• 4 Kumar A, Mishra P, Baskaran K, Shukla AK, Shasany AK, Sundaresan V. Higher efficiency of ISSR markers over plastid psbA-trnH region in resolving taxonomical status of genus Ocimum L. Ecol Evol 2016; 6: 7671-7682
• 5 Shasany AK. The Holy basil (Ocimum sanctum L.) and its genome. Indian J Hist Sci 2016; 51: 343-350
• 6 Prakash O, Jyoti AK, Kumar P, Manna NK. Adulteration and substitution in Indian medicinal plants: an overview. J Med Plants Stud 2013; 1: 127-132
• 7 Kumar SP. Adulteration and substitution in endangered ASU medicinal plants of India: a review. Int J Med Arom Plants 2014; 4: 56-73
• 8 Kumar A, Mishra P, Singh SC, Sundaresan V. Efficiency of ISSR and RAPD markers in genetic divergence analysis and conservation management of Justicia adhatoda L., a medicinal plant. Plant Syst Evol 2014; 300: 1409-1420
• 9 Kiran U, Khan S, Mirza KJ, Ram M, Abdin MZ. SCAR markers: a potential tool for authentication of herbal drugs. Fitoterapia 2010; 81: 969-976
• 10 Cheng JL, Yin ZC, Mei ZQ, Wei CL, Chen HC, Wu XS, Fu JJ. Development and significance of SCAR marker QG12–5 for Canarium album (Lour.) Raeusch by molecular cloning from improved RAPD amplification. Genet Mol Res 2016;
• 11 Hernandez P, Martin A, Dorado G. Development of SCARs by direct sequencing of RAPD products: a practical tool for the introgression and marker-assisted selection of wheat. Mol Breed 1999; 5: 245-253
• 12 Fu S, Cheng J, Wei C, Yang L, Xiao X, Zhang D, Stewart MD, Fu J. Development of diagnostic SCAR markers for genomic DNA amplifications in breast carcinoma by DNA cloning of high-GC RAMP-PCR fragments. Oncotarget 2017; 8: 43866-43877
• 13 Zietkiewicz E, Rafalski A, Labuda D. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics 1994; 20: 176-183
• 14 Mishra P, Kumar LD, Kumar A, Gokul S, Ravikumar K, Shukla AK, Sundaresan V. Population dynamics and conservation implications of Decalepis arayalpathra (J. Joseph and V. Chandras.) Venter., a steno endemic species of Western Ghats, India. Appl Biochem Biotechnol 2015; 176: 1413-1430
• 15 Chowdhury MA, Vandenberg B, Warkentin T. Cultivar identification and genetic relationship among selected breeding lines and cultivars in chickpea (Cicer arietinum L.). Euphytica 2002; 127: 317-325
• 16 Gupta M, Verma B, Kumar N, Chahota RK, Rathour R, Sharma SK, Bhatia S, Sharma TR. Construction of intersubspecific molecular genetic map of lentil based on ISSR, RAPD and SSR markers. J Genet 2012; 91: 279-287
• 17 Yuan XF, Dai ZH, Wang XD, Zhao B. Assessment of genetic stability in tissue-cultured products and seedlings of Saussurea involucrata by RAPD and ISSR markers. Biotechnol Lett 2009; 31: 1279-1287
• 18 Joshi K, Chavan P, Warude D, Patwardhan B. Molecular markers in herbal drug technology. Curr Sci 2004; 87: 159-165
• 19 Lee MY, Doh EJ, Chae HP, Young HK, Eung SK, Ko BS, Oh SE. Development of SCAR marker for discrimination of A. princeps and A. argyi from other Artemisia herbs. Biol Pharm Bull 2006; 29: 629-630
• 20 Dnyaneshwar W, Preeti C, Kalpana J, Bhushan P. Development and application of RAPD-SCAR marker for identification of Phyllanthus emblica L. Biol Pharm Bull 2006; 29: 2313-2316
• 21 Jain N, Shasany AK, Singh S, Khanuja SPS, Kumar S. SCAR markers for correct identification of Phyllanthus amarus, P. fraternus, P. debilis and P. urinaria used in scientific investigations and dry leaf bulk herb trade. Planta Med 2008; 74: 296-301
• 22 Theerakulpisut P, Kanawapee N, Maensiri D, Bunnag S, Chantaranothai P. Development of species-specific SCAR markers for identification of three medicinal species of Phyllanthus . J Syst Evol 2008; 46: 614-621
• 23 Wang J, Ha WY, Ngan FN, But PPH, Shaw PC. Application of sequence characterised amplified region (SCAR) analysis to authenticate Panax species and their adulterants. Planta Med 2001; 67: 781-783
• 24 Choi YE, Ahn CH, Kim BB, Yoon ES. Development of species specific AFLP-derived SCAR marker for authentication of Panax japonicus C. A. MEYER. Biol Pharm Bull 2008; 31: 135-138
• 25 Huh MK, Bang KH. Identification of Atractylodes japonica and Amacrocephala by RAPD analysis and SCAR markers. Silvae Genet 2006; 55: 101-105
• 26 Adinolfi B, Chicca A, Martinotti E, Breschi MC, Nieri P. Sequence characterized amplified region (SCAR) analysis on DNA from three medicinal Echinacea species. Fitoterapia 2007; 78: 43-45
• 27 Devaiah KM, Padma V. Development of SCAR marker for authentication of Pueraria tuberose (Roxb. ex. Willd.) DC. Curr Sci 2008; 94: 1306-1309
• 28 Choo BK, Moon BC, Yunui JI, Kim BB, Choi G, Yoon T, Kim HK. Development of SCAR markers for the discrimination of three species of medicinal plants, Angelica decursiva (Peucedanum decursivum), Peucedanum praeruptorum and Anthricus sylvestris, based on the internal transcribed spacer (ITS) sequence and random amplified polymorphic DNA (RAPD). Biol Pharm Bull 2009; 32: 24-30
• 29 Basha SD, Francis G, Makkar HPS, Becker K, Sujatha M. A comparative study of biochemical traits and molecular markers for assessment of genetic relationships between Jatropha curcas L. germplasm from different countries. Plant Sci 2009; 176: 812-823
• 30 Anuntalabhochai S, Sitthiphrom S, Thongtaksin W, Sanguansermsri M, Cutler RW. Hybrid detection and characterization of Curcuma species using sequence characterized DNA markers. Sci Hort 2007; 111: 389-393
• 31 Mishra P, Kumar A, Nagireddy A, Mani DN, Shukla AK, Tiwari R, Sundaresan V. DNA barcoding: an efficient tool to overcome authentication challenges in the herbal market. Plant Biotechnol J 2016; 14: 8-21
• 32 Bhamra S, Heinrich M, Howard C, Johnson M, Slater A. DNA authentication of tulsi (Ocimum tenuiflorum) using the nuclear ribosomal internal transcribed spacer (ITS) and the chloroplast intergenic spacer trnH-psbA . Planta Med 2015; 81: PW_20
• 33 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Bio 1990; 215: 403-410
• 34 Khanuja SPS, Shasany AK, Darokar MP, Kumar S. Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oils. Plant Mol Biol Report 1999; 17: 1-7
• 35 Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG. Primer3 – new capabilities and interfaces. Nucleic Acids Res 2012; 40: e115