American Journal of Life Sciences 2016; 4(5): 118-128 http://www.sciencepublishinggroup.com/j/ajls doi: 10.11648/j.ajls.20160405.14 ISSN: 2328-5702 (Print); ISSN: 2328-5737 (Online) Identification of Nigella sativa Seed and Its Adulterants Using DNA Barcode Marker Sudhir S. P. 1, * , Alagappan Kumarappan 2 , Lalit K. Vyas 3 , Divya Shrivastava 1 , Padma Deshmukh 4 , H. N. Verma 1 1 Department of Life Science, Jaipur National University, Jaipur, India 2 Department of Microbiology, Marine Biology, and Virology, University of Modern Sciences, Dubai, UAE 3 Vidyabahrti Mahavidyalaya, Department of Cosmetic Technology, Amravati, India 4 Department of Microbiology, Smt. C.H.M. College of Arts, Commerce and Science, Ulhasnagar, Mumbai, India Email address: spsjaipurnationaluniversity@gmail.com (Sudhir S. P.) * Corresponding author To cite this article: Sudhir S. P., Alagappan Kumarappan, Lalit K. Vyas, Divya Shrivastava, Padma Deshmukh, Prof H. N. Verma. Identification of Nigella sativa Seed and Its Adulterants Using DNA Barcode Marker. American Journal of Life Sciences. Vol. 4, No. 5, 2016, pp. 118-128. doi: 10.11648/j.ajls.20160405.14 Received: September 15, 2016; Accepted: September 28, 2016; Published: October 19, 2016 Abstract: Adulteration, misidentification, and substitution are the biggest challenges in maintaining safety and therapeutic efficacy of medicinal herbs. Nigella sativa seed, which is well known medicinal herb susceptible to adulteration or substitution due to its great therapeutic value. Adulteration and substitution by morphologically similar seeds are the primary concern in commercially available Nigella sativa seed. In this study, we have used DNA barcode marker to find out adulteration, misidentification, and substitution of Nigella sativa seed sold in various markets. We collected 10 samples, which were labelled as Black seed/Nigella sativa seed from open markets in India (1 No.), Pakistan (1 No.), Saudi Arabia(1 No.), Egypt (2 No.), Turkey (1 No.), Syria (1 No.), Tunisia(2 No.) and Oman (1 No.). All samples collected from different geographies were studied morphologically. Although few samples were quickly identified as Nigella sativa seeds, few were tough to detect and differentiate accurately. This is where DNA barcode marker proved to be useful. Plant DNA were obtained from seed coat cells of samples, was amplified by PCR with forward and reverse rbcl and matK primers as recommended by CBOL (The Consortium for the Barcode of Life). PCR amplification of plastid genome with matK was not very successful, while PCR amplification with rbcl primers was quite successful. We used rbcl sequences for alignment and further analysis. PCR products obtained were subjected to electrophoresis on 1.5% agarose plate. PCR products were sent to Macrogen (Seoul, South Korea) for DNA sequencing. DNA reads obtained with rbcl sequences were aligned and analyzed for nucleotide composition, conserved sites, variable sites, singleton sites and parsimony-informative sites, genetic distance and phylogenetic tree using MEGA 7. The phylogenetic tree was constructed using UPGMA method. NCBI Blast along with phylogenic tree and nucleotide characteristic were used to identify Nigella sativa seeds from different geographies and discriminate two adulterants as Allium cepa seed and Clitoria guianensis seed. Both of these adulterants are different regarding their active medicinal contents and therapeutic utility from Nigella sativa seed. This study proved the utility of DNA marker, especially rbcl loci in accurately identifying medicinal herb and its adulterants. Keywords: Nigella sativa, Kalongi, DNA Barcoding, Molecular Markers, rbcl, Matk, Adulteration, Misidentification 1. Introduction Worldwide trade of medicinal herb is about $ 60 billion dollar business annually. There are about 1000 companies from different countries involved in the trading of medicinal herbs, Business of medicinal herbs is growing at the rate of 15 to 20% per year [1]. This growth in the trade of herbal medicine is due to significant demand for natural, safe and reliable therapeutic agents. Patients want a more safe, secure and natural way of treatment of diseases.