POPULATION STRUCTURE OF KAEMPFERIA GALANGA L. FROM EASTERN INDIA Original Article REENA PARIDA, SUJATA MOHANTY, SANGHAMITRA NAYAK Centre for Biotechnology, SPS, Siksha ‘O’ Anusandhan University, Bhubaneswar, Odisha, India Email: ms.reenas@gmail.com Received: 07 Dec 2018 Revised and Accepted: 26 Jan 2019 ABSTRACT Objective: India has been a producer of a large number of aromatic medicinal plants which serves as a valuable genetic resource for future quality improvement to meet the ever-growing demand of human essential products. Thus, an urgent need arises for germplasm conservation of these high yielding varieties to help the pharmaceutical and other industries. For this understanding, the population structure is essential in order to explore their genetic identification by fingerprinting and molecular characterization. Methods: In the present study DNA was isolated using modified Cetyl Trimethyl Ammonium Bromide (CTAB) method and Polymerase Chain Reaction (PCR) was performed according to standardized method along with its data analysis. This study was undertaken to characterize the highly medicinal Kaempferia galanga collected from 4 different populations of Odisha using the molecular markers as Random Amplified Polymorphic DNA and Inter-Simple Sequence Repeats for the first time. Results: A dendrogram constructed through Sequential Agglomerative Hierarchical and Nested (SAHN) clustering and Unweighted Pair Group Method with Arithmetic mean (UPGMA) analysis showed an average similarity of 0.993 ranging between 0.967 to 1.000. Jaccard’s similarity coefficient of combined markers segregated the genotypes into two main clusters, 1 with six samples and the others at 0.98 similarity coefficient. Conclusion: Hence, the molecular analysis could be further used for the identification of important novel gene present in Kaempferia galanga which can be utilized for future crop improvement as well as pharmacological activities. Keywords: Kaempferia galanga, Polymerase Chain Reaction, Random Amplified Polymorphic DNA, Inter-Simple Sequence Repeats © 2019 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) DOI: http://dx.doi.org/10.22159/ijpps.2019v11i3.31226 INTRODUCTION Kaempferia galanga L. (Zingiberaceae) an important medicinal herb is distributed in Asia and Africa [1]. Kaempferia galanga locally known as ‘gandhasunthi’ is famous as a health-promoting herb used in ayurvedic drugs, perfumery, cosmetic industries and as spice ingredients [2-5]. The plants have handsome, showy flowers and foliage due to their arching form and shining leaves and are cultivated as ornamentals. The plant stays close to the ground achieving a height of 3 inches, no central stem and its leaves just grow right off the rhizomes up to 6 inches. The plant blossoms with small white fragrant flowers with a splash of purple at the center but lasts only for a few hours. Leaves are used to flavour foodstuffs, mouthwashes possessing antioxidant, antinociceptive and anti- inflammatory activities [6, 7]. The rhizomes are used to treat piles, tumours, coughs, epilepsy, asthma, spleen disorders, fever, abdominal pain, toothache, flatulence and anti-food-borne bacteria [8-9]. Natural sunscreens and Sun Protection Factor (SPF) boosters, a natural material that protects from Ultra Violet (UV) rays also isolated from Kaempferia galanga has been reported. This plant is economically important having a price value of Rs.300/Kg in dry rhizomes whereas its oil is US$ 700/Kg internationally and is used by tribal and pharmaceuticals. The plant is conventionally propagated by rhizome with very low multiplication rate. Also due to demand and depletion of this highly valued medicinal plant, it is decreasing naturally and being recognized as an endangered [10]. The taxonomic identification of Kaempferia is difficult without its floral parts due to the problem in its morphological similarity with other related Zingiberaceae species. This paper aims the novelty of steps taken to identify the Kaempferia galanga species in molecular level present in various places of eastern India which is not yet reported. The species after identification could be used for various activities testing by the pharmaceutical industries and others to be used as medicines. This technique could further help in multiplication and conservation of the plant to fulfil the ever- growing human demand. MATERIALS AND METHODS Plant sample collection and molecular analysis Kaempferia galanga were collected from the wild habitats of Koraput (18 °82' N, 82 °72' E), Rourkela (22 °25' N, 85 °00' E), Mayurbhanj (21 °93' N, 86 °73' E) and Jagatsinghpur population (20 °16' N, 86 °10' E) of Odisha. DNA isolation from fresh 2 gm of leaves was done by Doyle and Doyle method [11]. Polymerase Chain Reaction (PCR) techniques namely Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeats (ISSR) were done [12,13]. RAPD primers (Operon Tech., Alameda, USA) were dissolved in double sterilized T 10E 1 buffer, pH 8.0 to the working concentration of 15 ng/µl. 19 RAPD primers as per banding pattern were selected namely A4, A7, A9, A18, C2, C5, C11, D3, D7, D8, D12, D18, D20, N4, N16, N18, AF5, AF14 and AF15. 9 ISSR primers similarly used as (GAC) 5 , (GTGC) 4 , (GACA) 4 , (AGG) 6 , (GA) 9 T, T(GA) 9, (GTG) 5 , (GGA) 4 and (CAA) 5 Data scoring from Bangalore Genei Pvt. Ltd, India. PCR product for both RAPD and ISSR was electrophoresed with 1.5% and 2% agarose gel and ethidium bromide at 60 volts for three hours. These were visualized in UV-transilluminator (BioRad, USA) and gel documented in Gel Documenting System (Bio-Rad, USA) for band details. The scoring of bands as ‘1’ and ‘0’ for presence and absence of amplified products was done [14]. Statistical analysis Resolving power (Rp) and Primer Index (PI) Rp of both were calculated as Rp=ΣIB (IB (Band informativeness) = 1-[2×(0.5-P)], P is the proportion of species containing band [15]. P I was calculated from the polymorphic index. The polymorphic index value was calculated as PIC = 1-∑P 2 i , P i is the frequency of band of i th allele [16]. In both cases, PIC was 1-p 2 -q 2 , where p is the frequency International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 11, Issue 3, 2019