National Conference on Green Engineering and Technologies for Sustainable Future-2014 Journal of Chemical and Pharmaceutical Sciences ISSN: 0974-2115 Genetic variation of Dioscorea oppositifolia L. From four different regions of , R.Uma Maheswari a, R.Selvarajan b, V.NandaGopalanc, A.Lakshmi Prabhaa* aDepartment of Biotechnology, Pavendar Bharathidasan College of Arts and Science, Tiruchirappalli. bDepartment of Molecular Virology, National Centre for Banana Research. cDepartment of Botany, National College, Tiruchirappalli. a*Department of Plant Science, Bharathidasan University, Tiruchirappalli. *Corresponding Author: Email: [email protected] ABSTRACT In 2050, the population around the Earth is expected to reach a maximum of 9 billion and healthcare is being an important issue because of the increasing demand for the drugs. Among the monocots the biosynthesise of steroidal saponins are authentically reported in Agavaceae (Agave), Dioscoreaceae (Dioscorea) and Liliaceae (Allium, Asparagus, Lillium). Many species of the Dioscorea are considered as an important source for pharmaceutical industry and medicine especially for steroidal saponin which is the raw material for the synthesis of sex hormones and corticosteroid. Dioscorea oppositifolia L. were collected from four different regions of Tamilnadu –, Pachaimalai, Sirumalai and Pulivalam Plains.DNA were isolated and PCR Reactions were carried out using RAPD and ISSR Markers. Population genetics were identified by constructing dendrogram through NTSYS software. This is the first report to analyse the genetic variation in Dioscorea oppositifolia collected from four different regions in Tamilnadu for pharmaceutical purposes. Key words: Dioscorea oppositifolia L., Kolli hills, Pachamalai, Sirumalai and Pulivalam, RAPD and ISSR Markers. INTRODUCTION The world average annual yield of yams was 10.2 tons per hectare in 2010. The most productive yam farms in the world were in Colombia, where nationwide average annual yield was 28.3 tons per hectare. Dioscorea (true yams) is a large genus that contains more than 600 species, some are important for food (with edible tubers) and others are rich sources of bioactive compounds used in a wide range of applications. Dioscorea is a major staple food in West Africa and the Pacific islands, and are widely used as a famine food. Twenty–six species of Dioscorea have been found in India. Kolli Hills constituting the of South India covering about 280 km and are located at latitude: 10°12′ - 11°7 ′ N, longitude: 76°-77°17′ E at altitudes ranging from 1000 to 1300m above MSL. Sirumalai hills is located in district of Tamilnadu between 10 ̊07’ 10 ̊18’N Latitude and 77 ̊55’-78 ̊ 12’E longitude. Located near 11°11′N 78°21′E / 11.18°N 78.35°E / 11.18; 78.35. Dioscorea oppositifolia L. (Dioscoreaceae) is used traditionally for antiseptics, ulcers and asthma. The traditional uses of Dioscorea oppositifolia L. rhizome are prescribed for indigestion, anorexia, diarrhea, and diabetes. It also contains dioscin, diosgenin, phytic acid, allantoin, dopamine, batatasin, dioscorea-mucilage B, and sterols. The extracted diosgenin is used for the commercial synthesis of cortisone, pregnenolone, progesterone, and other steroid products. In Tamilnadu 7 species –Dioscorea alata, D.ulbifera, D.esculenta, D.oppositifolia, D.pentaphylla, D.tomentosa and D. wallichi were found as edible tubers are used as food by the tribal Kanikkars of Kanyakumari district, Tamilnadu, India in times of food shortage but only after extensive detoxification processes. Recently, molecular-based techniques, such as the random amplified polymorphic DNA technique and ISSR analysis of DNA by polymerase chain reaction (PCR) have been recognized as the most efficient tools to detect genetic diversity and assess phylogenic relationships. The literature survey shows that the four Dioscorea oppositifolia L. under investigation have not been previously genetically profiled. The objective of the present study was to analyse genetic profile of four Dioscorea oppositifolia from Tamilnadu, India. MATERIALS AND METHODS All the chemicals and reagents were purchased from Sigma Adrich, USA..Plants were collected from different altitudes of various hills like Kolli Hills, Pachamalai, Sirumalai, and Pulivalam (Ref No. UM001, UM002, UM003 and UM004) were authenticated in Botanical Survey of India, T.N.A.U, Coimbatore,Tamilnadu,India. DNA Preparation: DNA were isolated as per the protocol described by and then the isolated DNA was air dried and dissolved in 200 μL nuclease free water. RAPD and ISSR Reactions: Fourty random 10-base primers were used in this study .PCR was carried out in a 25 μL reaction volume containing 10 μL buffer, 2.0 mM of each dNTP (dATP, dCTP, dGTP, and dTTP), 2.5 μM of primer (Operon), 0.25μM of TaqDNA polymerase (Boehringer Mannheim), MgCl2 0.5 μM and 5ng template DNA. Samples were subjected to 40 cycles consisting of denaturation for 3 min at 95°C, annealing for 1 min at 50°C, extension for 1.30 min at 72°C, and one final extension cycle at 72°C for 10min. After completion of the PCR, 10 μL of reaction mixture was loaded into a 1.2% agarose gel, containing 0.5 μg/ mL ethidium bromide for electrophoresis and subsequent visualization by UV transillumination. For ISSR marker,after completion of the PCR, 10 μL of reaction mixture was loaded into a 2% High Resolution agarose gel, containing 0.5 μg/mL ethidium bromide for electrophoresis and subsequent visualization by UV transillumination. The PCR amplified products from individual plants were scored as either presence or absence. Analysis of similiarity matrix within the NTSYS program. RESULTS AND DISCUSSION Twenty two primers produced polymorphic products and one hundred thirty seven amplified markers were generated by using the eighteen primers, 164 of which (96.2%) were polymorphic. Of twenty two primers, Primer OPP-03 resulted in eleven distinct fingerprints, (Figure: 1) could be clearly differentiated and Phylogenetic tree were constructed (Figure: 3 and Figure: 4). OPA 3 had generated eleven different RAPD patterns than others. Results showed that a high degree of genetic variability existed within same species of Dioscorea oppositifolia L., which originated from four different places of Tamilnadu Pulivalam species of Dioscorea JCHPS Special Issue 4: December 2014 www.jchps.com Page 36 National Conference on Green Engineering and Technologies for Sustainable Future-2014 Journal of Chemical and Pharmaceutical Sciences ISSN: 0974-2115 oppositifolia were classified into different subgroups on the basis of RAPD and ISSR data, and could be clearly distinguished by RAPD as well as ISSR analysis by combination of both RAPD and ISSR primers. The literature survey shows that the four Dioscorea oppositifolia L. under investigation here have not been previously genetically profiled.

1 2 3 4 5 6 7 8 9 10 11 12 M 1 2 3 4 5 6 7 8 9 10 11 12 M

Figure 1: Polymorphism patterns RAPD-OPA-03 Primer 36 - Figure 2: Polymorphism patterns – ISSR CTCTCTCTCTCTCTCTA (Lane1-3 Kolli Hills Lane 4-6- Pachamalai samples Lane 7-9 Sirumalai Samples Lane 10-12 Pulivalam,M-Marker)

Dendrogram showing genetic diversity Figure.3 RAPD and Figure.4. ISSR Markers

CONCLUSION The main investigation of our study is there is a strong correlation of secondary metabolite contents with that of RAPD and ISSR data among the four Dioscorea oppositifolia L. from Tamilnadu, India. So, we conclude that Kolli hills in Tamilnadu are the best one for producing of high medicinal properties containing plants which can be used for pharmacology purposes with high genetic diversity nature. ACKNOWLEDGEMENTS The authors thank Department of Molecular Virology, National Centre for Banana Research (NRCB),ICAR for providing lab facilities to carry the research successfully and also thanks UGC for providing Rajiv Gandhi fellowship for financial support. REFERENCES Caetano-Anolles G, Bassam BJ and Gresshoff PM, Primer-template interactions during DNA amplification fingerprinting with single arbitrary oligonucleotides. Mol. Gen. 235, 1992, 157-165. Chang, H. C., Na, C., Hsieh, M. T., Kan, W. S., Chang, Y. S., Liu, S. Y., Liou, P. C, Study on new varieties of DioscoreaalataL. in Taiwan and its isozymes, Proceeding of a Symposium on Development and Utilization of Resources of Medicinal Plants in Taiwan, TARI special publication No. 48, 1995, 49-68. Cheng, K. T. and Liu, S. Y, Genetic relationships in Dioscoera alata revealed by RAPD analysis. Chin. Pharm. J, 48, 1996, 197-205. Djerassi C, Steroid research at Syntex: "the pill" and cortisone, Steroids, 57 (12), 1992, 631–41. Gawel, N. J. and Jarret, R. L, Cytoplasmic genetic diversity in banana and plantain, Euphytica, 52, 1991, 19-23. Geary, S. J., Forsyth, M. H., Saoud, S. A., Wang, G., Berg, D. E. and Berg, C. M, Mycoplasmagallisepticum strain differentiation by arbitrary primer (RAPD) fingerprinting. Mole. Cell Probe, 8, 1994, 311-316.

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