AJPS  Vol.4 No.8 , August 2013
Assessment of Genetic Diversity in Nepalese Populations of Swertia chirayita (Roxb. Ex Fleming) H. Karst Using RAPD-PCR Technique
Abstract: Owing to the high demand, Swertia chirayita populations in the wild are being depleted beyond its regeneration capacity. S. chirayita is one of the most valuable medicinal plants of Nepal in trade. Present Molecular investigation was undertaken to understand the level of genetic diversity in five S. chirayita populations of Nepal using Polymerase Chain Reaction (PCR)-based Random amplified polymorphic DNA (RAPD) technique. Thirty four accessions of S. chirayita along with six outlier accessions were analyzed using 26 arbitrary primers. Of the total 285 amplified bands scored for S. chirayita, 263 bands (92.28%) were polymorphic. Two major clusters were revealed in the phenogram generated from cluster analysis using NTSYS-PC software (version 2.21i) for the geographic populations under study. Principal Coordinate Analysis further substantiated the results of the phenograms. Swertia chirayita populations from Sankhuwasabha and Terathum were found to be genetically closest (68%, similar) whilst Nagarjun and Terathum were found to be most distant (33%, similar).The high genetic polymorphism reflected in S. chirayita populations indicates the good survival potentiality and adaptability in changing environmental scenario. The results thus produced might be helpful to plant breeders for elite cultivar development. The RAPD-PCR technique is found to be the rapid and effective tool for genetic diversity assessment in S. chirayita populations and generated insights for the formulation of conservation strategy of this vulnerable species together with its phytochemical distinctiveness.
Cite this paper: J. Shrestha, T. Bhattarai, J. Sijapati, N. Rana, J. Maharjan, D. Rawal, B. Raskoti and S. Shrestha, "Assessment of Genetic Diversity in Nepalese Populations of Swertia chirayita (Roxb. Ex Fleming) H. Karst Using RAPD-PCR Technique," American Journal of Plant Sciences, Vol. 4 No. 8, 2013, pp. 1617-1628. doi: 10.4236/ajps.2013.48196.

[1]   S. K. Ghimire, “Medicinal Plants in the Nepal Himalaya: Current Issues, Sustainable Harvesting, Knowledge Gaps and Research Priorities,” In: P. K. Jha, S. B. Karmacharya, M. K. Chhetri, C. B. Thapa and B. B. Shrestha, Eds., Medicinal Plants in Nepal: Anthology of Contemporary Research, Ecological Society (ECOS), Kathmandu, Nepal, 2008, pp. 25-42.

[2]   DPR, “Prioritized Medicinal Plants of Nepal for Research and Cultivation,” Department of Plant Resources Publication, Ministry of Forests and Soil Conservation, Government of Nepal, 2005.

[3]   L. Struwe and V. A. Albert, “Gentianaceae—Systematics and Natural History,” Cambridge University Press, Cambridge, 2002.

[4]   K. Joshi, “Molecular Differentiation and Phylogeny of Swertia (Gentianaceae) of the Himalayan Region, Nepal,” International Journal of Biotechnology and Biochemistry, Vol. 7, No. 2, 2011, pp. 265-277.

[5]   J. R. Press, K. K. Shrestha and D. A. Sutton, “Annotated Checklist of the Flowering Plants of Nepal,” The Natural History Museum, London, 2000, pp. 117-119.

[6]   D. P. Rijal, “Taxonomic Study of Some Medicinally Important Species of Swertia L. (Gentianaceae) in Nepal,” Botanica Orientalis—Journal of Plant Science, Vol. 6, 2009, pp. 18-24.

[7]   T. P. Barakoti, T. Chapagain, Y. Thapa and C. Bhusal, “Chiraito Conservation and Cultivation Workshop and Achievement,” Nepal Agriculture Research Centre, Pakhribas, 1999.

[8]   T. P. Barakoti, “Commercial Cultivation and Production Management of Chiraito: Scheme Guide,” ARS Pakhribas, Nepal Agricultural Research Council, Nepal, 2002, pp. 1-50.

[9]   K. Joshi and A. Joshi, “Swertia L. (Gentianaceae) in Nepal Himalaya: Checklist, Phytogeography, Ethnobotany and Conservation Status,” Ethnobotanical Leaflets, Vol. 12, 2008, pp. 361-372.

[10]   T. P. Barakoti, “Attempts Made for Domestication, Conservation and Sustainable Development of Chiretta (Swertiachirayita),” Nepal Agriculture Research Centre (NARC), Dhankuta, 2004.

[11]   S. Phoboo, P. K. Jha and P. C. Bhowmik, “Biology and Phytochemistry of Swertiachirayita,” In: P. K. Jha, S. B. Karmacharya, M. K. Chhetri, C. B. Thapa and B. B. Shrestha, Eds., Medicinal Plants in Nepal: Anthology of Contemporary Research, Ecological Society (ECOS), Kathmandu, 2008, pp. 203-211.

[12]   M. B. Bajpai, R. K. Asthana, N. K. Sharma, S. K. Chatterji and S. K. Mukherjee, “Hypoglycemic Effect of Swerchirin from the Hexane Fraction of Swertia chirayita,” Planta Medica, Vol. 57, No. 2, 1991, pp. 102-104. doi:10.1055/s-2006-960041

[13]   N. Pant, D. C. Jain and R. S. Bhakuni, “Phytochemicals from Genus Swertia and Their Biological Activities,” Indian Journal of Chemistry, Vol. 39, 2000, pp. 565-586.

[14]   P. Joshi and V. Dhawan, “Swertia chirayita—An Overview,” Current Science, Vol. 89, No. 4, 2005, pp. 635-640.

[15]   P. Saha, S. Mandal, A. Das, P. C. Das and S. Das, “Evaluation of Anti-carcinogenic Activity of Swertia chirata Buch. Ham, an Indian Medicinal Plant on DMBA-Induced Mouse Skin Carcinogenesis Model,” Phytotherapy Research, Vol. 18, No. 5, 2004, pp. 373-378. doi:10.1002/ptr.1436

[16]   H. Verma, P. R. Patil, R. M. Kolhapure and V. Gopalkrishna, “Antiviral Activity of the Indian Medicinal Plant Extract, Swertia chirata against Herpes Simplex Virus: A Study by in Vitro and Molecular Approach,” Indian Journal of Medical Microbiology, Vol. 26, No. 4, 2008, pp. 322-326. doi:10.4103/0255-0857.43561

[17]   S. Phoboo and P. K. Jha, “Trade and Sustainable Conservation of Swertia chirayita (Roxb. Ex Fleming) H. Karst in Nepal,” Nepal Journal of Science and Technology, Vol. 11, 2010, pp. 125-132.

[18]   S. Phoboo, M. D. S. Pinto, A. C. L. Barbosa, D. Sarkar, P. C. Bhowmik, P. K. Jha, et al., “Phenolic-Linked Biochemical Rationale for the Anti-Diabetic Properties of Swertia chirayita (Roxb. Ex Flem) Karst,” Phytotherapy Research, Vol. 27, No. 2, 2012, pp. 227-235.

[19]   IUCN, “National Register of Medicinal and Aromatic Plants,” IUCN Nepal Publication, 2004.

[20]   B. Y. Wang, L. Shi, Z. Y. Ruan and J. Deng, “Genetic Diversity and Differentiation in Dalbergiasissoo (Fabaceae) as Revealed by RAPD,” Genetics and Molecular Research, Vol. 10, No. 1, 2011, pp. 114-120. doi:10.4238/vol10-1gmr995

[21]   O. H. Frankel, “The Place of Management in Conservation,” In: C. M. Schonewald-Cox, S. M. Chambers, B. MacBryde and L. Thomas, Eds., Genetics and Conservation: A Reference Manual for Managing Wild Animals and Plant Populations, Benjamin/Cummings, Menlo Park, 1993, pp. 1-14.

[22]   V. Arya, S. Yadav and J. P. Yadav, “Intra-Specific Genetic Diversity of Different Accessions of Cassia occidentalis by RAPD Markers,” Genetic Engineering and Biotechnology Journal, Vol. 22, 2011, pp. 1-8.

[23]   T. N. Khoshoo and S. R. Tandon, “Cytological, Morphological and Pollination Studies on Some Himalayan Species of Swertia,” Caryologia, Vol. 16, 1963, pp. 445-477.

[24]   S. Chakraborty, D. Mukherjee and T. Dasgupta, “Cytological Study on Chromosome Behaviour and New Report on Nature of Mode of Pollination of Swertia chirayita, a High Value Endangered Medicinal Plant of North Eastern Himalayan Region,” Caryologia, Vol. 62, No. 1, 2009, pp. 43-52.

[25]   J. C. Avise, “Molecular Markers Natural History and Evolution,” 2nd Edition, Sinauer Associates Inc. Publishers, Sunderland, 2004.

[26]   K. Weising, H. Nybom, K. Wolff and G. Kahl, “DNA Fingerprinting in Plants Principles, Methods and Applications,” 2nd Edition, Taylor & Francis Group, Boca Raton, London, New York, Singapore, 2005. doi:10.1201/9781420040043

[27]   J. G. K. Williams, A. R. Kubelik, K. J. Livak, J. A. Rafalski and S. V. Tingey, “DNA Polymorphisms Amplified by Arbitrary Primers Are Useful as Genetic Markers,” Nucleic Acids Research, Vol. 18, No. 22, 1990, pp. 6531-6535. doi:10.1093/nar/18.22.6531

[28]   T. E. Dowling, C. Moritz, J. D. Palmer and L. H. Rieseberg, “Nucleic Acids III: Analysis of Fragments and Restriction Sites,” In: D. M. Hillis, C. Moritz and B. K. Mable, Eds., Molecular Systematic, 2nd Edition, Sinauer Associates Inc., Sunderland, 1996, pp. 249-282.

[29]   J. P. Clapp, “Species Diagnostics Protocols PCR and Other Nucleic Acid Methods,” Humana Press, Totowa, 1996, pp. 25-38.

[30]   K. Joshi, P. Chavan, D. Warude and B. Patwardhan, “Molecular Markers in Herbal Drug Technology,” Current Science, Vol. 87, No. 2, 2004, pp. 159-165.

[31]   S. Shrestha, S. W. Adkins, G. C. Graham and D. S. Loch, “An Identification Tool for the Australian Weedy Sporobolus Species Based on Random Amplified Polymorphic DNA (RAPD) Profiles,” Australian Journal of Agricultural Research, Vol. 56, No. 2, 2005, pp. 157-167. doi:10.1071/AR04180

[32]   H. H. Salem, B. A. Ali, T. H. Huang, D. N. Qin, X. M. Wang and Q. D. Xie, “Use of Random Amplified Polymorphic DNA Analysis for Economically Important Food Crops,” Journal of Integrated Plant Biology, Vol. 49, No. 12, 2007, pp. 1670-1680. doi:10.1111/j.1744-7909.2007.00582.x

[33]   N. J. Sucher and M. C. Carles, “Genome Based Approaches to the Authentication of Medicinal Plants,” Planta Medica, Vol. 74, No. 6, 2008, pp. 603-623. doi:10.1055/s-2008-1074517

[34]   S. S. Sandhu, C. Colombo, C. R. Bastos and W. J. Siqueira, “DNA Tagging of Blast Resistant Gene(s) in Three Brazilian Rice Cultivars,” Genetics and Molecular Biology, Vol. 26, No. 4, 2003, pp. 473-477. doi:10.1590/S1415-47572003000400011

[35]   K. P. S. Chandel and B. S. Joshi, “Multivariate Analysis in Green-Seeded Pea,” Indian Journal of Agricultural Science, Vol. 53, No. 4, 1983, pp. 198-200.

[36]   G. C. Graham, P. Mayers and R. J. Henry, “A Simplified Method for the Preparation of Fungal Genomic DNA for PCR and RAPD Analysis,” BioTechniques, Vol. 16, No. 1, 1994, pp. 48-50.

[37]   K. Yu and K. P. Pauls, “Optimisation of the PCR Program for RAPD Analysis,” Nucleic Acids Research, Vol. 20, No. 10, 1992, p. 2606. doi:10.1093/nar/20.10.2606

[38]   K. J. Edwards, “Randomly Amplified Polymorphic DNAs (RAPDs),” In: A. Karp, P. G. Isaac and D. S. Ingram, Eds., Molecular Tools for Screening Biodiversity—Plants and Animals, Chapman and Hall, London, 1998, pp. 171-179. doi:10.1007/978-94-009-0019-6_34

[39]   D. K. Transue, D. J. Fairbanks, L. R. Robison and W. R. Andersen, “Species Identification by RAPD Analysis of Grain Amaranth Genetic Resources,” Crop Science, Vol. 34, No. 5, 1994, pp. 1385-1389. doi:10.2135/cropsci1994.0011183X003400050044x

[40]   D. Botstein, R. L. White, V. Skolnick and V. Davis, “Construction of a Genetic Linkage Map in Man Using Restriction Fragment Length Polymorphisms,” American Journal of Human Genetics, Vol. 32, No. 3, 1980, pp. 314-331.

[41]   A. Prevost and M. J. Wilkinson, “A New System of Comparing PCR Primers Applied to ISSR Fingerprinting of Potato Cultivars,” Theoretical and Applied Genetics, Vol. 98, No. 1, 1999, pp. 107-112. doi:10.1007/s001220051046

[42]   P. H. A. Sneath and R. R. Sokal, “Numerical Taxonomy, the Principle and Practice of Numerical Classification, W H Freeman and Co., San Francisco, 1973.

[43]   R. R. Sokal and C. D. Michener, “A Statistical Method for the Evaluating Systematic Relationships,” University of Kansas, Kansas, 1958.

[44]   L. R. Dice, “Measures of the Amount of Ecologic Association between Species,” Ecology, Vol. 26, No. 3, 1945, pp. 297-302. doi:10.2307/1932409

[45]   P. Jaccard, “Nouvelles Recherché sur la Distribution Florale. Bulletin,” Societe Vaudoise Sciences Naturelles, Vol. 44, 1908, pp. 223-270.

[46]   N. A. Mantel, “The Detection of Disease Clustering and a Generalized Regression Approach,” Cancer Research, Vol. 27, No. 2, 1967, pp. 209-220.

[47]   R. R. Sokal, “Ecological Parameters Inferred from Spatial Correlograms,” In: G. P. Patil and M. Rosenzweig, Eds., Contemporary Quantitative Ecology and Related Econometrics, ICPH, 1979, pp. 167-196.

[48]   B. D. Singh, “Plant Breeding-Principle and Metods,” Kalyani Publishers, Ludhiana, 2000.

[49]   C. E. Thormann and T. C. Osborn, “Use of RAPD and RFLP Markers for Germplasm Evaluation,” In: Applications of RAPD Technology to Plant Breeding, Joint Plant Breeding Symposia Series, Minneapolis, 1992, pp. 9-11.

[50]   A. Arif, M. A. Bakir, H. A. Khan, A. H. Al Farhan, A. A. Al Homaidan, A. H. Bahkali, et al., “Application of RAPD for Molecular Characterization of Plant Species of Medicinal Value from an Arid Environment,” Genetics and Molecular Research, Vol. 9, No. 4, 2010, pp. 2191-2198. doi:10.4238/vol9-4gmr848

[51]   W. C. Black, “Statistical Analysis of Arbitrarily Primed PCR Patterns in Molecular Taxonomic Studies,” In: J. P. Clapp, Ed., Species Diagnostic Protocols: PCR and Other Nucleic Acid Methods, Humana Press Inc., Totowa, 1996, pp. 39-56.

[52]   P. Joshi and V. Dhawan, “Analysis of Genetic Diversity among Swertia chirayita Genotypes,” Biologia Plantarum, Vol. 51, No. 4, 2007, pp. 764-768. doi:10.1007/s10535-007-0156-z

[53]   D. Zhang, S. Chen, S. Hen, D. Zhang and Q. Gao, “Patterns of Genetic Variation in Swertia przewalskii, an Endangered Endemic Species of the Qinghai-Tibet Plateau,” Biochemical Genetics, Vol. 45, No. 1-2, 2007, pp. 33-50.

[54]   A. Teklewold and H. C. Becker, “Geographic Pattern of Genetic Diversity among 43 Ethiopian Mustard (Brassica carinata A. Braun) Accessions as Revealed by RAPD Analysis,” Genetic Resources and Crop Evolution, Vol. 53, No. 6, 2006, pp. 1173-1185. doi:10.1007/s10722-005-2011-4

[55]   W. Zheng, L. Wang, L. Meng and J. Liu, “Genetic Variation in the Endangered Anisodus tanguticus (Solanaceae), an Alpine Perennial Endemic to the Qinghai-Tibetan Plateau,” Genetica, Vol. 132, No. 2, 2008, pp. 123-129. doi:10.1007/s10709-007-9154-5

[56]   S. Sundaram and S. Purwar, “Assessment of Genetic Diversity among Fenugreek (Trigonella foenum-graecum L.), Using RAPD Molecular Markers,” Journal of Medicinal Plants Research, Vol. 5, No. 9, 2010, pp. 1543-1548.

[57]   C. Luce, J. L. Noyer, D. Tharreau, N. Ahmadiand and H. Feyt, “The Use of Microsatellite Markers to Examine the Genetic Markers to Examine the Genetic Resources of Rice (Oryza sativa L.) Adapted to European Conditions,” Acta Horticulturae, Vol. 546, 2001, pp. 221-234.

[58]   V. A. Sebastian, L. D. Cruz, R. B. Subramanian and V. J. Braganza, “Assessment of Genetic Diversity within and among Populations of Tylophora rotundifolia Using RAPD Markers,” Gene Conserve, Vol. 9, No. 37, 2010, pp. 94-117.

[59]   J. C. Rief, A. E. Melchinger and M. Frisch, “Genetical and Mathematical Properties of Similarity and Dissimilarity Coefficients Applied in Plant Breeding and Seed Bank Management,” Crop Science, Vol. 45, No. 1, 2005, pp. 1-7. doi:10.2135/cropsci2005.0001

[60]   M. Nei and W. H. Li, “Mathematical Model for Studying Genetic Variation in Terms of Restriction Endonucleases,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 76, No. 10, 1979, pp. 5269-5273.doi:10.1073/pnas.76.10.5269

[61]   K. V. Bhat, P. B. Prashant and S. Lakhanpaul, “Study of Genetic Diversity in Indian and Exotics Sesame (Sesamum indicum L.) Germplasm Using Random Amplified Polymorphic DNA (RAPD) Markers,” Euphytica, Vol. 110, No. 1, 1999, pp. 21-33. doi:10.1023/A:1003724732323

[62]   S. P. J. Prakash, K. R. Biji, S. M. Gomez, K. G. Murthy and R. C. Babu, “Genetic Diversity Analysis of Sorghum (Sorghum bicolor L. Moench) Accessions Using RAPD Markers,” Indian Journal of Crop Science, Vol. 1, No. 1-2, 2006, pp. 109-112.

[63]   M. Slatkin, “Gene Flow and Geographic Structure of Natural Populations,” Science, Vol. 236, No. 4803, 1987, pp. 787-792. doi:10.1126/science.3576198

[64]   D. Tilman, “Causes, Consequences and Ethics of Biodiversity,” Nature, Vol. 405, 2000, pp. 208-211. doi:10.1038/35012217