Abstract
Cymbopogon is an important genus of family Poaceae, cultivated mainly for its essential oils which possess high medicinal and economical value. Several cultivars of Cymbopogon species are available for commercial cultivation in India and identification of these cultivars was conceded by means of morphological markers and essential oil constitution. Since these parameters are highly influenced by environmental factors, in most of the cases, it is difficult to identify Cymbopogon cultivars. In the present study, Random amplified polymorphic DNA (RAPD) and Inter-simple sequence repeat (ISSR) markers were employed to discriminate nine leading varieties of Cymbopogon since prior genomic information is lacking or very little in the genus. Ninety RAPD and 70 ISSR primers were used which generated 63 and 69 % polymorphic amplicons, respectively. Similarity in the pattern of UPGMA-derived dendrogram of RAPD and ISSR analysis revealed the reliability of the markers chosen for the study. Varietal/cultivar-specific markers generated from the study could be utilised for varietal/cultivar authentication, thus monitoring the quality of the essential oil production in Cymbopogon. These markers can also be utilised for the IPR protection of the cultivars. Moreover, the study provides molecular marker tool kit in both random and simple sequence repeats for diverse molecular research in the same or related genera.
References
Jagadishchandra, K. S. (1975). Recent studies on Cymbopogon Spreng. (aromatic grasses) with special reference to Indian taxa: taxonomy, cytogenetics, chemistry and scope. Journal of Plantation Crops, 3, 43–57.
Pinder, A. R. (1960). Chemistry of terpenes. (pp. 38–40). Chapman and Hall Ltd, London
Subrahmanyan, V., & Bose, S.M. (1955). Bactericidal property of Indian essential oils with special reference to lemongrass oil. In Essential oils and Aromatic chemicals (pp. 1958). A symposium held at Dehra Dun October 6–9, under Joint Auspices of CSIR and Forest Research Institute and College CSIR, New Delhi.
Tyagi, B. K., Shahi, A. K., & Koul, B. L. (1988). Evaluation of repellent activities of Cymbopogon essential oils against mosquito vectors of Malaria, Filariasis and Dengue fever. Phytomedicine, 5, 324–329.
Nybom, H., & Bartish, I. (2000). Effects of life history traits and sampling strategies on genetic diversity estimates obtained with RAPD markers in plants. Perspectives in Plant Ecology Evolution and Systatics, 3, 93–114.
Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A., & Tingey, S. V. (1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research, 18, 6531–6535.
Zietkiewicz, E., Rafalski, A., & Labuda, D. (1994). Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics, 20, 176–183.
Karatash, H., & Agaoglu, S. Y. (2008). Genetic diversity among Turkish local grape accessions (Vitis vinifera L.) using RAPD markers. Hereditas, 145, 58–63.
Ahmad, F., Khan, A. I., Awan, F. S., & Sadia, B. (2010). Genetic diversity of chickpea (Cicer arietinum L.) germplasm in Pakistan as revealed by RAPD analysis. Genetics and Molecular Research, 9, 1414–1420.
Geetha, K. A., Kawane, A., Bishoyi, A. K., Phurailatpam, A., Ankita, C., Malik, S. K., Srinivasan, R., & Bhat, S. R. (2013). Characterization of mode of reproduction of Commiphora wightii [(Arnott) Bhandari] reveals novel pollen-pistil interaction and occurrence of obligate sexual female plants. Trees Structure and Function, 27, 567–581.
Bishoyi, A. K., Pillai, V. V., Geetha, K. A., & Maiti, S. (2014). Assessment of genetic diversity in Clitoria ternatea populations from different parts of India by RAPD and ISSR markers. Genetic Resources and Crop Evolution, 6, 1597–1609.
Ratnaparkhe, M. B., Santra, D. K., Tullu, A., & Muehlbauer, F. J. (1998). Inheritance of inter-simple-sequence-repeat polymorphisms and linkage with a fusarium wilt resistance gene in chickpea. Theory and Applied Genetics, 96, 348–353.
Desai, P., Gajera, B., Mankad, M., Shah, S., Patel, A., Patil, G., Narayanan, S., & Kumar, N. (2015). Comparative assessment of genetic diversity among Indian bamboo genotypes using RAPD and ISSR markers. Molecular Biology Reports, 42, 1265–1273.
Long, Y., Cheng, J., Mei, Z., Zhao, L., Wei, C., Fu, S., Khan, M. A., & Fu, J. (2015). Genetic analysis of litchi (Litchi chinensis Sonn.) in southern China by improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR). Molecular Biology Reports, 42, 159–166.
Sangwan, N. S., Yadav, U., & Sangwan, R. S. (2001). Molecular analysis of genetic diversity in elite Indian cultivars of essential oil trade types of aromatic grasses (Cymbopogon species). Plant Cell Reports, 20, 437–444.
Shasany, A. K., Lal, R. K., Patra, N. K., Darokar, M. P., Garg, A., Kumar, S., & Khanuja, S. P. S. (2000). Phenotypic and RAPD diversity among Cymbopogon winterianus Jowitt accessions in relation to Cymbopogon nardus Rendle. Genetic Resources and Crop Evolution, 47, 553–559.
Khanuja, S. P. S., Shasany, A. K., Pawar, A., Lal, R. K., Darokar, M. P., Naqvi, A. A., Rajkumar, S., Sundaresan, V., Lal, N., & Kumar, S. (2005). Essential oil constituents and RAPD markers to establish species relationship in Cymbopogon Spreng. (Poaceae). Biochemical Systematic and Ecology, 33, 171–186.
Ganjewala, D. (2008). RAPD characterization of three selected cultivars OD-19, GRL-1 and Krishna of East Indian Lemongrass (Cymbopogon flexuosus Nees ex Steud) Wats. American Eurasian Journal of Botany, 1, 53–57.
Adhikari, S., Bandopadhyay, T. K., & Ghosh, P. D. (2013). Assessment of genetic diversity in certain Indian elite clones of Cymbopogon species through RAPD analysis. Indian Journal of Biotechnology, 12, 109–114.
Kumar, J., Verma, V., Shahi, A. K., Qazi, G. N., & Balyan, H. S. (2007). Development of simple sequeme repeats markers in Cymbopogon species. Planta Medica, 73, 262–266.
Kumar, J., Verma, V., Goyal, A., Shahi, A. K., Sparoo, R., Sangwan, R. S., & Qazi, G. N. (2009). Genetic diversity analysis in Cymbopogon species using DNA markers. Plant Omics Journal, 2, 20–29.
Shiran, B., Amirbakhtiar, N., Kiani, S., Mohammadi, S., Sayed-Tabatabaei, B. E., & Moradi, H. (2007). Molecular characterization and genetic relationship among almond cultivars assessed by RAPD and SSR markers. Scientia Horticulture, 111, 280–290.
Rohlf, F. J. (1998). NTSYS-PC, numerical taxonomy and multivariate analysis system, version 2.02a. Setauket, New York: Exeter Publications.
Jaccard, P. (1908). Nouvelles recherches sur la distribution florale. Bulletin de la Societe vaudoise des sciences naturelles, 44, 223–270.
Sneath, P. H. A., & Sokal, R. R. (1973). Numerical taxonomy—the principles and practice of numerical classification. W. H., San Francisco: Freeman.
Prevost, A., & Wilkinson, M. J. (1999). A new system of comparing PCR primers applied to ISSR fingerprinting. Theory and Applied Genetics, 98, 107–112.
Powell, W., Morgante, M., Andre, C., Hanafey, M., Vogel, J., Tingey, S., & Rafalski, A. (1996). The unity of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Molecular Breeding, 2, 25–238.
Anderson, J. A., Churchill, G. A., Autrique, J. E., Tanksley, S. D., & Sorrells, M. E. (1993). Optimizing parental selection for genetic linkage maps. Genome, 36, 181–186.
Manly, B. F. J. (1997). Randomization, bootstrap and Monte Carlo methods in biology. London: Chapman & Hall.
Divaret, I., Margale, E., & Thomas, G. (1999). RAPD markers on seed bulks efficiently assess the genetic diversity of Brassica oleracea L. collection. Theory and Applied Genetics, 98, 1029–1035.
Kumar, L. S., Sawant, A. S., Gupta, V. S., & Ranjekar, P. K. (2001). Comparative analysis of genetic diversity among Indian populations of Scirpophaga incertulas by ISSR-PCR and RAPD-PCR. Biochemical Genetics, 39, 297–309.
Ngezahayo, F., Dong, Y., & Liu, B. (2007). Somaclonal variation at the nucleotide sequence level in rice (Oryza sativa L.) as revealed by RAPD and ISSR markers, and by pairwise sequence analysis. Journal of Applied Genetics, 48, 329–336.
Maslova, E. V. (2008). Differentiation of two hemp nettle species (Galeopsis bifida Boenn. and G. tetrahit L.) inferred from morphological characters and DNA markers. Russian Journal of Genetics, 44, 312–319.
Mohanty, S., Das, A. B., Ghosh, N., Panda, B. B., & Smith, D. W. (2010). Genetic diversity of 28 wild species of fodder legume Cassia using RAPD, ISSR, and SSR markers: a novel breeding strategy. Journal of Biotechnology Research, 2, 44–55.
Chandrashekar, R., Prasad, B., Vankudothu, N., Jyothi, C. P., Lakshmi, B. N., Rama, R. Y., & Kudle, K. R. (2015). A comparative genetic diversity study among elite Bt and Non-Bt cotton varieties of Andhra Pradesh by RAPD markers. Discover Biotechnology, 16, 30–37.
Khaled, K. A., El-Demardash, I. S., & Amer, E. A. M. (2015). Genetic polymorphism among some sugarcane germplasm collections as revealed by RAPD and ISSR analyses. Life Science Journal, 12, 159–167.
Thangjam, R. (2014). Inter-simple sequence repeat (ISSR) marker analysis in Parkia timoriana (DC.) Merr. populations from Northeast India. Applied Biochemistry and Biotechnology, 172, 1727–1734.
Mishra, P., Kumar, L. D., Kumar, A., Gokul, S., Ravikumar, K., Shukla, A. K., & Sundaresan, V. (2015). Population dynamics and conservation implications of Decalepis arayalpathra (J. Joseph and V. Chandras.) Venter., a steno endemic species of Western Ghats, India. Applied Biochemistry and Biotechnology, 176, 1413–1430.
Mattioni, C., Casasoli, M., Gonzalez, M., & Ipinza, R. (2002). Comparison of ISSR and RAPD markers to characterize three Chilean Nothofagus species. Theory and Applied Genetics, 104, 1064–1070.
Mukherjee, A., Sikdar, B., Ghosh, B., Banerjee, A., Ghosh, E., Bhattacharya, M., & Roy, S. C. (2013). RAPD and ISSR analysis of some economically important species, varieties, and cultivars of the genus Allium (Alliaceae). Turkish Journal of Botany, 37, 605–618.
Blair, M. W., Panaud, O., & McCouch, S. R. (1999). Inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L.). Theory and Applied Genetics, 98, 780–792.
Acknowledgment
The authors are thankful to the ICAR-Directorate of Medicinal and Aromatic Plants Research (ICAR-DMAPR) for providing facilities for this study. We also acknowledge the encouragement and support provided by Dr. Satyabrata Maiti, former Director, ICAR-DMAPR, for conducting the study.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of Interest
The authors declare that they have no conflict of interest.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary Table 1
(DOC 17 kb)
Supplementary Table 2
(DOC 85 kb)
Supplementary Table 3
(DOC 93 kb)
Supplementary Table 4
(DOC 45 kb)
Supplementary Table 5
(DOC 30 kb)
Supplementary Table 6
(DOC 20 kb)
Supplementary Table 7
(DOC 20 kb)
Supplementary Table 8
(DOC 19 kb)
Rights and permissions
About this article
Cite this article
Bishoyi, A.K., Sharma, A., Kavane, A. et al. Varietal Discrimination and Genetic Variability Analysis of Cymbopogon Using RAPD and ISSR Markers Analysis. Appl Biochem Biotechnol 179, 659–670 (2016). https://doi.org/10.1007/s12010-016-2022-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12010-016-2022-y