Genetic Variabilities of Stevia rebaudiana Bertoni Cultivated in Malaysia as Revealed by Morphological, Chemical and Molecular Characterisations

Halimaton Saadiah Othman, Mohamad Osman, Zarina Zainuddin


Stevia rebaudiana Bertoni originally from Paraguay belongs in Asteraceae family. It is an alternative source of non-caloric sweetener due to the sweet steviol glycosides contained in the leaves. As an introduced species in Malaysia, it is important to elucidate the genetic variabilities and relatedness among stevia accessions in order to broaden the genetic basis for future stevia breeding. This study described morphological and chemical variations and investigates genetic relationships among stevia accessions derived from across Malaysia and Paraguay using inter simple sequence repeats (ISSR) markers. HPLC (high-performance liquid chromatography) analysis also revealed high variability with stevioside content between 4.54 % (Taman Pertanian) to 20.36 % (Bangi) and rebaudioside A content varied between 0.3 % (Nilai) to 2.04 % (MNQ). From 32 ISSR markers, a total of 332 bands were scored, of which 264 (78 %) were polymorphic. The dendrogram from UPGMA (Unweighted Pair Group Method with Arithmetic Mean) cluster analysis separated 17 stevia accessions into 3 main groups. Rawang and Nilai were found to be closely related. The wide genetic variabilities among stevia accessions are a promising indicator towards the development of new stevia varieties. This valuable information will be able to assist parental selection in future stevia breeding programmes.


Genetic Variabilities; ISSR Marker; Morphology; Stevia rebaudiana; Steviol glycosides

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Abdullateef, R. A., & Osman, M. (2011). Influence of genetic variation on morphological diversity in accessions of Stevia rebaudiana Bertoni. International Journal of Biology, 3(3), 66–72. crossref

Anami, E. T., Poletine, J. P., Gonçalves-Vidigal, M. C., Vidigal Filho, P. S., Lacanallo, G. F., Kvitschal, M. V., & Gonela, A. (2010). Characterization and genetic divergence in Stevia rebaudiana (Bert.) Bertoni clones based in agronomical and morphological characteristics. Journal of Food, Agriculture and Environment, 8(3–4 PART 1), 463–469. Retrieved from website

Bajpai, P. K., Warghat, A. R., Sharma, R. K., Yadav, A., Thakur, A. K., Srivastava, R. B., & Stobdan, T. (2014). Structure and genetic diversity of natural populations of Morus alba in the trans-Himalayan Ladakh region. Biochemical Genetics, 52(3–4), 137–152. crossref

Balcázar-Vargas, M. P., Peñuela-Mora, M. C., van Andel, T. R., & Zuidema, P. A. (2012). The quest for a suitable host: Size distributions of host trees and secondary hemiepiphytes search strategy. Biotropica, 44(1), 19–26. crossref

Barthe, S., Gugerli, F., Barkley, N. A., Maggia, L., Cardi, C., & Scotti, I. (2012). Always look on both sides: Phylogenetic information conveyed by simple sequence repeat allele sequences. PLoS ONE, 7(7), e40699. crossref

Behera, M. S., Verma, O. P., Mahapatra, P. K., Singandhupe, R. B., Kumar, A., Kannan, K., & Brahmanand, P. S. (2013). Effect of fertigation on stevia (Stevia rebaudiana) under drip irrigation. Indian Journal of Agronomy, 58(2), 243–250. crossref

Brandle, J. (1999). Genetic control of rebaudioside A and C concentration in leaves of the sweet herb, Stevia rebaudiana. Canadian Journal of Plant Science, 79(1), 85-91. crossref

Brandle, J. E., & Telmer, P. G. (2007). Steviol glycoside biosynthesis. Phytochemistry, 68(14), 1855–1863. crossref

Ceunen, S., Werbrouck, S., & Geuns, J. M. C. (2012). Stimulation of steviol glycoside accumulation in Stevia rebaudiana by red LED light. Journal of Plant Physiology, 169(7), 749–752. crossref

Chotikadachanarong, K., & Dheeranupattana, S. (2013). Micropropagation and acclimatization of Stevia rebaudiana Bertoni. Pakistan Journal of Biological Sciences, 16(17), 887–890. crossref

Deka, S. D., Dadlani, M., & Sharma, R. (2016). Diversity study in capsicum using numerical taxonomy. Sabrao Journal of Breeding and Genetics, 48(3), 277–284. Retrieved from PDF

Goettemoeller, J., & Ching, A. (1999). Seed germination in Stevia rebaudiana. In J. Janick (Ed.), Perspectives on new crops and new uses (pp. 510–511). Alexandria, VA: ASHS Press.

Guichoux, E., Lagache, L., Wagner, S., Chaumeil, P., Léger, P., Lepais, O., … Petit, R. J. (2011). Current trends in microsatellite genotyping. Molecular Ecology Resources, 11(4), 591–611. crossref

Gupta, E., Purwar, S., Sundaram, S., & Rai, G. K. (2013). Nutritional and therapeutic values of Stevia rebaudiana: A review. Journal of Medicinal Plants Research, 7(46), 3343-3353. crossref

Gupta, S., Srivastava, M., Mishra, G. P., Naik, P. K., Chauhan, R. S., Tiwari, S. K., … Singh, R. (2008). Analogy of ISSR and RAPD markers for comparative analysis of genetic diversity among different Jatropha curcas genotypes. African Journal of Biotechnology, 7(23), 4230-4243. Retrieved from website

Heikal, A. H., Badawy, O. M., & Hafez, A. M. (2008). Genetic relationships among some Stevia (Stevia rebaudiana Bertoni) accessions based on ISSR analysis. Research Journal of Cell and Molecular Biology, 2(1), 1-5. Retrieved from website

JECFA. (2008). Steviol glycosides. FAO JECFA Monographs 5. Retrieved from PDF

Lata, H., Chandra, S., Techen, N., Wang, Y.-H., & Khan, I. A. (2013). Molecular analysis of genetic fidelity in micropropagated plants of Stevia rebaudiana Bert. using ISSR marker. American Journal of Plant Sciences, 4(May), 964–971. crossref

Lin, T., Zhu, G., Zhang, J., Xu, X., Yu, Q., Zheng, Z., … Huang, S. (2014). Genomic analyses provide insights into the history of tomato breeding. Nature Genetics, 46(11), 1220–1226. crossref

Madan, S., Ahmad, S., Singh, G. N., Kohli, K., Kumar, Y., Singh, R., & Garg, M. (2010). Stevia rebaudiana (Bert.) Bertoni - A review. Indian Journal of Natural Products and Resources, 1(3), 267–286. crossref

Martins-Lopes, P., Lima-Brito, J., Gomes, S., Meirinhos, J., Santos, L., & Guedes-Pinto, H. (2007). RAPD and ISSR molecular markers in Olea europaea L.: Genetic variability and molecular cultivar identification. Genetic Resources and Crop Evolution, 54(1), 117–128. crossref

Mehta, B. K., Jain, D. C., Misra, H., Soni, M., Silawat, N., & Mehta, D. (2011). Antidiabetic activity of medium-polar extract from the leaves of Stevia rebaudiana Bert. (Bertoni) on alloxan-induced diabetic rats. Journal of Pharmacy and Bioallied Sciences, 3(2), 242. crossref

Mogra, R., & Dashora, V. (2009). Exploring the use of Stevia rebaudiana as a sweetener in comparison with other sweeteners. Journal of Human Ecology, 25(2), 117–120. crossref

Moraes, R. M., Donega, M. A., Cantrell, C. L., Mello, S. C., & McChesney, J. D. (2013). Effect of harvest timing on leaf production and yield of diterpene glycosides in Stevia rebaudiana Bert: A specialty perennial crop for Mississippi. Industrial Crops and Products, 51, 385–389. crossref

Nazneen, H., Reddy, P. V., & Reddy, S. K. (2016). Molecular analysis of genetic fidelity of Stevia rebaudiana Bert. using RAPD markers. World Journal of Pharmaceutical Research, 5(4), 750-759. crossref

Ning, H., Wang, W., Zheng, C., Li, Z., Zhu, C., & Zhang, Q. (2014). Genetic diversity analysis of sedges (Carex spp.) in Shandong, China based on inter-simple sequence repeat. Biochemical Systematics and Ecology, 56, 158–164. crossref

Owens, A., Cieslak, M., Hart, J., Classen-Bockhoff, R., & Prusinkiewicz, P. (2016). Modeling dense inflorescences. ACM Transactions on Graphics, 35(4), 1–14. crossref

Pal, P. K., Mahajan, M., Prasad, R., Pathania, V., Singh, B., & Ahuja, P. S. (2015). Harvesting regimes to optimize yield and quality in annual and perennial Stevia rebaudiana under sub-temperate conditions. Industrial Crops and Products, 65, 556–564. crossref

Parris, C. A., Shock, C. C., & Qian, M. (2016). Dry leaf and steviol glycoside productivity of Stevia rebaudiana in the Western United States. HortScience, 51(10), 1220–1227. crossref

Pereira, C., Storck, L., Lopes, S. J., Martin, T. N., & Bisognin, D. A. (2016). Dry biomass and glycosides yield from Stevia rebaudiana leaves under different harvesting times. Bioscience Journal, 32(6), 1462-1471. crossref

Prakash, I., Markosyan, A., & Bunders, C. (2014). Development of next generation stevia sweetener: Rebaudioside M. Foods, 3, 162-175. Retrieved from website

Raina, R., Bhandari, S. K., Chand, R., & Sharma, Y. (2013). Strategies to improve poor seed germination in Stevia rebaudiana, a low calorie sweetener. Journal of Medicinal Plants Research, 7(24), 1793–1799. crossref

Rajasekaran, T., Giridhar, P., & Ravishankar, G. A. (2007). Production of steviosides in ex vitro and in vitro grown Stevia rebaudiana Bertoni. Journal of the Science of Food and Agriculture, 87(3), 420–424. crossref

Rashid, R. A., Mohamad, A., Awang, M. R., Mutaat, H. H., Mohamad, S. A., Hasan, A. A., ... Mohd Nahar, S. K. (2013). Isolation and optimization of inter-simple sequence repeat (ISSR) technique for Pleurotus sajor caju towards enviromental study in support of the country’s nuclear power programme. Jurnal Sains Nuklear Malaysia, 25(1), 1-8. Retrieved from website

SAS Institute Inc. (1999). SAS/STAT® 8.02 User’s Guide. Cary, NC: SAS institute, Inc.

Serfaty, M., Ibdah, M., Fischer, R., Chaimovitsh, D., Saranga, Y., & Dudai, N. (2013). Dynamics of yield components and stevioside production in Stevia rebaudiana grown under different planting times, plant stands and harvest regime. Industrial Crops and Products, 50, 731–736. crossref

Sharma, N., Kaur, R., & Era, V. (2016). Potential of RAPD and ISSR markers for assessing genetic diversity among Stevia rebaudiana Bertoni accessions. Indian Journal of Biotechnology, 15, 95-100. Retrieved from PDF

Singh, B. D., & Singh, A. K. (2015). Marker-assisted plant breeding: Principles and practices. Marker-Assisted Plant Breeding: Principles and Practices. New Delhi, IN: Springer. crossref

Singh, P., & Dwivedi, P. (2014). Two-stage culture procedure using thidiazuron for efficient micropropagation of Stevia rebaudiana, an anti-diabetic medicinal herb. 3 Biotech, 4(4), 431–437. crossref

Steinmetz, W. E., & Lin, A. (2009). NMR studies of the conformation of the natural sweetener rebaudioside A. Carbohydrate Research, 344(18), 2533–2538. crossref

Tan, S. L., Ghawas, M. M., Najib, M. Y. M., & Zawayi, M. (2008). Preliminary evaluation and selection of stevia under Malaysian conditions. Journal of Tropical Agriculture and Food Science, 36(2), 1–7. Retrieved from PDF

Tateo, F., Mariotti, M., Bononi, M., Lubian, E., Martello, S., Cornara, L. (1998). Stevioside content and morphological variability in a population of Stevia rebaudiana (Bertoni) Bertoni from Paraguay. Italian Journal of Food Science,10, 261-267.

Tavarini, S., & Angelini, L. G. (2013). Stevia rebaudiana Bertoni as a source of bioactive compounds: the effect of harvest time, experimental site and crop age on steviol glycoside content and antioxidant properties. Journal of the Science of Food and Agriculture, 93(9), 2121–2129. crossref

Thiyagarajan, M., & Venkatachalam, P. (2015). Assessment of genetic and biochemical diversity of Stevia rebaudiana Bertoni by DNA fingerprinting and HPLC analysis. Annals of Phytomedicine, 4(1), 79-85. Retrieved from PDF

Tucak, M., Popović, S., Čupić, T., Grljušić, S., Bolarić, S., & Kozumplik, V. (2008). Genetic diversity of alfalfa (Medicago spp.) estimated by molecular markers and morphological characters. Periodicum Biologorum, 110(3), 243–249. Retrieved from website

Vasilakoglou, I., Kalfountzos, D., Gougoulias, N., & Reppas, C. (2016). Productivity of two stevia varieties under reduced irrigation and fertilization inputs. Archives of Agronomy and Soil Science, 62(4), 457–472. crossref

Vouillamoz, J. F., Wolfram-Schilling, E., Carron, C.-A., & Baroffio, C. A. (2016, June 19-23). FSL 4: Agronomical and phytochemical evaluation of Stevia rebaudiana genotypes. Paper presented at 6th International Symposium Breeding Research on Medicinal and Aromatic Plants, BREEDMAP 6, Quedlinburg, Germany. crossref

Wu, F. Q., Shen, S. K., Zhang, X. J., Wang, Y. H., & Sun, W. B. (2014). Genetic diversity and population structure of an extremely endangered species: the world’s largest Rhododendron. AoB PLANTS, 7(0), plu082. crossref

Yadav, A. K., Singh, S., Dhyani, D., & Ahuja, P. S. (2011). A review on the improvement of stevia [Stevia rebaudiana (Bertoni)]. Canadian Journal of Plant Science, 91(1), 1–27. crossref



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