RAPD Analysis for Genetic Variability Detection of Mutant Soybean (Glycine max (L.) Merr)


Didik Wahyudi(1*), Lia Hapsari(2), S. Sundari(3)

(1) Biology Department, Faculty of Science and Technology, State Islamic University of Maulana Malik Ibrahim
(2) Purwodadi Botanic Garden – Indonesian Institute of Sciences
(3) Department of Biology Education, Universitas Khairun
(*) Corresponding Author


This study aimed to detect and evaluate the genetic mutation from mutagenized soybean by RAPD markers. Soybean seeds of “Grobogan” variety were treated with two different concentrations of EMS (0.5% and 1%) and three incubation times (4, 6 and 8 h). DNA whole-genome was isolated from young leaf seedling with the Qiagen DNeasy Plant Mini Kit. Twenty OPA primers (OPA-1 to OPA-20) were used for DNA amplification. The results showed that EMS treatments successfully generated genetic variation in soybean, which indicated by high values of PIC, EMR, and MI. RAPD primers that effective to detect the mutation were OPA-2, OPA-07, OPA-10, OPA-11, OPA-12, OPA-13, OPA-14, OPA-15, OPA-16, OPA-18 and OPA-20. The band expression of those primers was exhibited a stronger intensity along with increasing of EMS concentration and incubation time used in this study. Treatment of 0.5% EMS in 6 hours incubation was successfully generated soybean mutants with the lowest genetic similarity compared to the wild-type. Thus, this study provides a new approach to generate genetic variability in soybean and has the potential to improve for soybean breeding program.


ethyl methane sulphonate; genetic mutation; RAPD; soybean

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Aisyah, S.I., Hapsari L. & Herlina, D., 2005, Induced mutation on jasmine (Jasminum spp.) through gamma irradiation, Journal of Agriculture and Rural Development in the Tropics and Subtropics Beheift 83, 120 – 127.

Arif, I.A., Bakir, M.A., Khan, H.A., Al Farhan, A.H., Al Homaidan, A.A., Bahkali, A.H.,. Al Sadoon M. & Shobrak M., 2010, Application of RAPD for molecular characterization of plant species of medicinal value from an arid environment, Genetics and Molecular Research 9, 2191-2198.

Ashraf, M., Cheema, A.A., Zıa-Ul-Qamar & Rashid M., 2007, Detection of Polymorphism In Rice Germplasm Using RAPD Marker, Pakistan Journal of Botany 39, 2483-2493.

Atienzar, F.A., Conradi, M., Evenden, A.J., Jha, A.N. & Depledge M.H., 2010, Qualitative  assessment of genotoxicity using  random amplified polymorphic DNA: comparison of genomic template stability with key fitness parameter in Daphnia magna exposed to benzo[a] pyrene, Environmental Toxicology and Chemistry 18, 2275–2282.

Atienzar, F.A. & Jha A.N., 2006, The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: A critical review, Mutation Research 613, 76–102.

Balitbangtan, 2005, Prospek dan Arah Pengembangan agrıbısnıs kedelai, Departemen Pertanian RI, Jakarta.

Balitbangtan, 2016, Grobogan, Panen Kedelai 3 ton/ha, Viewed July 11, 2018, from http://www.litbang.pertanian.go.id/press/one/49/pdf/Grobogan,%20Panen%20Kedelai%203%20ton/ha.pdf.

Balitkabi, 2016, Deskripsi Varietas Unggul Aneka Kacang dan Umbi. Balai Penelitian Tanaman Aneka Kacang dan Umbi, Badan Penelitian dan Pengembangan Pertanian, Kementerian Pertanian Republik Indonesia, Jakarta.

Behera, M., Panigrahi, J., Mishra, R.R. & Rath S.P., 2012, Analysis of EMS induced in vitro mutants of Asteranctha longifolia (L.) Nees using RAPD Markers, Indian Journal of Biotechnology 11, 39-47.

Biswas, M.K., Xu, Q. & Deng, X-X., 2010, Utility of RAPD, ISSR, IRAP and REMAP markers for the genetic analysis of Citrus spp., Scientia Horticulturae 124, 254–261.

Daryono, B.S., Aristya, G.R. &. Kasiamdari, R.S., 2011, Development of Random Amplifed Polymorphism DNA Markers Linked to Powdery Mildew Resistance Gene in Melon, Indonesian Journal of Biotechnology 16, 76-82.

Dhakshanamoorthy, D., Selvaraj, R. & Chidambaramb, A., 2014, Utility  of  RAPD  marker  for  genetic  diversity  analysis  in  gammarays  and  ethyl  methane  sulphonate  (EMS)-treated  Jatropha curcas plants, Comptes Rendus Biologies 338, 75-82.

Fei, Y., Tang, W., Shen, J., Tianjing, Z., Rui, Q., Xiao, B., Zhou, C., Liu, Z. & Anna, Y.T., 2014, Application of random amplified polymorphic DNA (RAPD) markers to identify Taxus chinensis var. mairei cultivars associated with parthenogenesis, African Journal of Biotechnology 13, 2385-2393.

Garcia, V., Bres, C., Just, D., Fernandez, L., Tai, F.W., Mauxion, J.P., Le Paslier, M.C., Bérard, A., Brunel, D., Aoki, K., Alseekh, S., Fernie, A.R., Fraser, P.D. & Rothan, C., 2016, Rapid identification of causal mutations in tomato EMS populations via mapping-by-sequencing, Nature Protocols 11, 2401-2418.

Girija, M., Dhanavel, D. & Gnanamurthy, S., 2013, Gamma rays and EMS induced flower color and seed mutants in cowpea (Vigna unguiculata L. Walp), Advances in Applied Science Research 4, 134-139.

Greene, E.A., Codomo, C.A., Taylor, N.E., Henikoff, J.G., Till, B.J., Reynolds, S.H., Enns, L.C., Burtner, C., Johnson, J.E., Odden, A.R., Comai, L. & Henikoff, S., 2003, Spectrum of Chemically Induced Mutations From a Large-Scale Reverse-Genetic Screen in Arabidopsis, Genetics 164, 731–740.

Hammer, Ø., Harper, D.A.T. & Ryan, P.D., 2001, PAST: Paleontological Statistics Software Package for Education and Data Analysis. Palaeontol, Palaeontologia Electronica 4, 1-9.

Kawar, P.G., Devarumath, R.M. & Nerkar Y., 2009, Use of RAPD Marker for assessment of Genetic Diversity in Sugarcane cultivar, Indian Journal of Biotechnology 8, 67-71.

Khan, M.H. & Tyagi, S.D., 2013, A review on induced mutagenesis in soybean, Journal of Cereals and Oilseeds 4, 19-25.

Khan, M.H. & Tyagi, S.D., 2010, Induced morphological mutants in soybean [Glycine max (L.) Merrill], Frontiers of Agriculture in China 4, 175–180.

Kolade, O.A., Olowolafe, M.O. & Fawole, I., 2016, Characterization of mutant cowpea [Vigna unguiculata (L) Walp] lines using random amplified polymorphic DNAs (RAPDs) and amplified fragment length polymorphism (AFLP) markers, African Journal of Biotechnology 15, 2530-2537.

Krisdiana, R., 2013, Penyebaran Varietas Unggul Kedelai dan Dampaknya terhadap Ekonomi Perdesaan, Penelıtıan Pertanıan Tanaman Pangan 33, 61-69.

Kristanti, N.E., Rahmawati, F. & Maksum, M., 2017, Analysis of Productivity of Soybean [Glycine max (L.) Merr.] for Production for Farmers in Indonesia, Proceedings of The 3rd International Conference on Agro-Industry 2016 “Competitive & Sustainable AgroIndustry: Value Creation in Agribusiness” (3rd IcoA 2016), Bangkok, Thailand, pp. 237–246.

Kumari, N. & Thakur, S.K., 2014, Randomly Amplified Polymorphic DNA-a Brief Review, American Journal of Animal and Veterinary Sciences 9, 6-13.

Laurentin, H. & Karlovsky, P., 2007, AFLP fingerprinting of sesame (Sesamum indicum L.) cultivars: identification, genetic relationship and comparison of AFLP  informativeness  parameters, Genetic Resources and Crop Evolution 54, 1437–1446.

Li, Z., Jiang, L., Ma, Y., Wei, Z., Hong, H., Liu Z., Lei, J., Liu, Y., Guan, R., Guo. Y., Jin, L., , Zhang, L., Li, Y., Ren, Y., He, W., Liu, M., Htwe, N.M.P.S., Liu, L., Guo, B., Song, J., Tan, B., Liu, G., Li, M., Zhang, X., Liu, B., Shi X., Han, S., Hua, S., Zhou, F., Yu, L., Li., Y., Wang, S., Wang, J., Chang, R., & Qiu, L., 2017, Development and utilization of a new chemically-induced soybean library with a high mutation density, Journal of Integrative Plant Biology 59(1), 60-74

Maia, S.H.Z., Mangolin, C.A., Collet, S.A. & Machado, M.F., 2009, Machado Genetic diversity in somatic mutants of grape (Vitis vinifera) cultivar Italia based on random amplifed polymorphic DNA, Genetics and Molecular Research 8, 28-38.

Mishra, K.K., Fougat, R.S., Ballani, A., Thakur, V., Jha, Y. & Bora, M., 2014, Potential and application of molecular markers techniques for plant genome analysis, International Journal of Pure & Applied Bioscience 2, 169-188.

Nagy, S., Poczai, P., Cerna´k, I., Gorji, A.M., Hegedus, G. & Taller J., 2012, PICcalc: An Online Program to Calculate Polymorphic Information Content for Molecular Genetic Studies, Biochemical Genetics 50, 670–672.

Nikmah, I.A., Azrianingsih, R., Wahyudi, D., 2016. Genetic diversity of porang populations (Amorphophallus muelleri Blume) in Central Java and West Java based on LEAFY second intron marker, Journal of Tropıcal Lıfe Scıence 6 (1), 23-27.

Oladosu, Y., Rafii, M.Y., Abdullah, N., Hussin, G., Ramli, A., Rahim, H.A., Miah, G. & Usman M., 2016, Principle and application of plant mutagenesis in crop improvement: a review, Agriculture and Environmental Biotechnology 30, 1-16.

Pusdatin, 2015, Outlook Komoditas Pertanian Tanaman Pangan Kedelai, Kementerian Pertanian RI, Jakarta.

Rahajeng, W. & Adie, M.M., 2013, Varietas kedelai umur genjah, Buletin Palawija 26, 91–100.

Setiawan A, 2013, RI Belum Lepas Ketergantungan Impor Kedelai Tahun Ini, viewed March 3, 2018 from: http://finance.detik.com/read/.

Shah, T.M., Mirza, J.I., Haq, M.A. & Atta, B.M., 2009, Screening of chickpea (Cicer arietinum) induced mutants against Fusarium wilt, Pakistan Journal of Botany 41, 1945‒1955.

Singh, G., Sareen, P.K, Saharan, R.P. & Singh, A., 2001, Induced variability in mungbean (Vigna radiata L), İndian Journal of Genetics and Plant Breeding. 61, 281-282.

Soeranto, H., Manurung, S. & Masrizal, 2001, The use of physical/chemical mutagens for crop improvements in Indonesia, Proceedings of the 8th workshop on plant mutation breeding, Japan, pp. 90–101.

Sreeju, S.N., Babu, M.M., Mariappan, C. & Selvamohan, T., 2011, Effect of physical and chemical mutagens on biopolmer producing strains and RAPD analysis of mutated strains, Archives of Applied Science Research 3, 233-246.

Sunaryo, W., Wıdoretno, W., Nurhasanah & Sudarsono, 2016, Drought tolerance selection of soybean lines generated from somatic embryogenesis using osmotic stress simulation of poly-ethylene glycol (PEG), Nusantara Bioscience 8, 45-54.

Suherman M, 2014, Optimis Tiga Tahun Swasembada Kedelai, Viewed July 7, 2018, from: http://agroindonesia.co.id/2014/11/18/optimis-tiga-tahun-swasemba da-kedelai.

Sun, M. & Wong, K.C., 2001,  Genetic  structure  of  three  orchid  species  with contrasting  breeding  systems  using  RAPD  and  allozyme  markers, American Journal of Botany 88, 2180–2188.

Sundari, Arumingtyas, E.L., Hakim, L., Azrianingsih, R. and Wahyudi D., 2017, Genetic variability of Local Durian (Durio zibethinus murr.) In Ternate Island Based on Rapd Markers, Plant Cell Biotechnology and Molecular Biology 18, 68-75.

Thilagavathi, C. & Mullainathan L., 2009, Isolation of Macro Mutants and Mutagenic Effectiveness, Efficiency in Black Gram (Vigna mungo (L.) Hepper), Global Journal of Molecular Sciences 4(2), 76-79.

Varshney, R.K., Chabane, K., Hendre, P.S., Aggarwal, R.K. & Graner A., 2007, Comparative assessment of EST-SSR, EST-SNP and AFLP markers for evaluation of genetic  diversity and conservation of genetic resources using wild, cultivated and elite barleys, Plant Science 173, 638–649.

Wahyudi, D., Azrianingsih, R., & Mastuti, R., 2013, Genetic variability of Porang Populations (Amorphophallus muelleri) in West Java and Central Java Based on TrnL İntron Sequences. Journal of Biodiversity and Environmental Sciences 3, 31-41.

Wang, L., Zhang, B., Li, J., Yang, X. & Ren, Z., 2014, Ren Ethyl Methanesulfonate (EMS)-Mediated Mutagenesis of Cucumber (Cucumis sativus L.), Agricultural Sciences 5, 716-721.

Wulandari, S.S., 2016, Kebijakan Pemerintah Indonesia Dalam Melindungi Petani Lokal Dari Ancaman Impor Kedelai Amerika Serikat Tahun 2012-2016, JOM FISIP 4, 1-15.

DOI: https://doi.org/10.22146/jtbb.53653

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