High Quality DNA Extraction Protocol for Cotyledonary and Mature Leaves of Rhododendron arboreum Sm.
DOI:
https://doi.org/10.36808/if/2021/v147i11/157680Keywords:
Rhododendron arboreum, Cotyledonary Leaves, Charcoal, Genomic DNA.Abstract
The extraction of high quality DNA from different plant source is a limiting factor in genetic diversity studies of any tree species. DNA extraction is the preliminary and essential step for almost all molecular marker based genetic studies. High-quality DNA extraction from cotyledonary and mature leaves of Rhododendron arboreum is extremely difficult due to the presence of high concentration of polyphenols and polysaccharides. These contaminants, if present in the extracted DNA severely affects the PCR amplification. Therefore, a method of DNA extraction from the cotyledonary and mature leaves has been developed. Solubility of polysaccharides was minimised with high salt concentrations (3M) and polyphenols removed by adding, Polyvinylpyrrolidone (2%) and activated charcoal (1%) in the DNA extraction buffer. Additionally, RNase-H was used to remove RNA contamination. The extracted DNA was quantified using bio-spectrophotometer. DNA was obtained with concentration ranging from 2.30 to 4.70μg with the absorbance ratio of 1.80 to 1.88, per milligram of cotyledonary leaves and 4.25 to 7.70μg with the absorbance ratio of 1.80 to 1.88, per milligram of mature leaves. Further, quality of extracted genomic DNA was assessed by random amplified fragment length polymorphism (RAnd simplePD) a sequence repeat (SSR) markers.References
Aboul-Maaty N.A.F. and Oraby H.A.S. (2019). Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Bulletin of the National Research Centre, 43(1): 1-10.
Ali Q., Salisu I.B., Raza A., Shahid A.A., Rao A.Q. and Husnain T. (2019). A modified protocol for rapid DNA isolation from cotton (Gossypium spp.). MethodsX, 6, 259-264.
Bhattacharyya D. and Sanjappa M. (2008). Rhododendron habitats in India. J. Amer. Rhodo. Soc., 62: 14-18.
Choudhary S., Thakur S., Saini R.G. and Bhardwaj P. (2014). Development and characterization of genomic microsatellite markers in Rhododendron arboreum. Conservation genetics resources, 6(4): 937-940.
Dayanandan S., Bawa K.S. and Kesseli R. (1997). Conservation of microsatellites among tropical trees (Leguminosae). American Journal of Botany, 84(12): 1658-1663.
Doyle J.J. and Doyle J.L. (1990). Isolation of plant DNA from fresh tissue. Focus, 12(13): 39-40.
Ginwal H.S. and Maurya S.S. (2010). Evaluation and optimization of DNA extraction method for Dalbergia sissoo leaf. Indian Journal of Biotechnology, 09(1): 69-73
Hamza N.B., Habeballa R.S. and Abdalla I.E. (2009). Phylogenetic relationships within indigenous Sudanese Cassia senna (L.) using RAPD molecular markers. African Journal of Biotechnology, 8(19): 4824-4829.
Heikrujam J., Kishor R. and Mazumder P.B. (2020). The chemistry behind plant DNA isolation protocols. Biochemical Analysis Tools–Methods for Bio-Molecules Studies, 8.
Hosseinzadeh-Colagar A., Haghighatnia M.J., Amiri Z., Mohadjerani M., and Tafrihi M. (2016). Microsatellite (SSR) amplification by PCR usually led to polymorphic bands: Evidence which shows replication slippage occurs in extend or nascent DNA strands. Molecular biology research communications, 5(3): 167.
Idrees M. and Irshad M. (2014). Molecular markers in plants for analysis of genetic diversity: a review. European academic research, 2(1): 1513-1540.
John M.E. (1992). An efficient method for isolation of RNA and DNA from plants containing polyphenolics. Nucleic Acids Research, 20(9): 2381.
Mabberley D.J. (2008). Rhododendrons Mabberley's PlantBook: A portable dictionary of plants, their classification and uses. Cambridge University Press, Cambridge, England Ed.3: 737.
Mingyuan F., Ruizheng F., Mingyou H.E., Linzhen H.U., Hanabi Y. and Chamber-Lain D.F. (2005). Rhododendron L. Flora of China. 14 (Ericaceae): 260-455. Science Press, Beijing and Missouri Botanical Garden Press, St. Louis.
Negi V.S., Maikhuri R.K., Rawat L.S. and Chandra A. (2013). Bioprospecting of Rhododendron arboreum for livelihood enhancement in central Himalaya, India. Environment & We: International Journal of Science and Technology, 8: 61-70.
Panda S. and Kirtania I. (2016). Variation in Rhododendron arboreum Sm. complex (Ericaceae): insights from exomorphology, leaf anatomy and pollen morphology. Modern Phytomorphology, 9: 27-49.
Paul A., Khan M.L., Arunachalam A. and Arunachalam K. (2005). Biodiversity and conservation of Rhododendrons in Arunachal Pradesh in the Indo-Burma biodiversity hotspot. Current Science, 89(4): 623-634.
Prakash T., Fadadu S.D., Sharma U.R., Surendra V., Goli D., Stamina P., and Kotresha D. (2008). Hepatoprotective activity of leaves of Rhododendron arboreum in CCl4 induced hepatotoxicity in rats. Journal of Medicinal Plants Research, 2(11): 315-320.
Raza S., Farooqi S., Shoaib M.W. and Mubeen H. (2015). Role of molecular markers and their significance. American Journal of Pharmacy and Health Research, 3: 23-30.
Rosazlina R., Jacobsen N., orgaard M. and Othman A.S. (2015). Utilizing next generation sequencing to characterize microsatellite loci in a tropical aquatic plant species Cryptocoryne cordata var. cordata (Araceae). Biochem. Syst. Ecol., 61: 385-389.
Saavedra J., Silva T.A., Mora F. and Scapim C.A. (2013). Bayesian analysis of the genetic structure of a Brazilian popcorn germplasm using data from simple sequence repeats (SSR). Chilean journal of agricultural research, 73(2): 99-107.
Sekar K.C. and Srivastava S.K. (2010). Rhododendrons in Indian Himalayan region: diversity and conservation. American Journal of Plant Sciences, 1(02): 131-137.
Sharma G., Singh A., Ali V. and Bharadwaj P. (2017). Development and characterization of UGMS markers for genetic diversity analysis in Rhododendron arboreum. In Proceedings: International Conference on Recent Innovations in Science, Agriculture, Engineering and Management, Punjab 20th November, 2017, University College of Computer application Guru Kashi University, Bhatinda, Punjab, 1057-1070.
Singh K.K., Kumar S., Rai L.K. and Krishna A.P. (2003). Rhododendrons conservation in the Sikkim Himalaya. Current Science, 85(5): 602-606.
Singh K.K., Rai L.K. and Gurung B. (2009). Conservation of Rhododendrons in Sikkim Himalaya: an overview. World Journal of Agricultural Sciences, 5(3): 284-296.
Srivastava P. (2012). Rhododendron arboreum: An overview. Journal of Applied Pharmaceutical Science, 2(1): 158-162.
Taheri S., Lee Abdullah T., Yusop M.R., Hanafi M.M., Sahebi M., Azizi P. and Shamshiri R.R. (2018). Mining and development of novel SSR markers using next generation sequencing (NGS) data in plants. Molecules, 23(2): 399.
Tiwari S., Tomar R.S., Tripathi M.K. and Ahuja A. (2017). Modified protocol for plant genomic DNA isolation. Indian Res. J. Genet. & Biotech, 9(4): 478-485.
Varshney R.K., Graner A. and Sorrells M.E. (2005). Genic microsatellite markers in plants: features and applications. Trends in Biotechnology, 23(1): 48-55.
Verma N., Kashyap P., Mishra D., Dutta D. and Nath A. (2017), Burans (Rhododendron arboretum Smith). 253-276.
Verma N., Singh A.P., Gupta A., Sahu P.K. and Rao C.V. (2011). Antidiarrheal potential of standardized extract of Rhododendron arboreum Smith flowers in experimental animals. Indian journal of pharmacology, 43(6): 689.
Youssef M., Valdez-Ojeda R., Ku-Cauich J.R. and EscobedoGraciaMedrano R.M. (2015). Enhanced protocol for isolation of plant genomic DNA. Journal of Agriculture and Environmental Sciences, 4(2): 172-180.
Zhang D.X. and Hewitt G.M. (2003). Nuclear DNA analyses in genetic studies of populations: practice, problems and prospects. Molecular ecology, 12(3): 563-584.
Zhao D.W., Yang J.B., Yang S.X., Kato K. and Luo J.P. (2014). Genetic diversity and domestication origin of tea plant Camellia taliensis (Theaceae) as revealed by microsatellite markers. BMC Plant Biol, 14: (1).
Downloads
Downloads
Additional Files
Published
How to Cite
Issue
Section
License
Unless otherwise stated, copyright or similar rights in all materials presented on the site, including graphical images, are owned by Indian Forester.
