Extraction of High Quality Genomic DNA for Genetic Diversity and Molecular Marker Studies from Leaves of Makhana (Euryale ferox)

Genetic diversity study of plants through molecular markers depends on both quality and quantity of DNA. The standard protocols and commercially available kits failed to isolate high quality DNA from leaves tissue of Makhana (Euryale ferox Salisb.) due to the occurrence of the high amount of polysaccharides, polyphenols and proteins. A cetyl trimethyl ammonium bromide (CTAB) protocol has been optimized for isolation of high quality genomic DNA from the leaves of E. ferox. The optimized protocol yielded up to 4081 ng per gram leaves tissue of good quality DNA, which was free of polysaccharide, polyphenols and protein. The problems of DNA degradation, contamination, and low yield due to irreversible binding of phenolic compounds and co-precipitation of polysaccharides with DNA were avoided by this method. The isolated DNA has excellent spectral qualities, and efficiently digested by restriction endonucleases and it is also suitable for development of molecular markers such as RAPD and ISSR. *Address for correspondence: Dr. Nitish Kumar Centre of Biological Sciences, (Biotechnology), School of Earth, Biological and Environmental Sciences, Central University of Bihar, Patna 800014, Bihar, India E-mail: nitish@cub.ac. in INTRODUCTION Makhana (Euryale ferox Salisb.), a prickly floating plant of Nymphaeaceae family, is one of the most important food crops of north Bihar, India (Mandal et al. 2010). The commercial value of E. ferox seeds lies in their popped form. The starchy white puffs are very nutritious and tasty, and marketed as a premium dry fruit commodity of E. ferox (Nath and Chakraborty 1985). Seeds of E. ferox are also reported for its medicinal properties, and it is widely used in Ayurveda and Chinese preparations for the treatment of a number of human ailments involving respiratory, circulatory, digestive, excretory and reproductive systems (Kumar et al. 2016). The cultivation E. ferox is still neglected, probably because of lack of awareness among the rural masses and paucity of suitable improved varieties. In order to take effective measure to encourage genetic conservation and suitable use of E. ferox resources, the development and application of molecular markers in E. ferox are imperative. For molecular marker studies, there is need of high quality DNA. Many standard protocols are available for extracting plant genomic DNA, however, problems of DNA extraction is quit a vital concern in the area of plant molecular biology, as the quality of template DNA influences the accomplishment of subsequent downstream experiments (Sika et al. 2015). High levels of polysaccharides, many types of secondary metabolites; polyphenols and other organic compounds affect DNA purification, the quality of the DNA sample and also inhibit enzymatic reactions, such as polymerase chain reaction (PCR) while DNA is used as a template (Abdel Latif and Osman 2017). The E. ferox leaf is rich in pigments as the underside of the leaf is dark purplish. The leaf also contains intercalating ribs underside and upper surface is covered by sharp prickles. It seems leaves are rich in proteins, polysaccharides and polyphenolic compounds are the problem for DNA isolation. J Biodiversity, 8(2): 87-92 (2017) 10.1080/09766901.2018. 1425081 DOI: 88 NITISH KUMAR, HEMANT KUMAR AND SWATI KUMARI