An Efficient DNA Isolation Method from Nigella sativa L. (Ranunculaceae) Seeds for RAPD and ISSR Analysis

Öz

Nigella sativa L. (Ranunculaceae) seeds (black cumin) that contain high oil, terpene, protein, alkaloids are an economically important for medicine and food industry. Lack of an efficient DNA isolation procedure is a limiting factor for any molecular studies of these plant seeds. We have used a genomic DNA isolation protocol for Nigella sativa seeds based on a hexadecyltrimethylammonium bromide (CTAB) method described for other plant species. The method is a modified CTAB extraction using high salt concentrations and polyvinyl pyrrolidone (PVP), and successive isoamyl alcohol chloroform extractions and involves mortar grinding of black seeds. Black seeds include much little DNA despite high amounts nutrients as polysaccharides and oils. Therefore, the yield was approx., 20 ng DNA per 150 mg of dry seed material. The genomic DNA obtained by this method was suitable to be used in inter-simple sequence repeat (ISSR) and random amplified-polymorphic DNA (RAPD) reactions. This extraction method should facilitate the molecular analysis as genotypic selection via use of dry seeds instead of germination of N. sativa seeds.

Anahtar Kelimeler

• CTAB, DNA Extraction, ISSR, Nigella sativa L., RAPD, Seeds

Kaynakça

1. [1] Burits, M. and Bucar, F., “Antioxidant Activity of Nigella sativa Essential Oil” ,Phytother. Res., vol. 14, pp. 323– 328, 2000.
2. [2] Zaoui, A., Cherrah, Y., Mahassini, N., Alaoui, K., Amarouch, H. and Hassar, M., “Acute and chronic toxicity of Nigella sativa fixed oil,” Phytomedicine, vol. 9, pp. 69-74, 2002.
3. [3] Gali-Muhtasıb, H., El-Najjar, N. and Schneider-Stock, R., “The medicinal potential of black seed (Nigella sativa) and its components,” Lead Molecules from Natural Products, pp.133-153, 2006.
4. [4] Abdel-Zaher, A.O., Abdel-Rahman, M.S. and Elwasei, F.M., “Protective effect of Nigella sativa oil against tramadol-induced tolerance and dependence in mice: Role of nitric oxide and oxidative stress,” NeuroToxicology, 32, pp. 725-733, 2011.
5. [5] Harzallah, H.J., Kouidhi, B., Flamini, G., Bakhrouf, A. and Mahjoub T., “Chemical composition, antimicrobial potential against cariogenic bacteria and cytotoxic activity of Tunisian Nigella sativa essential oil and thymoquinone,” Food Chemistry, vol. 129, pp.1469-1474, 2011.
6. [6] Rogozhin, E.A, Oshchepkova, Y.I., Odintsova, T.I., Khadeeva N.V., Veshkurova, O.N., Egorov, T.A., Grishin, E. and Salikhov, S.I., “Novel antifungal defensins from Nigella sativa L. seeds,” Plant Physiology and Biochemistry, vol. 49, pp. 131-137, 2011.
7. [7] Salem, M.L. and Hossain, M.S., “Protective effect of black seed oil from Nigella sativa against murine cytomegalovirus infection,” International Journal of Immunopharmacology, vol. 22, pp. 729-740, 2000.
8. [8] Ilhan, A., Gurel, A., Armutcu, F., Kamisli, S. and Iraz, M., “Antiepileptogenic and antioxidant effects of Nigella sativa oil against pentylenetetrazol-induced kindling in mice,” Neuropharmacology, vol. 49, pp. 456-464, 2005.

9. [9] Mahmoud, M.R., El-Abhar, H.S. and Saleh, S., “The effect of Nigella sativa oil against the liver damage induced by Schistosoma mansoni infection in mice,” Journal of Ethnopharmacology, vol.79, pp. 1-11, 2002.
10. [10] Al-Naggar, T.B., Gómez-Serranillos, M.P., Carretero, M.E., Villar, A.M., “Neuropharmacological activity of Nigella sativa L. extracts,” Journal of Ethnopharmacology, vol. 88, pp. 63–68, 2003.
11. [11] Swamy, S.M.K. and Tan, B.K.H., “Cytotoxic and immunopotentiating effects of ethanolic extract of Nigella sativa L. seeds,” Journal of Ethnopharmacology, vol.70, pp. 1–7, 2000.
12. [12] Doulis, A.G., Harfouche, A.L. and Aravanopoulos, F.A., “Rapid, high quality DNA isolation from cypress (Cupressus sempervirens L.) needles and optimization of the RAPD’s marker technique,” Plant Mol. Biol. Rep., 17, pp. 1-14, 2000.
13. [13] Sozen, E. and Poyraz, I., “Rapid and High Quality DNA Isolation from Origanum onites for RAPD and ISSR Analysis,” Zeitschrift für Naturforschung C., vol. 63c, pp. 595-598, 2008.
14. [14] Khanuja, S.P.S., Shasany, A.K., Darokar, M.P. and Kumar ,S., “Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oil,” Plant Mol. Biol. Rep., vol. 17, pp. 1-7, 1999.
15. [15] Al-Huqail, A. and Al-Saad, F., “DNA Fingerprinting and Genotyping of Four Black Seed (Nigella sativa L.) Taxa,” Met. Env. & Arid. Land Agric. Sci., vol. 21/1, pp. 93-108, 2010.
16. [16] Doyle, J.J. and Doyle, J.L., “A rapid DNA isolation procedure from small quantities of fresh leaf tissue,” Phytochem. Bull., vol. 19, pp. 11-15, 1987.
17. [17] Fisher, P.J., Gardner, R.C. and Richardson, T.E., “Single locus microsatellites isolated using 5’ anchored PCR,” Nucleic Acids Res., vol.24, pp. 4369-4371, 1996.
18. [18] Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. and Tingey, S.V., “DNA polymorphisms amplified by arbitrary primers are useful as genetic markers,” Nucleic Acids Res., vol.18, pp. 6531-6535, 1990.
19. [19] Khan, M.A., “Chemical composition and medicinal properties of Nigella sativa Linn.,” Inflammopharmacology, vol. 7/1, pp. 15-35, 1999.
20. [20] Fang, G., Hammer, S. and Groumet, R., “A quick and inexpensive method for removing polysaccharides from plant genomic DNA,” Biofeedback, vol.13, pp. 52-54, 1992.
21. [21] Manoj, K., Tushar, B. and Sushama, C., “Isolation and purification of genomic DNA from black plum (Eugenia jambolana Lam.) for analytical applications,” Int. J. Biotech. Biochem. C., vol.3, pp. 49-55, 2007.
22. [22] Sarwat, M., Negi, M.S., Lakshmikumaran, M., Tyagi, A.G., Das, S. and Srivastava, P.S., “A standardized protocol for genomic DNA isolation from Terminalia arjuna for genetic diversity analysis,” Electron. J. Biotechn., vol.9, pp. 86-91, 2006.
23. [23] Remya, R., Syamkumar, S. and Sasikumar, B., “Isolation and amplification of DNA from turmeric powder,” Br. Food J., 106, pp. 673-678, 2004.
24. [24] Yu, S.T., Wang, C.T., Yu, S.L., Wang, X.Z., Tang, Y.Y., Chen, D.X. and Zhang, J.C., “Simple method to prepare DNA templates from a slice of peanut cotyledonary tissue for Polymerase Chain Reaction,” Electronic Journal of Biotechnology, vol.13/4, pp. 3-4, 2010.
25. [25] Fernández, M.E., Figueiras, A.M. and Benito, C., “The use of ISSR and RAPD markers for detecting DNA polymorphism, genotype identification and genetic diversity among barley cultivars with known origin,” Theor. Appl. Genet., vol. 104, pp. 845–851, 2002.
26. [26] Kalpana, D., Choi, S.H., Choi, T.K., Senthil, K. and Lee, Y.S., “Assessment of genetic diversity among varieties of mulberry using RAPD and ISSR fingerprinting,” Scientia Horticulturae, 134, pp. 79–87, 2012.
27. [27] Zhang, Q.X., Shen, Y.K., Shao, R.X., Fang, J., He, Y.Q., Ren, J.X., Zheng, B.S. and Chen, G.J., “Genetic diversity of natural Miscanthus sinensis populations in China revealed by ISSR markers,” Biochemical Systematics and Ecology, vol. 48, pp. 248–256, 2013.
28. [28] Yin, Y., Liu, Y., Wang, S., Zhao, S. and Xu, F., “Examining genetic relationships of Chinese Pleurotus ostreatus cultivars by combined RAPD and SRAP markers,” Mycoscience, vol.54, pp. 221-225, 2013.