Optimization of DNA Isolation and PCR Protocol for Analysis and Evaluation of Genetic Diversity of the Medicinal Plant, Anemopsis Californica Using RAPD

Abstract

Anemopsis californica is a perennial herbaceous plant that has been utilized as a medicinal plant for the treatment of various diseases. The present work was carried out with the objective of optimizing a method of extraction of the genomic DNA of A. californica and a PCR protocol and later to evaluate the existing genetic diversity among the genotypes deriving from different origins. For DNA extraction, we tested four procedures: with the CTA B-2 protocol, we obtained the highest yield (61.5±2.2 µg DNA/g of leaf tissues) and the best quality (A260/280 1.83±0.022). To estimate genetic variability, we utilized the randomly amplified polymorphism DNA (RAPD) technique, employing 20 oligonucleotides, of which only 18 generated reproducible banding patterns, producing 123 polymorphic bands generated, thus obtaining a polymorphism rate of 93.93% among the genotypes analyzed. The Jaccard similarity coefficient generated a variation ranging from 0.325-0.921, indicating a high level of genetic variation among the studied genotypes. An Unweighted pair-group method with arithmetic mean (UPGMA) group analysis indicated six distinct groups. The present optimized method for DNA isolation and RAPD protocol may serve as an efficient tool for further molecular studies.

References

  1. 1.

    Abu-Romman, S. (2011) Comparison of methods for isolating high quality DNA from sage (Salvia officinalis). J. Med. Plants Res. 5, 938–941.

    CAS  Google Scholar 

  2. 2.

    Adams, J. D., Garcia, C. (2006) Women’s health among the Chumash. eCAM 3, 125–131.

    PubMed  Google Scholar 

  3. 3.

    Agarwal, M., Shrivastava, N., Padh, H. (2008) Advances in molecular marker techniques and their applications in plant sciences. Plant Cell Rep. 27, 617–631.

    CAS  PubMed  Google Scholar 

  4. 4.

    Aljanabi, S. M., Forget, A., Dookun (1999) An improved and rapid protocol for the isolation of polysaccharide- and polyphenol-free sugarcane DNA. Plant Mol. Biol. Rep. 17, 1–8.

    Google Scholar 

  5. 5.

    Belaj, A., Trujilo, I., Rosa. R., Rallo, L., Gimenez, M. J. (2001) Polymorphism and discrimination capacity of randomly amplified polymorphic markers in an olive germplasm bank. J. Am. Soc. Hort. Sci. 126, 64–71.

    CAS  Google Scholar 

  6. 6.

    Calderón, R. G. (1996) Saururaceae. Flora del Bajío y Regiones Adyacentes 42, 1–5.

    Google Scholar 

  7. 7.

    Caldwell, D. G., McCallum, N., Shaw, P., Muehlbauer, G. J., Marshall, D. F., Waugh, R. (2004) A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.). Plant J. 40, 143–150.

    CAS  PubMed  Google Scholar 

  8. 8.

    Chalmers, K. J., Waugh, R., Sprent, J. I., Simons, A. J., Powell, W. (1992) Detection of genetic variation between and within populations of Gliricidia sepium and G. maculata using RAPD markers. Heredity 69, 465–472.

    Google Scholar 

  9. 9.

    Dellaporta, S., Wood, J., Hicks, J. B. (1983) A plant DNA minipreparation: version II. Plant Mol. Biol. Rep. 1, 19–21.

    CAS  Google Scholar 

  10. 10.

    Demeke, T., Jenkins, G. R. (2010) Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Anal. Bioanal. Chem. 396, 1977–1990.

    CAS  PubMed  Google Scholar 

  11. 11.

    Deshwall, R. P. S., Singh, R., Malik, K., Randhawa, G. J. (2005) Assessment of genetic diversity and genetic relationships among 29 populations of Azadirachta indica A. Juss. using RAPD markers. Genet. Resour. Crop Evol. 52, 285–292.

    Google Scholar 

  12. 12.

    Dhakshanamoorthy, D., Selvaraj, R. (2009) Extraction of genomic DNA from Jatropha sp. using modified CTAB method. Rom. J. Biol. - Plant Biol. 54, 117–125.

    Google Scholar 

  13. 13.

    Doyle, J. J., Doyle, J. L. (1990) Isolation of fresh DNA from fresh tissue. Focus 12, 13–15.

    Google Scholar 

  14. 14.

    Ganesh Ram, S., Parthiban, K. T., Senthil, K. R., Thiruvengadam, V., Paramathma, M. (2008) Geneticdiversity among Jatropha species as revealed by RAPD markers. Genet. Resour. Crop Evol. 55, 803–809.

    Google Scholar 

  15. 15.

    Gillies, A. C., Navarro, C., Lowe, A. J., Newton, A. C., Hernandez, M., Wilson, J., Cornelius, J. P. (1999) Genetic diversity in Mesoamerican populations of mahogany (Swietenia macrophylla) assessed using RAPDs. Heredity 83, 722–732.

    PubMed  Google Scholar 

  16. 16.

    Izumi, M., Kojima, E., Matsuda, M., Shimizu, T., Murakami, A., Tanabe, T., Murakami, K. (1997) Random amplified polymorphic DNA observed in Eucalyptus by PCR study with random primers. Nucleic Acids Symp. Ser. 37, 167–168.

    CAS  Google Scholar 

  17. 17.

    Jaccard, P. (1908) Nouvelles recherches sur la distribution florale. Bull. Soc. Vaud. Nat. 44, 223–270.

    Google Scholar 

  18. 18.

    Kaminsky, C. N., Ferrey, S. L., Lowrey, T., Guerra, L., Van Slambrouck, S., Steelant, W. F. A. (2010) In vitro anticancer activity of Anemopsis californica. Oncol. Lett. 1, 711–715.

    Google Scholar 

  19. 19.

    Kesari, V., Sudarshan, M., Das, A., Rangan, L. (2009) PCR amplification of the genomic DNA from the seeds or Ceylon ironwood, Jatropha, and Pongamia. Biomass Bioenergy 33, 1724–1728.

    CAS  Google Scholar 

  20. 20.

    Kumar, R. V., Tripathi, Y. K., Shukla, P., Ahlawat, S. P., Gupta, V. K. (2009) Genetic diversity and relationship among germoplasm of Jatropha curcas L. revealed by RAPDs. Trees 23, 1075–1079.

    CAS  Google Scholar 

  21. 21.

    Li, L. N. (1998) Biologically active components from traditional Chinese medicines. Pure Appl. Chem. 70, 547–554.

    CAS  Google Scholar 

  22. 22.

    Loveless, M. D. (1992) Isozyme variation in tropical trees:patterns of genetic organization. New For. 6, 67–94.

    Google Scholar 

  23. 23.

    Medina-Holguín, A. L., Holguín, F. O., Micheletto, S., Goehle, S., Simon, J. A., O’Connell, M. A. (2008) Chemotypic variation of essential oils in the medicinal plant, Anemopsis californica. Phytochemistry 69, 919–927.

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Medina-Holguín, A. L., Micheletto, S., Holguín, F. O., Rodriguez, J., O’Connell, M. A., Martin, C. (2007) Environmental influences on essential oils in roots of Anemopsis californica. HortScience 42, 1578–1583.

    Google Scholar 

  25. 25.

    Mejjad, M., Vedel, F., Ducreux, G. (1994) Improvement of DNA preparation and of PCR cycling in RAPD analysis of marine microalgae. Plant Mol. Biol. Rep. 12, 6–13.

    Google Scholar 

  26. 26.

    Nair, N. V., Nair, S., Sreenivasan, T. V., Mohan, M. (1999) Analysis of genetic diversity and phylogeny in Saccharum and related genera using RAPD markers. Genet. Resour. Crop Evol. 46, 73–79.

    Google Scholar 

  27. 27.

    Padmalatha, K., Prasad, M. N. V. (2006) Optimization of DNA isolation and PCR protocol for RAPD analysis of selected medicinal and aromatic plants of conservation concern from Peninsular India. Afr. J. Biotechnol. 5, 230–234.

    CAS  Google Scholar 

  28. 28.

    Parrotta, J. A. (2001) Healing Plants of Peninsular India. CABI Publishers, New York, USA, pp. 97–99.

    Google Scholar 

  29. 29.

    Pikkart, M. J., Villeponteau, B. (1993) Suppression of PCR amplification by high levels of RNA. Biotechniques 14, 24–25.

    Google Scholar 

  30. 30.

    Pirttilä, A. M., Hirsikorpi, M., Kamarainen, T., Jaakola, L., Hohtola, A. (2001) DNA isolation methods for aromatic and medicinal plants. Plant Mol. Biol. Rep. 19, 273a–273e.

    Google Scholar 

  31. 31.

    Pluhár, Z., Kocsis, M., Kuczmog, A., Csete, S., Simkó, H., Sárosi, S., Molnár, P., Horváth, G. (2012) Essential oil composition and preliminary molecular study of four Hungarian Thymus species. Acta Biol. Hung. 63, 81–96.

    PubMed  Google Scholar 

  32. 32.

    Rohlf, F. J. (1998) NTSYS-pc Numerical Taxonomy and Multivariate Analysis System, Ver. 2.2. Exeter Software, Setauket, New York.

    Google Scholar 

  33. 33.

    Sambrook, J., Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Google Scholar 

  34. 34.

    Schroeder, M. S., Weller, S. G. (1997) Self-incompatibility and clonal growth in Anemopsis californica. Plant Spec. Biol. 12, 55–59.

    Google Scholar 

  35. 35.

    Stöckigt, J., Obitz, P., Falkenhagen, H., Lutterbach, R., Endress, S. (1995) Natural products and enzymes from plant cell cultures. Plant Cell Tissue Organ Culture 43, 97–109.

    Google Scholar 

  36. 36.

    Storts, D. (1993) Promega Technical Manual 24 fmol DNA Sequencing System (TM024). Promega Corporation, Madison.

    Google Scholar 

  37. 37.

    Tan, S. C., Yiap, B. C. (2009) DNA, RNA and protein extraction: The past and the present. J. Biomed. Biotechnol. 1–10.

    Google Scholar 

  38. 38.

    Tripathi, L., Tripathi, J. N. (2003) Role of biotechnology in medicinal plants. Trop. J. Pharm. Res. 2, 243–253.

    Google Scholar 

  39. 39.

    Tripathi, Y. K., Gurha, P., Ghosh, D., Kumar, R. V., Goswami, H. K., Prakash, V. (2007) Determination of phylogenetic relationship among Isoetes species using random primers. Turk. J. Bot. 31, 1–6.

    Google Scholar 

  40. 40.

    Varma, A., Padh, H., Shrivastava, N. (2007) Plant genomic DNA isolation: an art or a science. Biotechnol. J. 2, 386–392.

    CAS  PubMed  Google Scholar 

  41. 41.

    Vural, H. C. (2009) Genomic DNA isolation from aromatic and medicinal plants growing in Turkey. Sci. Res. Essays 4, 59–64.

    Google Scholar 

  42. 42.

    Zhang, D. X., Hewitt, G. M. (2001) Special DNA Extraction Methods for Some Animal species. In: Karp, A., Isaac, P. G., Ingram, D. S. (eds.) Molecular Tools for Screening Biodiversity - Plants and Animals. Kluwer Academic Publishers.

    Google Scholar 

  43. 43.

    Zobel, B. J., Talbert, J. (1984) Applied Tree Improvement. John Wiley & Co, New York, pp. 503.

    Google Scholar 

Download references

Author information

Affiliations

Authors

Rights and permissions

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Reprints and Permissions

About this article

Cite this article

Lizette Del-Toro-Sánchez, C., Villaseñor-Alvarado, S., Zurita-Martínez, F. et al. Optimization of DNA Isolation and PCR Protocol for Analysis and Evaluation of Genetic Diversity of the Medicinal Plant, Anemopsis Californica Using RAPD. BIOLOGIA FUTURA 64, 184–195 (2013). https://doi.org/10.1556/ABiol.64.2013.2.5

Download citation

Keywords

  • Anemopsis californica
  • DNA isolation
  • RAPD analysis
  • genetic diversity