Skip to main content
Physiology and Molecular Biology of Plants logoLink to Physiology and Molecular Biology of Plants
. 2010 Sep 5;16(2):135–148. doi: 10.1007/s12298-010-0015-9

Assessment of genetic diversity through RAPD, ISSR and AFLP markers in Podophyllum hexandrum: a medicinal herb from the Northwestern Himalayan region

Pradeep Kumar Naik 1,, Md Afroz Alam 1, Harvinder Singh 1, Vinod Goyal 2, Swarup Parida 2, Sanjay Kalia 2, T Mohapatra 2
PMCID: PMC3550604  PMID: 23572963

Abstract

Total synthesis of podophyllotoxin is an expensive process and availability of the compound from the natural resources is an important issue for pharmaceutical companies that manufacture anticancer drugs. In order to facilitate reasoned scientific decisions on its management and conservation for selective breeding programme, genetic analysis of 28 populations was done with 19 random primers, 11 ISSR primers and 13 AFLP primer pairs. A total of 92.37 %, 83.82 % and 84.40 % genetic polymorphism among the populations of Podophyllum were detected using RAPD, ISSR and AFLP makers, respectively. Similarly the mean coefficient of gene differentiation (Gst) were 0.69, 0.63 and 0.51, indicating that 33.77 %, 29.44 % and 26 % of the genetic diversity resided within the population. Analysis of molecular variance (AMOVA) indicated that 53 %, 62 % and 64 % of the genetic diversity among the studied populations was attributed to geographical location while 47 %, 38 % and 36 % was attributed to differences in their habitats using RAPD, ISSR and AFLP markers. An overall value of mean estimated number of gene flow (Nm) were 0.110, 0.147 and 0.24 from RAPD, ISSR and AFLP markers indicating that there was limited gene flow among the sampled populations.

Keywords: Podophyllum hexandrum, RAPD, ISSR, AFLP

Full Text

The Full Text of this article is available as a PDF (631.8 KB).

Reference

  1. Ajmone, Marsan P., Castiglioni P., Fusari F., Kuiper M., Motto M. Genetic diversity and its relationship to hybrid performance in maize as revealed by RFLP and AFLP markers. Theor Appl Genet. 1998;96:219–227. doi: 10.1007/s001220050730. [DOI] [Google Scholar]
  2. Alam M.A., Pallavi G., Gulati A.K., Mishra G.P., Naik P.K. Characterization of genetic structure of Podophyllum hexandrum populations-an endangered medicinal herb of Northwestern Himalaya using ISSRPCR markers and its relatedness with podophyllotoxin content. African journal of Biotechnology. 2008;7(8):1028–1040. [Google Scholar]
  3. Anon (1970). Podophyllum hexandrum. The wealth of India: A Dictionary of Indian Raw Material and industrial products: Raw Materials. Vol. VIII: Ph.Re. Publication and Information Directorate. CSIR, New Delhi. pp. 170–174.
  4. Barker J.H.A., Matthes M., Arnold G.M., Edwards K.J., Ahman I., Larsson S., Karp A. Characterization of genetic diversity in potential biomass willows (Salix spp.) by RAPD and AFLP analyses. Genome. 1999;42:173–183. doi: 10.1139/gen-42-2-173. [DOI] [PubMed] [Google Scholar]
  5. Broomhead A.J., Dewick P.M. Tumor inhibitory aryltralin lignans in Podophyllum versipelle, Diphyllcia cymosa and Diphyllicia grayi. Phytochemistry. 1990;29:3831–3837. doi: 10.1016/0031-9422(90)85342-D. [DOI] [Google Scholar]
  6. Chatterjee R. Indian Podophyllum. Econ. Bot. 1952;6:342–354. [Google Scholar]
  7. De Knijff P., Denkers F., van Swelm N.D., Kuiper M. Genetic affinities within the herring gull Larus argentatus assemblage revealed by AFLP genotyping. J. Mol. Evol. 2001;52:85–93. doi: 10.1007/s002390010137. [DOI] [PubMed] [Google Scholar]
  8. Excoffier L., Smouse P.E., Quattro J.M. Analysis of molecular variance inferred from metric distance among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics. 1992;131:479–491. doi: 10.1093/genetics/131.2.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fahima T., Roder M.S., Wendehake K., Kirzhner V.M., Nevo E. Microsatellite polymorphism in natural population of wild emmer wheat, Trititum dicoccoides in Israel. Theor. Appl. Gent. 2002;104:17–29. doi: 10.1007/s001220200002. [DOI] [PubMed] [Google Scholar]
  10. Fay D.A., Ziegler H.W. Botanical source differentiation of Podophyllum resin by high performance liquid chromatography. J liq Chromatogr. 1985;8:1501–1506. doi: 10.1080/01483918508067160. [DOI] [Google Scholar]
  11. Felsenstein J. Phylogenies from restriction sites, a maximum likelihood approach. Evolution. 1995;46:557–574. doi: 10.1111/j.1558-5646.1992.tb01991.x. [DOI] [PubMed] [Google Scholar]
  12. Fritsch P.W., Rieseberg L.H. The use of random amplified polymorphic DNA (RAPD) in conservation genetics. In: Smith T., Wayne B., editors. Molecular Genetic Approaches in Conservation. New York: Oxford Univ. Press; 1996. pp. 54–73. [Google Scholar]
  13. Fu L.-G. Plant Red Book of China: Rare Threatened Plants. Beijing: Science Press; 1992. pp. 184–185. [Google Scholar]
  14. Fu Y.B., Phan A.T., Coulman B., Richards K.W. Genetic diversity in natural populations and corresponding seed collections of little bluestem as revealed by AFLP markers. Crop Sci. 2004;44:2254–2260. doi: 10.2135/cropsci2004.2254. [DOI] [Google Scholar]
  15. Hamrick J.L., Godt M.J.W. Conservation genetics of endemic plant species. In: Avise J.C., Hamrick J.L., editors. Conservation Genetics. New York: Chapman and Hall; 1996. pp. 281–304. [Google Scholar]
  16. Han T.H., Jeu M.D., Eck H.V., Jacobsen E. Genetic diversity of Chilean and Brazilian Alstroemeria species assessed by AFLP anaslysis. Heredity. 2000;84:564–569. doi: 10.1046/j.1365-2540.2000.00682.x. [DOI] [PubMed] [Google Scholar]
  17. Imbert T.F. Discovery of podophyllotoxins. Biochimie. 1998;80:207–22. doi: 10.1016/S0300-9084(98)80004-7. [DOI] [PubMed] [Google Scholar]
  18. Janssen P., Coopman R., Huys G., Swings J., Bleeker M., Vos P., Zabeau M., Kersters K. Evaluation of the DNA fingerprinting method AFLP as a new tool in bacterial taxonomy. Microbiol. 1996;142:1881–1893. doi: 10.1099/13500872-142-7-1881. [DOI] [PubMed] [Google Scholar]
  19. Kazan K., Manners J.M., Cameron D.F. Genetic variation in agronomically important species of Stylosanthes determine using random amplified polymorphic DNA markers. Theor. Appl. Genet. 1993;85:882–288. doi: 10.1007/BF00225033. [DOI] [PubMed] [Google Scholar]
  20. McGregor C.E., Lambert C.A., Greyliing M.M., Louw J.H., Warnich L. A comparative assessment of genetic diversity in dent and pocorn (Zea mays) inbred lines using inter-simple sequence repeat (ISSR) amplification. Mol. Breed. 2000;1:365–373. [Google Scholar]
  21. Myburg AA, O’Malley D, Sederoff RR and Whetten R (2000). Highthroughput multiplexed AFLP analysis of interspecific hybrids of Eucalyptus trees species. Plant & Animal Genome VIII conference, San Diego, CA, p 544.
  22. Na H.J., Um J.Y., Kim S.C., Koh K.H., Hwang W.J., Lee K.M., Kim C.H., Kim H.M. Molecular discrimination of medicinal Astragali radix by RAPD analysis. Immunopharmacol Immunotoxicol. 2004;26:265–272. doi: 10.1081/IPH-120037723. [DOI] [PubMed] [Google Scholar]
  23. Odat N., Jetschke G., Hellwig F.H. Genetic diversity of Ranunculus acris L. (Ranunculaceae) populations in relation to species diversity and habitat type in grassland communities. Mol. Breed. 2004;2:225–238. doi: 10.1111/j.1365-294X.2004.02115.x. [DOI] [PubMed] [Google Scholar]
  24. Prevost A., Wilkinson M.J. A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theo Appl Genet. 1999;98:107–112. doi: 10.1007/s001220051046. [DOI] [Google Scholar]
  25. Russel J.R., Fuller J.D., Macaulay M., Hatz B.G.B., Jahoor A., Powell W., Waugh R. Direct comparison of level of genetic variation among barley accessions detected by RFLPs, AFLPs, SSRs and RAPDs. Theor. Appl. Genet. 1997;95:714–722. doi: 10.1007/s001220050617. [DOI] [Google Scholar]
  26. Saghai-Maroof M.A., Soliman K.M., Jorgensen R.A., Allard R.W. Ribosomal spacer length in barley: Mendelian inheritance, Chromosomal location and population dynamics. Proc. Natl. Acad. Sci. (USA) 1984;81:8104–8118. doi: 10.1073/pnas.81.24.8014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sharma S.K., Knox M.R., Ellis T.H.N. AFLP analysis of the diversity and phylogeny of Lens and its comparison with RAPDanalysis. Theoretical and Applied Genetics. 1996;93(5–6):751–758. doi: 10.1007/BF00224072. [DOI] [PubMed] [Google Scholar]
  28. Sharma T.R., Singh B.M., Sharma N.R., Chauhan R.S. Identificatin of high podophyllotoxin producing biotypes of Podophyllum hexandrum form north-western Himalaya. J. Plan Biochem. & Biotech. 2000;9:49–51. [Google Scholar]
  29. Slatin M. Gene flow and geographic structure of natural populations. Science (Washington, DC) 1987;236:787–792. doi: 10.1126/science.3576198. [DOI] [PubMed] [Google Scholar]
  30. Sneath PHA and Sokal K (1973). Numerical Taxonomy, pp. 100–308.
  31. Stahelin H.F., Wartburg A.V. The chemical and biological route form podophyllotoxin glucoside to etoposide: Ninth Cain Memorail Award Lecture. Cancer Res. 1991;51:5–15. [PubMed] [Google Scholar]
  32. Vos P.R., Hogers M., Bleeker M., van de Lee Reijans T., Hornes M., Fritjers A., Pot J., Peleman J., Kuiper M., Zabeau M. AFLP: a new concept for DNA fingerprinting. Nucleic Acids Res. 1995;23:4407–4414. doi: 10.1093/nar/23.21.4407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wang X., Li Y., Li H., Zhang Y., Zhao L., Yu Y. RAPD analysis of genuineness on source of Bupleurum chinense. Zhong Yao Cai. 2003;26:855–856. [PubMed] [Google Scholar]
  34. Williams J.G.K., Kubelik A.R., Livak K.J., Rafalski J.A., Tingey S.V. DNA Polymorphisms amplified by arbitrary primers and useful as genetic markers. Nucl Acids Res. 1990;18:6531–6535. doi: 10.1093/nar/18.22.6531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ying T.S. On Dysosma Woodson and Sinopodophyllum Ying, gen. nov. of the Berberidaceae. Acta Phytoxon. Sin. 1979;17:15–23. [Google Scholar]
  36. Zietkiewicz E., Rafalski A., Labuda D. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics. 1994;20:176–183. doi: 10.1006/geno.1994.1151. [DOI] [PubMed] [Google Scholar]

Articles from Physiology and molecular biology of plants : an international journal of functional plant biology are provided here courtesy of Springer

RESOURCES