Document Type : Final File


1 Department of Botany, Faculty of Sciences, University of Shahrekord, Shahrekord, Islamic Republic of Iran

2 Department of Plant Breeding, Faculty of Agriculture, University of Shahrekord, Shahrekord, Islamic Republic of Iran


This study concerns the genetic diversity and taxonomic status of Marrubium species from central and south-west of Zagros region, Iran. It is investigated by Inter-Simple Sequence Repeat analysis. A total of 68 accessions from five Marrubium species were collected from their natural habitats. Molecular analysis was approved with 17 primers, of which 12 were carried out in the reaction mixture. Moreover, a data matrix was designed to estimate genetic parameters. To determine the genetic structure and taxonomic status, analysis of molecular variance, clustering analysis with UPGMA (Unweighted Pair Group Method Average) and the Jaccard similarity coefficient were estimated using NT-SYS-pc and Gene Alex software. Supplementary morphological evidences of calyx teeth features were also provided. The results of this study revealed that both sections Marrubium, Microdonta, and Ballota aucheri displayed a high percentage of polymorphism (PP=100%). In addition, their genetic diversity (Gst=0.99), number of effective alleles (Ne=1.53) and Shannon information index (I=0.51) showed a high percentage. Notably, all of the 12 primers produced reproducible bands. Analysis of molecular variance detected low quantities of gene variation among species (18%) from which high proportion of variation presented among populations within species (82%). Based on cluster analysis, M. cuneatum, M. vulgare and M. anisodon were definitely separated. Moreover, M. crassidens and M. vulgare were closely grouped. The calyx teeth features of M. cuneatum and M. crassidens revealed high variations which is consistent with molecular results. In conclusion, high genetic diversity in Marrubium species and accessions presents a valuable genetic resource in Zagros region, Iran. 


Main Subjects

  1. Abu-Asab M.S. and Cantino P.D. Systematic implications of pollen morphology in subfamilies Lamioideae and Pogostemonoideae. Ann. Mo. Bot. Gard. 81: 653-686 (1994).
  2. Agostini G., Teixeira de Souza-Chies T. and Echeverrigaray S. Genetic diversity of Cunila incisa Benth. (Lamiaceae). Med. Aromatic Plants 1: 1-3 (2012).
  3. Ahvazi M., Jamzad Z., Balali G.R. and Saeidi H. Trichome micro-morphology in Marrubium L. (Lamiaceae) in Iran and the role of environmental factors on their variation. Iran. J. Bot. 22: 39-58 (2016).
  4. Akgul G., Ketenoglu O., Pinar N.M. and Kurt L. Pollen and seed morphology of the genus Marrubium (Lamiaceae) in Turkey. Ann. Bot. Fenn. 45: 1-10 (2008).
  5. Ansari S.A., Narayanan C., Wali S.A., Kumar R., Shukla N. and Rahangdale S.K. ISSR markers for analysis of molecular diversity and genetic structure of Indian teak (Tectona grandis L.f.) populations. Ann. For. Res. 55:11–23 (2012).
  6. Aytac Z., Akgul G. and Ekici M. A new species of Marrubium (Lamiaceae) from Central Anatolia, Turkey. Turk. J. Bot. 36: 443-449 (2012).
  7. Badfar-Chaleshtori S., Shiran B., Kohgard M., Mommeni H., Hafizi A., Khodambashi M., Mirakhorli N. and Sorkheh K. Assessment of genetic diversity and structure of Imperial Crown (Fritillaria imperialis L.) populations in the Zagros region of Iran using AFLP, ISSR and RAPD markers and implications for its conservation. Biochem. Syst. Ecol. 42: 35–48 (2012).
  8. Bentham G. Labiatarum Genera et Species. Ridgeway & Sons, London, 783 p. (1834). 
  9. Boissier P.E. Flora Orientalis. Regnum Academic Scientific, Basel, 1276 p. (1879).
  10. Briquet J. Labiatae. In: Engler A. and Pran H.K. (Eds.), Die Naturlichen Pflanzenfamilien, W. Engelmann, Leipzig, pp.183-375 (1896).
  11. Chen L., Chen F., He S. and Ma L. High genetic diversity and small genetic variation among populations of Magnolia wufengensis (Magnoliaceae), revealed by ISSR and SRAP markers. Electron. J. Biotechnol. 17: 268–274 (2014).
  12. Cullen J. MarrubiumL. In: Davis P.H. (Ed.), Flora of Turkey and the Aegean Islands, Edinburgh Univ. Press, Edinburgh, pp. 165-178 (1982).
  13. Dundar E., Akcicek E., Dirmenci T. and Akgun S. Phylogenetic analysis of the genus Stachys sect. Eriostomum (Lamiaceae) in Turkey based on nuclear ribosomal ITS sequences. Turk. J. Bot. 37: 14-23 (2013).
  14. El Bardai S., Morel S., Wibo N., Faber M., Llabers N.G., Lyoussi B. and Uetin-Leclercq J. The vasorlaxant activity of murrabenol and marrubin from M. vulgare. Planta Med. 69: 75-77 (2003).
  15. Erbano M., Schuhli G.S. and Pereira dos Santos E. Genetic Variability and Population Structure of Salvia lachnostachys: Implications for Breeding and Conservation Programs. Int. J. Mol. Sci. 16: 7839-7850. (2015).
  16. Hou Y.C., Yan Z.H., Wei Y.M. and Zheng Y.L. Genetic diversity in barley from west China based on RAPD and ISSR analysis. Barley Genet. Newsl. 35: 9-22 (2005).
  17. Jamzad Z. Lamiaceae. In: Asadi M., Masoumi A.A. and Mozafarian V. (Eds.), Flora Iran, Research Institute of Forest and Rangelands, Tehran, pp.152-251 (2012).
  18. Karioti A., Heilmannb J. and Skaltsa H. Secondary Metabolites from Marrubium velutinum, Growing Wild in Greece. Z. Naturforsch. 60b: 328-332 (2005).
  19. 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 oils. Plant Mol. Biol. Rep. 17: 1-7 (1999).
  20. Kharazian N., Rahimi S. and Shiran B. Genetic diversity and morphological variability of fifteen Stachys (Lamiaceae) species from Iran using morphological and ISSR molecular markers. Biologia 70: 438-452 (2015).
  21. Kharazian N. and Hashemi M. Chemotaxonomy and morphological studies in five Marrubium L. species in Iran. Iran. J. Sci. Technol. 41: 17-31 (2017).
  22. Knorring O.F. Marrubium L. In: Schischkin B.K. (Ed.), Flora of the USSR, Israel Program for Scientific Translations, Jerusalem, pp.155-165 (1954).
  23. Kochieva E.Z., Khussein I.A., Legkobit M.P. and Khadeeva N.V. The detection of genome polymorphismin Stachys species using RAPD. Russ. J. Genet. 38: 516–520 (2002).
  24. Kochieva E.Z., Ryzhova N.N., Legkobit M.P. and Khadeeva N.V. RAPD and ISSR analyses of species and populations of the genus Stachys. Russ. J. Genet. 42: 723–727 (2006).
  25. Liu B. and Wendel J.F. Inter simple sequence repeat (ISSR) polymorphisms as a genetic marker system in cotton. Mol. Ecol. Notes. 1: 205-208 (2001).
  26. Liu J., Wang L., Geng Y., Wang Q., Luo L. and Zhong Y.G. Genetic diversity and population structure of Lamiophlomis rotata (Lamiaceae), an endemic species of Qinghai–Tibet Plateau. Genetica 128: 385-394 (2006).
  27. Mathiesen C., Scheen A.C.  and Lindqvist C. Phylogeny and biogeography of the Lamioid genus Phlomis (Lamiaceae). Kew Bull. 66: 83-89 (2012).
  28. Meimberg H., Abele T., Brauchler Ch., McKay J.K., Perez de Paz P.L. and Heubl G. Molecular evidence for adaptive radiation of Micromeria Benth. (Lamiaceae) on the Canary Islands as inferred from chloroplast and nuclear DNA sequences and ISSR fingerprint data. Mol. Phylogenet. Evol. 41: 566-578 (2006).
  29. Milbourne D., Meyer R., Bradshaw J.E., Baird E., Bonar N., Provan J., Powell W. and Waugh R. Comparison of PCR based marker systems for the analysis of genetic relationships in cultivated potato. Mol. Breeding 3:127-136 (1997).
  30. Moresco R.M., Maniglia T.C., De Oliveira C. and Margarido V.P. The pioneering use of ISSR (Inter Simple Sequence Repeat) in Neotropical anurans: preliminary assessment of genetic diversity in populations of Physalaemus cuvieri (Amphibia, Leiuperidae). Biol. Res. 46: 53-57 (2013).
  31. Muhamed N.H. Anticancer activity of Marrubium alysson L. and its phenolic constituents. In: Awaad A.S., Govil J.N. and Singh V.K. (Eds.), Drug Plants, Stadium Press LLC, USA, pp.185-193 (2010).
  32. Nei M. Analysis of gene diversity in subdivided populations. In: Proceeding of National Academic Sciences of the USA, pp. 3321-3323 (1973).
  33. Peakall R. and Smouse P. Gen AlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research – an update. Bioinformatics 28: 2537–2539 (2012).
  34. Puppo P., Curto M., Gusmao-Guedes J., Cochofel J., Perez de Paz P.L., Brauchler C. and Meimberg H. Molecular phylogenetics of Micromeria (Lamiaceae) in the Canary Islands, diversification and inter-island colonization patterns inferred from nuclear genes. Mol. Phylogenet. Evol. 89: 160-70 (2015).
  35. Rodrigues L., van den Berg C., Povoa O. and Monteiro A. Low genetic diversity and significant structuring in the endangered Mentha cervina populations and its implications for conservation. Biochem. Syst. Ecol. 50: 51-61 (2013).
  36. Rohlf F.J. NTSYS-pc: Numerical Taxonomy and Multivariate Analysis System. Exeter software, Setauket, New York, ver. 2.1. (2000).
  37. Saidi M., Movahedi K., Mehrabi A.A. and Kahrizi D. Molecular genetic diversity of Satureja bachtiarica. Mol. Biol. Rep. 40: 6501–6508 (2013).
  38. Sajadi S., Shiran B., Kharazian N., Houshmand S. and Sorkheh K. Genetic diversity of Salvia species from Chaharmahal va Bakhtiari and Isfahan province using AFLP molecular markers. J. Hort. Sci. 40: 79-88 (2010).
  39. Salmaki Y., Zarre S., Rydin O., Lindqvist C., Scheunert A., Brauchler C. and Heubl G. Phylogeny of the tribe Phlomideae with special focus on Eremostachys and Phlomioides: new insights from nuclear and chloroplast sequences. Taxon 61:161-179 (2012b).
  40. Seybold S. Marrubium L.In: Rechinger K.H. (Ed.), Flora Iranica, Akademische Druck- und Verlagsanstalt, Graz, pp. 88-108 (1982).
  41. Smolik M., Czak D.J. and Głowczyk A. Assessment of morphological and genetic variability in chosen Nepeta accessions. Herba Pol. J. 54: 68-78 (2008).
  42. Song Z., Li X., Wang H. and Wang J. Genetic diversity and population structure of Salvia miltiorrhiza Bge. In China revealed by ISSR and SRAP. Genetica 138: 241-249 (2010).
  43. Stankovic M.S. Total phenolic content, flavonoid concentration and antioxidant activity of Marrubium peregrinum L. extracts. Kragujevac J. Sci. 33: 63-72 (2011).
  44. Tero N., Aspi J., Siikamaki P., Jakalaniemi A. and Tuomi J. Genetic structure and gene flow in a metapopulation of an endangered plant species, Silene tatarica. Mol. Ecol. 12: 2073-2085 (2003).
  45. Wink M. and Kufmann M. Phylogenetic relationships between some members of the subfamily Lamioidae inferred from nucleotide sequence of the rbcl gene. Bot. Acta 109: 139-148 (1996).
  46. Wu F.Q., Shen Sh.K., Zhang X.J., Wang Y.H. and Sun W.B. Genetic diversity and population structure of an extremely endangered species: the world’s largest Rhododendron. AoB Plants 7:1-9 (2014).
  47. Yousefi Azarkhanian M., Asghari A., Ahmadi J., Asghari B. and Jafari A.A. Genetic diversity of Salvia species assessed by ISSR and RAPD markers. Not. Bot. Horti Agrobo. 44:431-436 (2016).
  48. Yuzbasıoglu E. and Dadand M.Y. Phylogenetic relationships among species of the subsection Dendrophlomis Bentham. Electron. J. Biotechno. 11: 1-9 (2008).
  49. Zaouali Y., Chograni H., Trimech R. and Boussaid M. Genetic diversity and population structure among Rosmarinus officinalis L. (Lamiaceae) varieties: var. typicus Batt. and var. troglodytorum Maire. based on multiple traits. Ind. Crop Prod. 38: 166-176 (2012).
  50. Zaghloul M.S., Hamrick J.L., Moustafa A.A., Kamel W.M. and El-Ghareeb R. Genetic diversity within and among Sinai populations of three Ballota species (Lamiaceae). J. Hered. 97: 45-54 (2006).