Investigation of the genetic variation of anise (Pimpinella anisum L.) using RAPD and ISSR markers

  • R. Refika Akçali GiachinoEmail author
Research Article


Anise (Pimpinella anisum L.) is an annual spice and medicinal plant belonging to the family Apiaceae. Only a limited number of anise breeding studies are available. Therefore, genetic variation among anise landraces has not been extensively examined with molecular markers. In this study, the molecular diversity of 15 Turkish anise landraces collected from different regions of Turkey and four foreign landraces obtained from Cyprus, Syria and France were analyzed using randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers. The discriminatory power of these markers was assessed by calculating various marker parameters, namely percent polymorphism, polymorphism information content (PIC), resolving power (RP), and marker index (MI). The genomic DNA of 19 anise landraces was amplified with nine RAPD primers that generated 71 polymorphic bands and five ISSRs primers that produced 45 polymorphic bands. The percentage of polymorphic loci was 59.8% in RAPD profiling and 51.5% in ISSR profiling. The average PIC values obtained using RAPD and ISSR markers were 0.40 and 0.38, respectively. The mean RP (2.90 and 2.67) and MI values (1.23 and 1.05) of the RAPD assay were found to be somewhat higher than those of the ISSR assay, but ISSR was more favorable than the RAPD techniques in terms of presenting the phylogenetic relationships in anise landraces. A UPGMA cluster grouped the 19 anise landraces into two major clusters based on both markers. The RAPD and ISSR marker systems were found to be useful for determining genetic diversity in P. anisum L. and identifying variation among landraces.


Anise Pimpinella anisum L. RAPD ISSR markers Molecular characterization Fingerprinting 



I thank the Commission of Scientific Research Projects of Ege University for financial support to carry out this study (Project No: 2013/OMYO/001). I am grateful to Dr. Tansel Kaygısız Aşçıoğul and Dr. Dilşat Yeğenoğlu for their assistance in statistical calculations of the data presented in this manuscript. I also thank Elif Denizaslanı and Christine Taylor for their suggestions to improve the language of the text. Finally, I would like to thank the reviewers for providing useful comments on the previous draft of this article.

Compliance with ethical standards

Conflict of interest

The author declares that they have no conflict of interest.


  1. Anderson JA, Churchill GA, Autrique JE, Tanksley SD, ME, Sorrells (1993) Optimizing parental selection for genetic linkage maps. Genome 36:181–186PubMedCrossRefPubMedCentralGoogle Scholar
  2. Arif M, Zaidi NW, Singh YP (2009) A comparative analysis of ISSR and RAPD markers for study of genetic diversity in Shisham (Dalbergia sissoo). Plant Mol Biol Rep 27:488–495CrossRefGoogle Scholar
  3. Arslan N, Gürbüz B, Sarihan EO, Bayrak A, Gümüşçü A (2004) Variation in essential Oil Content and composition in Turkish anise (Pimpinella anisum L.) Populations. Turk J Agric For 28:173–177Google Scholar
  4. Baruah J, Pandeya SK, Begum T, Sarma N, Paw M, Lal M (2019) Molecular diversity assessed amongst high dry rhizome recovery Ginger germplasm (Zingiber officinale Roscoe) from NE-India using RAPD and ISSR markers. Ind Crops Prod 129:463–471CrossRefGoogle Scholar
  5. Baser KHC (1997) Tıbbi ve Aromatik Bitkilerin Ilaç ve Alkollü içki Sanayinde Kullanımı. Istanbul Chamber of Commerce, Istanbul, p 33Google Scholar
  6. Bayram E (1992) Türkiye kültür anasonları (Pimpinella anisum L.) üzerine agronomic ve teknolojik araştırmalar. Dissertation, University of Ege, p 136Google Scholar
  7. Bishoyi AK, Sharma A, Kavane A, Geetha KA (2016) Varietal discrimination and genetic variability analysis of cymbopogon using RAPD and ISSR markers analysis. Appl Biochem Biotechnol 179:659–670. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Bolaric S, Barth S, Melchinger AE, Posselt UK (2005) Genetic diversity in European perennial ryegrass cultivars investigated with RAPD markers. Plant Breed 124:161–166CrossRefGoogle Scholar
  9. Chen S-Y, Dai T-X, Chang Y-T, Wang S-S, Ou S-L, Chuang W-L, Chuang C-Y, Lin Y-H, Lin Y-Y, Ku H-M (2013) Genetic diversity among Ocimum species based on ISSR, RAPD and SRAP markers. AJCS 7(10):1463–1471 ISSN: 1835–2707Google Scholar
  10. Chesnokov YV, Artemyeva A (2015) Evaluation of the measure of polymorphism information of genetic diversity. Agri Biol 50(5):571–578Google Scholar
  11. De Riek J, Calsyn E, Everaert I, Van Bockstaele E, De Loose M (2001) AFLP-based alternatives for the assessment of distinctness, uniformity and stability of sugar beet varieties. Theor Appl Genet 103:1254–1265CrossRefGoogle Scholar
  12. Dice LR (1945) Measures of the amount of ecologic association between species. Ecology 26:297–302CrossRefGoogle Scholar
  13. Ebrahimi M, Farajpour M, Rahimmalek M (2012) Inter-and intra-specific genetic diversity of Iranian yarrow species Achillea santolina and Achillea tenuifolia based on ISSR and RAPD markers. Genet Mol Res 11(3):2855–2861PubMedCrossRefPubMedCentralGoogle Scholar
  14. Elmeer K, Alghanem M, Al-Latifi L, Alhemairi H (2017) Efficiency of RAPD and ISSR markers for the detection of polymorphisms and genetic relationships in date palm. Biotechnology 16:19–26. CrossRefGoogle Scholar
  15. Fu YB (2002) Redundancy and distinctness in flax germplasm as revealed by RAPD. Plant Genet Resour 4:117–124CrossRefGoogle Scholar
  16. Fu YB, Guerin S, Peterson GW, Diederichsen A, Rowland GG, Richards KW (2003) RAPD analysis of genetic variability of regenerated seeds in the Canadian flax cultivar CDC Normandy. Seed Sci Technol 1:207–211CrossRefGoogle Scholar
  17. Gajera BB, Kumara N, Singha AS, Punvara BS, Ravikirana R, Sub-hasha N, Jadeja GC (2010) Assessment of genetic diversity in castor (Ricinus communis L.) using RAPD and ISSR markers. Ind Crops Prod 32:491–498. CrossRefGoogle Scholar
  18. Gajera HP, Tomar RS, Patel SV, Viradia RR, Golakiya BA (2011) Comparison of RAPD and ISSR markers for genetic diversity analysis among different endangered Mangifera indica genotypes of Indian Gir forest region. J. Plant Biochem Biotechnol 20(2):217–223CrossRefGoogle Scholar
  19. Giachino RRA, Sönmez Ç, Tonk FA, Bayram E, Yüce S, Telci I, Furan MA (2014) RAPD and essential oil characterization of Turkish basil (Ocimum basilicum L.). Plant Syst Evol. CrossRefGoogle Scholar
  20. Glemin S, Bazin E, Charlesworth D (2006) Impact of mating systems on patterns of sequence polymorphism in flowering plants. Proc Biol Sci 273:3011–3019CrossRefGoogle Scholar
  21. Gülçin İ, Oktay M, Kireçci E, Küfrevioğlu Öİ (2003) Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum) seed extracts. Food Chem 83(3):371–382CrossRefGoogle Scholar
  22. Gürbüz B (2010) Keyf Bitkileri-II. University of Ankara Faculty of Agriculture Edt., Ankara 1583–275 4–24p ISBN: 978–975–482–884–9Google Scholar
  23. Haider N, Nabulsi I, Mir Ali N (2012) Phylogenetic relationships among date palm (Phoenix dactylifera L.) cultivars in Syria using RAPD and ISSR markers. J Plant Biol Res 1:12–24Google Scholar
  24. Hamrick JL, Godt MJW (1996) Effects of life history traits on genetic diversity in plant species. Proc Biol Sci 351:1291–1298CrossRefGoogle Scholar
  25. İpek A, Demirayak Ş and Gürbüz B (2004) A study on the adaptation of some anise (Pimpinella anisum L.) population to Ankara conditions. Tarim Bilimleri Dergisi 10 (2): 202–205Google Scholar
  26. Iruela M, Rubio J, Cubero JI, Gill J, Millan T (2002) Phylogenetic analysis in the genus Cicer and cultivated chickpea using RAPD and ISSR markers. Theor Appl Genet 104:643–651PubMedCrossRefPubMedCentralGoogle Scholar
  27. Izzatullayeva V, Akparov Z, Babayeva S, Ojaghi J, Abbasov M (2014) Efficiency of using RAPD and ISSR markers in evaluation of genetic diversity in sugar beet. Turk J Biol. 38: 429–438 © TÜBİTAK. CrossRefGoogle Scholar
  28. Kayis SA, Erdogan EH and Emine P (2010) Comparison of Effectiveness of ISSR and RAPD Markers in Genetic Characterization of Seized Marijuana (Cannabis sativa L.) in Turkey Afr J Agric Res 5(21): 2925- 293Google Scholar
  29. Kumar M, Mishra GP, Singh R, Kumar J, Naik PK, Singh SB (2009) Correspondence of ISSR and RAPD markers for comparative analysis of genetic diversity among different apricot genotypes from cold arid deserts of trans-Himalayas. Physiol Mol Biol Plant 15:225–235CrossRefGoogle Scholar
  30. Kumar A, Mishra P, Singh SC, Sundaresan V (2014) Efficiency of ISSR and RAPD markers in genetic divergence analysis and conservation management of Justicia adhatoda L., a medicinal plant Subhash. Plant Syst Evol 300:1409–1420CrossRefGoogle Scholar
  31. Malik W, Ashraf J, Iqbal MZ, Khan AA, Qayyum A, Abid MA, Noor E, Ahmad MQ, Abbasi GH (2014) Molecular markers and cotton genetic improvement: current status and future prospects. Sci World J. CrossRefGoogle Scholar
  32. Manica-Cattani MF, Zacaria J, Pauletti G, Atti-Serafini L and Echeverrigaray S (2009) Genetic variation among South Brazilian accessions of Lippia alba Mill. (Verbenaceae) detected by ISSR and RAPD markers. Braz J Biol 69(2): 375–380CrossRefGoogle Scholar
  33. Mantel N (1967) The detection of disease clustering and a generalized regression approach. Cancer Res 27:175–178Google Scholar
  34. Maraklı S (2018) Transferability of barley retrotransposons (Sukkula and Nikita) to investigate genetic structure of Pimpinella anisum L. Marmara Fen Bilimleri Dergisi 3:299–304. CrossRefGoogle Scholar
  35. Milbourne D, Meyer R, Bradshaw JE, Baird E, Bonar N, Provan J, Powell W, Waugh R (1997) Comparison of PCR-based marker systems for the analysis of genetic relationships in cultivated Potato. Mol Breed 3(2):127–136CrossRefGoogle Scholar
  36. Mohd A, Zaidi NW, Singh YP, Haq QMR, Singh US (2009) A comparative analysis of ISSR and RAPD markers for study of genetic diversity in shisham (Dalbergiasissoo). Plant Mol Biol Rep 27:488–495CrossRefGoogle Scholar
  37. Moulin MM, Rodrigues R, Simões L, Gonçalves A, Sudré CP, Pereira MG (2012) A comparison of RAPD and ISSR markers reveals genetic diversity among sweet potato landraces (Ipomoea batatas (L.) Lam.). Maringá 4 (2):139–147Google Scholar
  38. Mukherjee A, Sikdar B, Ghosh B, Banerjee A, Ghosh E, Bhattacharya M, Roy S C (2013) RAPD and ISSR analysis of some economically important species, varieties, and cultivars of the genus Allium (Alliaceae). Turk J Bot (2013) 37: 605–618 © TÜBİTAK.
  39. Muthusamy S, Kanagarajan S, Ponnusamy S (2008) Efficiency of RAPD and ISSR markers system in accessing genetic variation of rice bean (Vigna umbellata) landraces. Electron J Biotechnol 11(3):15. ISSN: 0717–3458Google Scholar
  40. Naik A, Prajapat P, Krishnamurthy R, Pathak JM (2017) Assessment of genetic diversity in Costus pictus accessions based on RAPD and ISSR markers. 3Biotech 7:70Google Scholar
  41. Nurcahyanti AD, Nasser IJ, Sporer F, Graf J, Bermawie N, Reichling J & Wink M (2016) Chemical composition of the essential oil from aerial parts of Javanian Pimpinella pruatjan Molk and Its Molecular Phylogeny. Diversity 8(3): 15Google Scholar
  42. Nybom H (2004) Comparison of different nuclear DNA markersfor estimating intraspecific genetic diversity in plants. Mol Ecol 13:1143–1155CrossRefGoogle Scholar
  43. Özcan MM, Chalchat JC (2006) Chemical composition and antifungal effect of anise (Pimpinella anisum L.) fruit oil at ripening stage. Ann Microbiol 56:353–358CrossRefGoogle Scholar
  44. Parthasarathy U, Nandakishore OP, Kumar S, Parthasarathy VA (2013) Comparative effectiveness of inter-simple sequence repeat and randomly amplified polymorphic DNA markers to study genetic diversity of Indian Garcinia. Afr J Biotechnol 12:6443–6451CrossRefGoogle Scholar
  45. Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey S, Rafalski A (1996) The unity of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol Breed 2:25–238CrossRefGoogle Scholar
  46. Prevost A, Wilkinson MJ (1999) A new system of comparing PCR primers applied to ISSR fingerprinting. Theor Appl Genet 98:107–112CrossRefGoogle Scholar
  47. Rameshkumar R, Pandian S, Rathinapriya P, Selvi CT, Satish L, Gowrishankar S, Leung DWM (2019) Ramesh M (2019) Genetic diversity and phylogenetic relationship of Nilgirianthus ciliates populations using ISSR and RAPD markers: Implications for conservation of an endemic and vulnerable medicinal plant. Biocatal Agric Biotechnol 18:101072CrossRefGoogle Scholar
  48. Rana S and Das AB (2016) Assessment of genetic diversity in 48 landraces of Momordica dioica Roxb. ex Willd. from Odisha, India using RAPD and ISSR markers. Nucleus (2016) 59:107–114Google Scholar
  49. Rohlf FJ (2000) NTSYS-PC, Numerical taxonomy and multi-variate analysis system, version 2.1. Exeter Publications, Setauket, New YorkGoogle Scholar
  50. Saran PL, Choudhary R, Solanki IS, Patil P, Kumar S (2015) Genetic variability and relationship studies in new Indian papaya (Carica papaya L.) germplasm using morphological and molecular markers. Turk J Agric For 39:310–321CrossRefGoogle Scholar
  51. Sarı AO and Oguz B (2004) Activities on medicinal and aromatic plants at the Aegean Agricultural Research Institute, Baričevič, D., J. Bernáth, L. Maggioni and E. Lipman, compilers. Report of a Working Group on Medicinal and Aromatic Plants. Gozd Martuljek, Slovenia. International Plant Genetic Resources Institute, Rome, Italy.121–127Google Scholar
  52. Sivaprakash KR, Prasanth SR, Mohanty BP, Parida A (2004) Genetic diversity of black gram landraces as evaluated by AFLP markers. Curr Sci 86(10p):1411–1415Google Scholar
  53. Smýkal P, Bačová- Kerteszová N, Kalendar R, Corander J, Schulman AH, Pavelek M (2011) Genetic diversity of cultivated flax (Linum usitatissimum L.) germplasm assessed by retrotransposon-based markers. Theor Appl Genet 122:1385–1397PubMedCrossRefPubMedCentralGoogle Scholar
  54. Srilakshmi A, Ugraiah A, Gayatri MC and Rajanna L (2015) Assessment of genetic fidelity of micropropagated Pimpinella Candolleana Wight & Arn. By DNA markers. Int J Innov Appl Res 3(11):46–53Google Scholar
  55. Tabanca N, Douglas AW, Bedir E, Dayan FE, Kirimer N, Baser KHC, Aytac Z, Khan IA, Scheffler BE (2005) Patterns of essential oil relationships in Pimpinella (Umbelliferae) based on phylogenetic relationships using nuclear and chloroplast sequences. Plant Genet Resour 3(2):149–169CrossRefGoogle Scholar
  56. Tabanca N, Demirci B, Kirimer N, Baser KHC, Bedir E, Khan IA, Wedge DE (2006) Gas chromatographic–mass spectrometric analysis of essential oil from Pimpinella species gathered from Central and Northern Turkey. J Chromatogr A 1117:194–205PubMedCrossRefPubMedCentralGoogle Scholar
  57. Tagizad A, Ahmadi J, Haddad R, Zarrabi M (2010) A comparative analysis of ISSR and RAPD markers for studying genetic diversity in Iranian pistachio cultivars. Iran J Genet Plant Breed 1(1):9–13Google Scholar
  58. Tepe B, Akpulat AH, Sokmen M, Daferera D, Yumrutas O, Aydin E, Polissiou M, Sokmen M (2006) Screening of the antioxidative and antimicrobial properties of the essential oil of Pimpinella anisetum and Pimpinella flabellifolia from Turkey. Food Chem 97:719–724CrossRefGoogle Scholar
  59. Tirapelli CR, Andrade CR, Cassano AO, De Souza FA, Ambrosio SR, Costa FB, Oliveria AM (2007) Antispasmodic and relaxant effects of the hydroalcoholic extract of Pimpinella anisum (Apiaceae) on rat anococcygeous smooth muscle. J Enthopharmacol 110:23–29CrossRefGoogle Scholar
  60. Tonk FA, Yüce S, Bayram E, Giachino RRA, Sönmez Ç, Telci I, Furan MA (2010) Chemical and genetic variability of selected Turkish oregano (Origanum onites L.) clones. Plant Syst Evol 288:157–165CrossRefGoogle Scholar
  61. Tonk FA, Giachino RRA, Sönmez C, Yüce S, Bayram E, Telci I, Furan MA (2011) Characterization of various Hypericum perforatum clones by hypericin and RAPD analyses. Int J Agric Biol 13:31–37Google Scholar
  62. Touil L, Aike B, Suomin W, Ali F (2016) Genetic diversity of Tunisian and Chinese Alfalfa (Medicago sativa L.) revealed by RAPD and ISSR markers. Am J Plant Sci 7:967–979CrossRefGoogle Scholar
  63. Tripathi N, Saini N, Metho V, Kumar S, Tiwari S (2012) Assessment of Genetic Diversity among Withania somnifera collected from central India using RAPD and ISSR Analysis. Med Aromat Plant Sci Biotechnol 6(1):33–39Google Scholar
  64. TUİK (2018) Ministry of Food, Agriculture and Livestock,
  65. Tunc I, Sahinkaya S (1998) Sensitivity of two greenhouse pests to vapours of essential oils. Entomol Exp Appl 86:183–187CrossRefGoogle Scholar
  66. Wang ZX, Downie SR, Tan JB, Liao CY, Yu Y, He XJ (2014) Molecular Phylogenetics of Pimpinella and Allied Genera (Apiaceae), with Emphasis on Chinese Native Species, Inferred From nrDNA ITS and cpDNA Intron Sequence Data. Nord J Bot 32(5):642–657YXJCrossRefGoogle Scholar
  67. Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18:6531–6535PubMedPubMedCentralCrossRefGoogle Scholar
  68. Yilmaz KU, Ercisli S, Asma BM, Dogan Y, Kafkas S (2009) Genetic relatedness in Prunus genus revealed by inter-simple sequence repeat markers. HortScience 44:293–297CrossRefGoogle Scholar
  69. Zarini HN, Jafari H, Ramandi HD, Bolandi AR, Karimishahri MR (2019) A comparative assessment of DNA fingerprinting assays of ISSR and RAPD markers for molecular diversity of Saffron and other Crocus spp. in Iran. Nucleus. CrossRefGoogle Scholar
  70. Zietkiewicz E, Rafalski A, Labuda D (1994) Genome finger-printing by simple sequence repeat (SSR)-anchored poly-merase chain reaction amplification. Genomics 20:176–183CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Field Crops, Faculty of AgricultureEge UniversityIzmirTurkey

Personalised recommendations