Molecular Biology Reports

, Volume 45, Issue 5, pp 1405–1412 | Cite as

Rapid, simple and potentially universal method for DNA extraction from Opuntia spp. fresh cladode tissues suitable for PCR amplification

  • Joana Raimundo
  • Carlos Manuel Gaspar Reis
  • Maria Margarida RibeiroEmail author
Original Article


In Opuntia spp., the cladode tissues contain many polysaccharides and secondary metabolites that interfere with obtaining high-quality deoxyribonucleic acid (DNA), using currently available methods. To circumvent this problem, three commercial kits, three modified versions of the conventional cetyltrimethylammonium bromide method (CTAB) method and one combined method were tested in Opuntia ficus-indica, O. robusta, O. dillenii and O. elata species. We obtained a rapid and simple protocol that allows the extraction of DNA from all the tested species with good DNA yield and purity, namely, the combined method. With this method (DNeasy® Plant Mini Kit combined with the CTAB method), DNA yields from 13.2 ± 7.8 to 15.9 ± 11.3 µg g−1 of fresh tissue were obtained in the four Opuntia species. The purity, evaluated by the ratio A260/A280 ratio, ranged from 1.67 ± 0.12 to 2.01 ± 0.25, revealing low levels of problematic metabolites. The extracted DNA quality was confirmed by amplifying a set of nuclear microsatellites obtained for the genus. Reliable reproducible bands and electropherogram profiles were obtained. The combined method has potential to be universal for good-quality DNA extraction in cacti, particularly in the Opuntia genus and other difficult-to-extract species.


Cactus pear CTAB DNA extraction Polysaccharides Viscosity 



We acknowledge the Fundação para a Ciência e Tecnologia (Portugal) with grant UID/AGR/00239/2013, which funded MMR and grant UID/AMB/00681/2013, which funded MMR and CMGR, respectively. Our thanks are extended to Estelle Lerceteau-Köhler and Maria João Magalhães Gaspar for reviewing an earlier version of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

Cladode sampling of Opuntia samples was non-destructive. No specific permission was required to sample Opuntia species in Portugal. Field studies did not involve endangered or protected species.

Human and animal rights

No humans or animals were involved in the experiments.

Supplementary material

11033_2018_4303_MOESM1_ESM.docx (3.4 mb)
Supplementary material 1 (DOCX 3492 KB)


  1. 1.
    Kovats RS, Valentini R, Bouwer LM, Georgopoulou E, Jacob D, Martin E, Rounsvell M, Soussana JF (2014) Europe. In: Barros VR, Field CB, Dokken DJ, Mastrandrea MD, Mach KJ, Bilir TE, Chatterjee M, Ebi KL, Estrada YO, Genova RC, Girma B, Kissel ES, Levy AN, MacCracken S, Mastrandrea PR, White LL (eds) Climate Change 2014: Impacts, adaptation, and vulnerability. Part B: regional aspects. Contribution of working group II to the fifth assessment report of the intergovernmental panel on climate change. Cambridge University Press, Cambridge, pp 1267–1326Google Scholar
  2. 2.
    Nobel PS (1994) Remarkable agaves and cacti. Oxford University Press, OxfordGoogle Scholar
  3. 3.
    Mizrahi Y, Nerd A, Nobel PS (2010) Cacti as crops. In: Horticultural reviews. Wiley, pp 291–319.
  4. 4.
    Reis CMG, Gouveia C, Vitorino MC, Gazarini LC, Ribeiro MM, Peres F (2017) Bioactive compounds and morphology in Opuntia spp. fruits from Portuguese ecotypes. Bulg J Agric Sci 23:929–938Google Scholar
  5. 5.
    Lanuzza F, Occhiuto F, Monforte MT, Tripodo MM, D’Angelo V, Galati EM (2017) Antioxidant phytochemicals of Opuntia ficus-indica (L.) Mill. cladodes with potential anti-spasmodic activity. Pharmacogn Mag 13:S424–S429. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Kalegowda P, Chauhan AS, Nanjaraj Urs SM (2017) Opuntia dillenii (Ker-Gawl) Haw cladode mucilage: physico-chemical, rheological and functional behavior. Carbohydr Polym 157:1057–1064. CrossRefPubMedGoogle Scholar
  7. 7.
    Li H, Yuan Q, Zhou X, Zeng F, Lu X (2016) Extraction of Opuntia dillenii Haw. polysaccharides and their antioxidant activities. Molecules 21:1612CrossRefGoogle Scholar
  8. 8.
    Msaddak L, Abdelhedi O, Kridene A, Rateb M, Belbahri L, Ammar E, Nasri M, Zouari N (2017) Opuntia ficus-indica cladodes as a functional ingredient: bioactive compounds profile and their effect on antioxidant quality of bread. Lipids Health Dis 16:32CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Lorenzo FD, Silipo A, Molinaro A, Parrilli M, Schiraldi C, DÁgostino A, Izzo E, Rizza L, Bonina A, Bonina F, Lanzetta R (2017) The polysaccharide and low molecular weight components of Opuntia ficus-indica cladodes: structure and skin repairing properties. Carbohydr Polym 157:128–136. CrossRefPubMedGoogle Scholar
  10. 10.
    Shedbalkar UU, Adki VS, Jadhav JP, Bapat VA (2010) Opuntia and other cacti: applications and biotechnological insights. Trop Plant Biol 3:136–150. CrossRefGoogle Scholar
  11. 11.
    Nobel PS, Cavelier J, Andrade JL (1992) Mucilage in cacti: its apoplastic capacitance, associated solutes, and influence on tissue 5. J Exp Bot 43:641–648. CrossRefGoogle Scholar
  12. 12.
    Nobel PS (1988) Environmental biology of agaves and cacti. Cambridge Univ. Press, CambridgeGoogle Scholar
  13. 13.
    Mondragon-Jacobo C, Doudareva N, Bordelon BP (2000) DNA extraction from several cacti. HortScience 35:1124–1126Google Scholar
  14. 14.
    De la Cruz M, Ramirez F, Hernandez H (1997) DNA isolation and amplification from cacti. Plant Mol Biol Rep 15:319–325CrossRefGoogle Scholar
  15. 15.
    Guillemaut P, Maréchal-Drouard L (1992) Isolation of plant DNA: a fast, inexpensive, and reliable method. Plant Mol Biol Rep 10:60–65. CrossRefGoogle Scholar
  16. 16.
    Pandey RN, Adams RP, Flournoy LE (1996) Inhibition of random amplified polymorphic DNAs (RAPDs) by plant polysaccharides. Plant Mol Biol Rep 14:17–22. CrossRefGoogle Scholar
  17. 17.
    Wang XN, Felker P, Burow MD, Paterson AH (1998) Comparison of RAPD marker patterns to morphological and physiological data in the classification of Opuntia accessions. J Prof Assoc Cactus 3:3–14Google Scholar
  18. 18.
    Mondragon-Jacobo C, Bordelon BB (1996) Cactus pear (Opuntia spp. Cactaceae) breeding for fruit production. J Prof Assoc Cactus 1:19–35Google Scholar
  19. 19.
    Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  20. 20.
    Rowland LJ, Nguyen B (1993) Use of polyethylene glycol for purification of DNA from leaf tissue of woody plants. Biofeedback 14:735–736Google Scholar
  21. 21.
    Haymes KM (1996) Mini-prep method suitable for a plant breeding program. Plant Mol Biol Rep 14:280–284CrossRefGoogle Scholar
  22. 22.
    Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21. CrossRefGoogle Scholar
  23. 23.
    Keb-Llanes M, Gonzalez G, Chi-Manzanero B, Infante D (2002) A rapid and simple method for small scale DNA extraction in Agavaceae and other tropical plants. Plant Mol Biol Rep 20:299a–299eCrossRefGoogle Scholar
  24. 24.
    Cota-Sánchez JH, Remarchuk K, Ubayasena K (2006) Ready to use DNA extracted with a CTAB method adapted for herbarium specimens and mucilaginous plant tissue. Plant Mol Biol Rep 24:161–167CrossRefGoogle Scholar
  25. 25.
    Fehlberg SD, Allen JM, Church K (2013) A novel method of genomic DNA extraction for Cactaceae. Appl Plant Sci. PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Reis CMG, Gazarini LC, Fonseca TF, Ribeiro MM (2018) Above-ground biomass estimation of Opuntia ficus-indica (L.) Mill. for forage crop in a Mediterranean environment by using non-destructive methods. Exp Agric. CrossRefGoogle Scholar
  27. 27.
    Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
  28. 28.
    Ferreira ME, Grattapaglia D (1998) Introdução ao uso de marcadores moleculares em análise genética. Embrapa-Cenargen, BrasíliaGoogle Scholar
  29. 29.
    Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  30. 30.
    Helsen P, Verdyck P, Tye A, Desender K, Van Houtte N, Van Dongen S (2007) Isolation and characterization of polymorphic microsatellite markers in Galapagos prickly pear (Opuntia) cactus species. Mol Ecol Notes 7:454–456CrossRefGoogle Scholar
  31. 31.
    Erre P, Nieddu G, Chessa I (2011) Identification of microsatellite loci in Opuntia spp. and their characterization in cultivars and species. Acta Hortic 918:327–332CrossRefGoogle Scholar
  32. 32.
    Dennis ES, Ellis J, Green A, Llewellyn D, Morell M, Tabe L, Peacock WJ (2008) Genetic contributions to agricultural sustainability. Philos Trans R Soc B Biol Sci 363:591–609. CrossRefGoogle Scholar
  33. 33.
    Hoisington D, Khairallah M, Reeves T, Ribaut JM, Skovmand B, Taba S, Warburton M (1999) Plant genetic resources: what can they contribute toward increased crop productivity? Proc Natl Acad Sci USA 96:5937–5943. CrossRefPubMedGoogle Scholar
  34. 34.
    Arnholdt-Schmitt B, Girão LC, Llamoca-Zárate RM, Campos FAP (2001) Genome characterization of Opuntia ficus-indica: a simple and efficient micromethod. J Prof Assoc Cactus 4:57–65Google Scholar
  35. 35.
    Sánchez-Hernández C, Gaytán-Oyarzún JC (2006) Two mini-preparation protocols to DNA extraction from plants with high polysaccharide and secondary metabolites. Afr J Biotechnol 5(20):1864–1867Google Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Centro de Biotecnologia de Plantas da Beira InteriorEscola Superior Agrária de Castelo BrancoCastelo BrancoPortugal
  2. 2.Instituto Politécnico de Castelo BrancoEscola Superior AgráriaCastelo BrancoPortugal
  3. 3.Centro de Recursos Naturais, Ambiente e Sociedade (CERNAS) - Instituto Politécnico de Castelo BrancoCastelo BrancoPortugal
  4. 4.Forest Research Centre, School of AgricultureUniversity of LisbonLisbonPortugal

Personalised recommendations