Advertisement

Restriction Fragment Length Polymorphism of the 5S-rRNA-NTS Region: A Rapid and Precise Method for Plant Identification

  • Cinzia Margherita BerteaEmail author
  • Giorgio Gnavi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 862)

Abstract

Molecular genetic methods have several advantages over classical morphological and chemical analyses. The genetic method requires genotype instead than phenotype, therefore PCR-based techniques have been widely used for a rapid identification of plant species, varieties and chemotypes. Recently, the molecular discrimination of some higher plant species has been evaluated using sequences of a 5S-rRNA gene spacer region. The variation in the nontranscribed sequence (NTS) region has been used in a number of plant species for studying intraspecific variation, genome evolution, and phylogenetic reconstruction. Here, we describe a rapid method based on the use of the 5S-rRNA-NTS region as a tool for plant DNA fingerprinting, which combines PCR, sequencing and restriction fragment length polymorphism analyses.

Key words

DNA fingerprinting 5S-rRNA gene Nontranscribed sequence region PCR–RFLP High-resolution gel capillary electrophoresis Agilent 2100 Bioanalyzer 

References

  1. 1.
    Cai ZH, Li P, Dong TTX, Tsim KWK (1999) Molecular diversity of 5S-rRNA spacer domain in Fritillaria species revealed by PCR analysis. Planta Med 65:360–364PubMedCrossRefGoogle Scholar
  2. 2.
    Rubiolo P, Matteodo M, Bicchi C et al (2009) Chemical and biomolecular characterization of Artemisia umbelliformis Lam., an important ingredient of the alpine liqueur “Genepì”. J Agr Food Chem 57:3436–3443CrossRefGoogle Scholar
  3. 3.
    Bertea CM, Azzolin CMM, Bossi S et al (2005) Identification of an EcoRI restriction site for a rapid and precise determination of β-asarone-free Acorus calamus cytotypes. Phytochemistry 66:507–514PubMedCrossRefGoogle Scholar
  4. 4.
    Negi MS, Rajagopal J, Chauhan N et al (2002) Length and sequence heterogeneity in 5S rDNA of Populus deltoides. Genome 45:1181–1188PubMedCrossRefGoogle Scholar
  5. 5.
    Baker WJ, Hedderson TA, Dransfield J (2000) Molecular phylogenetics of Calamus (Palmae) and related rattan genera based on 5S nrDNA spacer sequence data. Mol Phylogenet Evol 14:218–231PubMedCrossRefGoogle Scholar
  6. 6.
    Trontin JF, Grandemange C, Favre JM (1999) Two highly divergent 5S rDNA unit size classes occur in composite tandem array in European larch (Larix decidua Mill.) and Japanese larch (Larix kaempferi (Lamb.) Carr.). Genome 42:837–848PubMedGoogle Scholar
  7. 7.
    Scoles GJ, Gill BS, Xin ZY et al (1988) Frequent duplication and deletion events in the 5S RNA genes and the associated spacer regions of the Triticeae. Plant Syst Evol 160:105–122CrossRefGoogle Scholar
  8. 8.
    Sugimoto N, Kiuchi F, Mikage M et al (1999) DNA profiling of Acorus calamus chemotypes differing in essential oil composition. Biol Pharm Bull 22:481–485PubMedCrossRefGoogle Scholar
  9. 9.
    Udovicic F, McFadden GI, Ladiges PY (1995) Phylogeny of Eucalyptus and Angophora based on 5S rDNA spacer sequence data. Mol Phylogenet Evol 4:247–256PubMedCrossRefGoogle Scholar
  10. 10.
    Foster LM, Kozak KR, Loftus MG et al (1993) The polymerase chain reaction and its application to filamentous fungi. Mycol Res 97:769–781CrossRefGoogle Scholar
  11. 11.
    Bertea CM, Luciano P, Bossi S et al (2006) PCR and PCR-RFLP of the 5S-rRNA-NTS region and salvinorin A analyses for the rapid and unequivocal determination of Salvia divinorum. Phytochemistry 67:371–378PubMedCrossRefGoogle Scholar
  12. 12.
    Gnavi G, Bertea CM, Usai M et al (2010) Comparative characterization of Santolina insularis chemotypes by essential oil composition, 5S-rRNA-NTS sequencing and EcoRV RFLP-PCR. Phytochemistry 71:930–936PubMedCrossRefGoogle Scholar
  13. 13.
    Ma XQ, Duan JA, Zhu DY et al (2000) Species identification of Radix Astragali (Huangqi) by DNA sequence of NTS 5S-rRNA spacer domain. Phytochemistry 54:363–368PubMedCrossRefGoogle Scholar
  14. 14.
    Gnavi G, Bertea CM, Maffei M (2010) PCR, sequencing and PCR–RFLP of the 5S-rRNA-NTS region as a tool for the DNA fingerprinting of medicinal and aromatic plants. Flavour Frag J 25:132–137CrossRefGoogle Scholar
  15. 15.
    Luciano P, Bertea CM, Temporale G et al (2007) DNA internal standard for the quantitative determination of hallucinogenic plants in plant mixtures. Forensic Sci Int Genet 1:262–266PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Plant Physiology Unit, Department of Plant BiologyUniversity of TurinTurinItaly

Personalised recommendations