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Planta Med 2017; 83(11): 946-953
DOI: 10.1055/s-0043-106585
Natural Product Chemistry and Analytical Studies
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

Phylogenetic Analysis and Molecular Characterization of Xanthium sibiricum Using DNA Barcoding, PCR-RFLP, and Specific Primers[*]

Salvatore Tomasello
Systematic Botany and Mycology, Department Biology I, Ludwig-Maximilians-University Munich (LMU) and GeoBio-Center (LMU), Munich, Germany
,
Günther Heubl
Systematic Botany and Mycology, Department Biology I, Ludwig-Maximilians-University Munich (LMU) and GeoBio-Center (LMU), Munich, Germany
› Author Affiliations
Further Information

Publication History

received 23 September 2016
revised 17 February 2017

accepted 13 March 2017

Publication Date:
10 April 2017 (online)

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Abstract

The fruits of Xanthium sibiricum have been widely used in traditional Chinese medicine for the treatment of nasal sinusitis and headaches. The genus Xanthium (cocklebur) is a taxonomically complex genus. Different taxonomic concepts have been proposed, some including several species, others lumping the different taxa in a few extremely polymorphic species. Due to the morphological similarities between species, the correct authentication of X. sibiricum is very difficult. Therefore, we established a polymerase chain reaction-restriction fragment length polymorphism method and diagnostic PCR based on nuclear internal transcribed spacer and chloroplast trnQ-rps16 barcodes to differentiate X. sibirium from related species.

Results from the phylogenetic analyses based on sequence information from four marker regions (plastidal psbA-trnH and trnQ-rps16 and nuclear ITS and D35) support those taxonomic concepts accepting a reduced number of species, as four to five major clades are revealed in the phylogenetic reconstructions. X. sibiricum, together with some accessions from closely related taxa, is always supported as monophyletic, constituting a well-defined genetic entity. Allele-specific primer pairs for ITS and trnQ-rps16 were designed to amplify diagnostic products from the genomic DNA of X. sibiricum. Specific PCR in combination with digestion using the restriction enzyme MseI allowed for the identification of X. sibiricum by producing specific restriction patterns. The results demonstrate that the applied techniques provide effective and accurate authentication of X. sibiricum.

Key words

Asteraceae - Xanthium - Xanthium sibiricum - phylogeny - Chinese medicine - internal transcribed spacer (ITS) - PCR- RFLP

* This publication is dedicated to Prof. Dr. Rudolf Bauer on the occasion of his 60th birthday.


Supporting information

    Individual gene trees from the phylogenetic analyses, a table listing the samples used and GenBank accession numbers, and detailed information on the DNA extraction and PCR amplification procedures are available as Supporting Information.

  • Supporting Information
 

  • References

  • 1 Parsons WT. Noxious Weeds of Victoria. Melbourne: Inkata Press; 1973: 300
    Google Scholar
  • 2 Reed CF, Hughes RD. Selected Weeds of the United States. U. S. Dep. Agric. Res. Serv. Agric. Handbook No. 366. Washington: U.S. Govt. Printing Office; 1970
    Google Scholar
  • 3 Martin RJ. Distribution, Ecology and Control of Xanthium Species [Dissertation]. Canberra: Australian National University; 1981
    PubMedGoogle Scholar
  • 4 Widder FJ. Die Arten der Gattung Xanthium. Beiträge zu einer Monographie. Rep Spec Nov Regni Veg 1923; 20: 1-222
    PubMedGoogle Scholar
  • 5 Sell PD, Murrell JG. Flora of Great Britain and Ireland, Vol. 4. Cambridge: Cambridge University Press; 2006
    Google Scholar
  • 6 Löve D, Dansereau P. Biosystematic studies on Xanthium: taxonomic appraisal and ecological status. Can J Bot 1959; 37: 173-208
    CrossrefPubMedGoogle Scholar
  • 7 Holm LG, Plucknett DL, Pancho JV, Herbergsr JP. The Worldʼs worst Weeds. Honolulu: University of Hawaii Press; 1977: 609
    Google Scholar
  • 8 Weawer SE, Lechowicz MJ. The biology of Canadian weeds. 56. Xanthium strumarium L. Can J Plant Sci 1983; 63: 211-225
    CrossrefPubMedGoogle Scholar
  • 9 Hicks JA. Systematic Studies of Xanthium (Compositae: Ambrosieae); the Cockleburs of Tazewell Country, Illinois [Dissertation]. Urbana-Champaign: University Illinois; 1971
    PubMedGoogle Scholar
  • 10 Nadeau LH. Etude biosystematique sur le genre Xanthium [unpubl. PhD thesis]. Montreal: University of Montreal; 1961
    PubMedGoogle Scholar
  • 11 Strother JR. Xanthium . In: Flora of North America Editorial Committee, eds. Flora of North America, vol. 21. New York, Oxford: Oxford University Press; 2006: 19-20
    Google Scholar
  • 12 Stuart BP, Cole RJ, Gosser HS. Cocklebur (Xanthium strumarium var. strumarium) intoxication in swine: review and redefinition of the toxic principle. Vet Pathol 1981; 18: 368-383
    CrossrefPubMedGoogle Scholar
  • 13 Witte ST, Osweiler GD, Stahr HM, Mobley G. Cocklebur toxicosis in cattle associated with the consumption of mature Xanthium strumarium . J Vet Diagn Invest 1990; 2: 263-267
    CrossrefPubMedGoogle Scholar
  • 14 Méndez MC, Santos RC, Riet-Correa F. Intoxication by Xanthium cavanillesii in cattle and sheep in southern Brazil. Vet Hum Toxicol 1998; 40: 144-147
    PubMedGoogle Scholar
  • 15 Cole RJ, Stuart BP, Lansden JA, Cox RX. Isolation and redefinition of the toxic agent from cocklebur Xanthium strumarium . J Agric Food Chem 1980; 28: 1330
    CrossrefPubMedGoogle Scholar
  • 16 Pfaff E, Klingenberg M, Heldt HW. Unspecific permeation and specific exchange of adenine nucleotides in liver mitochondria. Biochim Biophys Acta 1965; 104: 312-315
    CrossrefPubMedGoogle Scholar
  • 17 Vignais PV, Vignais PM, Defaye G. Gummiferin, an inhibitor of the adenine-nucleotide translocation. Study of its binding properties to mitochondria. FEBS Lett 1971; 17: 281-288
    CrossrefPubMedGoogle Scholar
  • 18 An HJ, Jeong HJ, Lee EH, Kim YK, Hwang QJ, Yoo SJ, Hong SH, Kim HM. Xanthii Fructus inhibits inflammatory responses in LPS-stimulated mouse peritoneal macrophages. Inflammation 2004; 28: 263-270
    CrossrefPubMedGoogle Scholar
  • 19 Hong SH, Jeong HJ, Kim HM. Inhibitory effects of Xanthii Fructus extract on mast cell-mediated allergic reaction in murine model. J Ethnopharmacol 2003; 88: 229-234
    CrossrefPubMedGoogle Scholar
  • 20 Song MY, Kim EK, Lee HJ, Park JW, Ryu DG, Kwon KB, Park BH. Fructus Xanthii extract protects against cytokine-induced damage in pancreatic beta-cells through suppression of NF-kappaB activation. Int J Mol Med 2009; 23: 547-553
    PubMedGoogle Scholar
  • 21 Pharmacopoeia Commission. Pharmacopoeia of the Peopleʼs Republic of China, Volume I. Beijing: China Medical Science Press; 2010: 461
    Google Scholar
  • 22 An J, Wang YD, Sheng CC, Wang GZ. Comparative analysis of carboxyatractyloside and atractyloside contents in Xanthii Fructus before and after processing. Chin J Pharm Anal 2013; 33: 1910-1913
    PubMedGoogle Scholar
  • 23 Nikles S, Heuberger H, Hilsdorf E, Schmücker R, Seidenberger R, Bauer R. Influence of processing on the content of toxic carboxyatractyloside and atractyloside and the microbiological status of Xanthium sibiricum fruits (Cangʼerzi). Planta Med 2015; 81: 1213-1220
    Article in Thieme ConnectPubMedGoogle Scholar
  • 24 Wallace LJ, Boilard SMAL, Eagle SHC, Spall JL, Shokaralla S, Hajibabaei M. DNA barcodes for everyday life: routine authentication of natural health products. Food Res Int 2012; 49: 446-452
    CrossrefPubMedGoogle Scholar
  • 25 Ferri G, Corradini B, Ferrari S, Santunione AL, Palazzoli F, Alú M. Forensic botany II, DNA barcode for land plants: which markers after the international agreement?. Forensic Sci Int 2015; 15: 131-136
    CrossrefPubMedGoogle Scholar
  • 26 Zhao X, Hu W. Application of ITS2 sequence as DNA barcode in Xanthium . Agric Biotechnol 2014; 3: 19-21
    PubMedGoogle Scholar
  • 27 Millspaugh CF, Sherrf EE. Revision of the North American species of Xanthium. Field Museum Natural History Publication 204. Botanical Series 1919; 4: 9-49
    PubMedGoogle Scholar
  • 28 Wang J, Liu X, Zhang Y, Song M, Lin Y, Ma X, Sun W, Xiang L, Hu Z, Wu L, Zhang X, Hu W. Identification of Xanthii Fructus and its adulterants based on ITS2 sequence. World Science and Technology-Modernization of Traditional Chinese Medicine and Materia Medica 2014; 16: 329-334
    PubMedGoogle Scholar
  • 29 Chapman MA, Chang J, Weisman D, Kesseli RV, Burke JM. Universal markers for comparative mapping and phylogenetic analysis in the Asteraceae (Compositae). Theor Appl Genet 2007; 115: 747-755
    CrossrefPubMedGoogle Scholar
  • 30 Meimberg H. Molekular-Systematische Untersuchungen an den Familien Nepenthaceae und Ancistrocladaceae sowie verwandter Taxa aus der Unterklasse Caryophyllidae s.l. [diploma thesis]. Munich: Ludwigs-Maximilian-Universität (LMU); 2002
    PubMedGoogle Scholar
  • 31 White TJ, Bruns T, Lee S, Taylor J. Amplification and direct Sequencing of fungal ribosomal RNA Genes for Phylogenetics. In: Innis MA, Gelfand DH, Sninski JJ, White TJ. eds. PCR Protocols. A Guide to Methods and Applications, UK eEdition. San Diego: Academic Press; 1990: 315-322
    Google Scholar
  • 32 Shaw J, Small RL. Addressing the ʼhardest puzzle in American pomologyʼ: phylogeny of Prunus sect. Prunocerasus (Rosaceae) based on seven noncoding chloroplast DNA regions. Am J Bot 2004; 91: 985-996
    CrossrefPubMedGoogle Scholar
  • 33 Calviño IC, Downie SR. Circumscription and phylogeny of Apiaceae subfamily Saniculoideae based on chloroplast DNA sequences. Mol Phylogenet Evol 2007; 44: 175-191
    CrossrefPubMedGoogle Scholar
  • 34 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013; 30: 2725-2729
    CrossrefPubMedGoogle Scholar
  • 35 Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994; 22: 4673-4680
    CrossrefPubMedGoogle Scholar
  • 36 Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 2012; 9: 772
    PubMedGoogle Scholar
  • 37 Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 2012; 61: 539-542
    CrossrefPubMedGoogle Scholar
  • 38 Miller MA, Pfeiffer W, Schwartz T. Creating the CIPRES Science Gateway for Inference of large phylogenetic Trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE). New Orleans: 2010: 1-8
    PubMedGoogle Scholar
  • 39 Simmons MP, Ochoterena H. Gaps as characters in sequence-based phylogenetic analyses. Syst Biol 2000; 49: 369-381
    CrossrefPubMedGoogle Scholar
  • 40 Young N, Healy J. GapCoder automates the use of indel characters in phylogenetic analysis. BMC Bioinformatics 2003; 4: 6
    CrossrefPubMedGoogle Scholar
  • 41 Rambaut A, Suchard MA, Xie D, Drummond AJ. Tracer v1.6. Available at. http://tree.bio.ed.ac.uk/software/tracer/ Accessed January 23, 2015
    PubMedGoogle Scholar
  • 42 Müller K. SeqState – primer design and sequence statistics for phylogenetic DNA datasets. Appl Bioinformatics 2005; 4: 65-69
    CrossrefPubMedGoogle Scholar
  • 43 Müller K. Incorporating information from length-mutational events into phylogenetic analysis. Mol Phylogenet Evol 2006; 38: 667-676
    CrossrefPubMedGoogle Scholar
  • 44 Vincze T, Posfai J, Roberts RJ. NEBcutter: a program to cleave DNA with restriction enzymes. Nucleic Acids Res 2003; 31: 3688-3691
    CrossrefPubMedGoogle Scholar