Design and evaluation of nematode 18S rDNA primers for PCR and denaturing gradient gel electrophoresis (DGGE) of soil community DNA

Ian S. Waite, Anthony G. O'Donnell, Andrew Harrison, John T. Davies, Stephanie R. Colvan, Klemens Ekschmitt, Hülya Dogan, Volkmar Wolters, Tom Bongers, Marina Bongers, G. Bakonyi, Péter Nagy, Efimia M. Papatheodorou, George P. Stamou, Sven Boström

Research output: Contribution to journalArticle

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Abstract

Consensus nematode 18S ribosomal DNA primers were designed by aligning available 18S sequences and identifying a variable region flanked by highly conserved regions. These primers were then used to amplify nematode 18S rDNA from whole soil community DNA extracted from a range of European grassland types. Cloning of the PCR amplicons (778 bp) followed by restriction digest analysis (RFLP) resulted in the recovery of 34 unique nematode sequences from the four grasslands studied. Comparison of these data with the limited number of 18S rDNA nematode sequences currently held in on-line databases revealed that all of the sequences could be assigned to known nematode taxa albeit tentatively in some cases. Two of the sequences recovered from the site in the Netherlands (wet, hay-grassland) were recovered in a clade that included a sequence of the genus Trichodorus whilst other sequences from this site showed similarity with 18S rDNA sequences of the genus Prismatolaimus (five sequences), Xiphinema (one sequence) and Enoplus (one sequence). Of the remaining sequences, two showed some affinity with Mylonchulus (UK, upland peat), four with Steinernema (UK) and one sequence with Mesorhabditis (Hungary, east European Steppe). Three sequences from the Netherlands and one from Hungary were recovered in a clade that included a sequence of the genus Pratylenchoides whilst three further sequences from the Netherlands and two from Hungary were recovered in a clade encompassing the genus Globodera. Of the remaining nine sequences, two (NL6, NL62) formed a distinct lineage within the Adenophorea with 90% bootstrap recovery in a paraphyletic clade that included sequences of Prismatolaimus and Trichodorus. Seven sequences (three from the Netherlands, three from the UK and one from Greece) were left unassigned though the tree topology suggested some relationship (58% bootstrap recovery) with the genus Cephalobus. To assess whether primers used to amplify 18S rDNA might be used to fingerprint genetic diversity in nematode communities in soil, the environmental sequence data were used to design a second set of primers carrying a GC-clamp. These primers amplified a 469 bp fragment internal to the region flanked by the primer set used to derive the nematode trees and were used to amplify 18S rDNA for subsequent analysis using denaturing gradient gel electrophoresis (DGGE). DGGE analysis of six major European grassland types revealed considerable genetic diversity between sites. However, the relationships seen with the DGGE data were inconsistent with previous studies where the same soils had been characterized with respect to functional and morphological diversity. To confirm that this second set of primers was amplifying nematode sequences, selected bands on the DGGE gels were extracted, PCR amplified and sequenced. The final alignment was 337 bases. These analyses revealed the presence of sequence signatures from the genera Paratrichodorus, Plectus, Steinernema, Globodera, Cephalobus and Pratylenchoides.

Original languageEnglish
Pages (from-to)1165-1173
Number of pages9
JournalSoil Biology and Biochemistry
Volume35
Issue number9
DOIs
Publication statusPublished - Sep 1 2003

Fingerprint

Denaturing Gradient Gel Electrophoresis
denaturing gradient gel electrophoresis
Ribosomal DNA
Electrophoresis
nematode
electrokinesis
Soil
gel
Gels
Nematoda
Soils
DNA
Netherlands
Polymerase Chain Reaction
Hungary
Pratylenchoides
soil
Cephalobus
grasslands
grassland

Keywords

  • 18S rDNA
  • Denaturing gradient gel electrophoresis
  • Grasslands
  • Molecular ecology
  • Nematodes
  • PCR
  • Soil community DNA

ASJC Scopus subject areas

  • Soil Science
  • Biochemistry
  • Ecology

Cite this

Waite, I. S., O'Donnell, A. G., Harrison, A., Davies, J. T., Colvan, S. R., Ekschmitt, K., ... Boström, S. (2003). Design and evaluation of nematode 18S rDNA primers for PCR and denaturing gradient gel electrophoresis (DGGE) of soil community DNA. Soil Biology and Biochemistry, 35(9), 1165-1173. https://doi.org/10.1016/S0038-0717(03)00177-9

Design and evaluation of nematode 18S rDNA primers for PCR and denaturing gradient gel electrophoresis (DGGE) of soil community DNA. / Waite, Ian S.; O'Donnell, Anthony G.; Harrison, Andrew; Davies, John T.; Colvan, Stephanie R.; Ekschmitt, Klemens; Dogan, Hülya; Wolters, Volkmar; Bongers, Tom; Bongers, Marina; Bakonyi, G.; Nagy, Péter; Papatheodorou, Efimia M.; Stamou, George P.; Boström, Sven.

In: Soil Biology and Biochemistry, Vol. 35, No. 9, 01.09.2003, p. 1165-1173.

Research output: Contribution to journalArticle

Waite, IS, O'Donnell, AG, Harrison, A, Davies, JT, Colvan, SR, Ekschmitt, K, Dogan, H, Wolters, V, Bongers, T, Bongers, M, Bakonyi, G, Nagy, P, Papatheodorou, EM, Stamou, GP & Boström, S 2003, 'Design and evaluation of nematode 18S rDNA primers for PCR and denaturing gradient gel electrophoresis (DGGE) of soil community DNA', Soil Biology and Biochemistry, vol. 35, no. 9, pp. 1165-1173. https://doi.org/10.1016/S0038-0717(03)00177-9
Waite, Ian S. ; O'Donnell, Anthony G. ; Harrison, Andrew ; Davies, John T. ; Colvan, Stephanie R. ; Ekschmitt, Klemens ; Dogan, Hülya ; Wolters, Volkmar ; Bongers, Tom ; Bongers, Marina ; Bakonyi, G. ; Nagy, Péter ; Papatheodorou, Efimia M. ; Stamou, George P. ; Boström, Sven. / Design and evaluation of nematode 18S rDNA primers for PCR and denaturing gradient gel electrophoresis (DGGE) of soil community DNA. In: Soil Biology and Biochemistry. 2003 ; Vol. 35, No. 9. pp. 1165-1173.
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AU - Davies, John T.

AU - Colvan, Stephanie R.

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