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

• Published in: Soil biology & biochemistry Vol. 35; no. 9; pp. 1165 - 1173
• Main Authors: 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, Gabor, Nagy, Péter, Papatheodorou, Efimia M., Stamou, George P., Boström, Sven
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01-09-2003; New York, NY Elsevier Science
• Subjects: 16s ribosomal-rna; 18S rDNA; Adenophorea; Agronomy. Soil science and plant productions; bacterial communities; Biochemistry and biology; biodiversity; Biological and medical sciences; Cephalobus; Chemical, physicochemical, biochemical and biological properties; Denaturing gradient gel electrophoresis; diversity; Enoplus; Fundamental and applied biological sciences. Psychology; genes; Globodera; grassland soils; Grasslands; hydrothermal vent; identification; Mesorhabditis; Molecular ecology; Mylonchulus; Nematoda; Nematodes; Paratrichodorus; PCR; Physics, chemistry, biochemistry and biology of agricultural and forest soils; Plectus; populations; Pratylenchoides; Prismatolaimus; quantification; Soil community DNA; Soil science; Steinernema; Trichodorus; Xiphinema; Zoology (interactions between soil fauna and agricultural or forest soils); Molecular ecology; Grasslands; Soil community DNA; Nematodes; 18S rDNA; Denaturing gradient gel electrophoresis; PCR; Meiofauna; Fauna; Ribosomal DNA; Primer; Polymerase chain reaction; Soil fauna; Animal community; Helmintha; Nemathelminthia; Invertebrata; Nematoda
• ISSN: 0038-0717; 1879-3428
• DOI: 10.1016/S0038-0717(03)00177-9

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.