Comparisons of different hypervariable regions of rrs genes for fingerprinting of microbial communities in paddy soils

The use of molecular approaches based on 16S rDNA-PCR in microbial ecology has revealed a tremendous prokaryotic diversity in environmental samples. However, there is little or no systematic evaluation of the impacts of hypervariable (V) regions of rrs genes choice on microbial community analysis in soil samples, especially the detailed information about the dominant groups preferentially amplified by different primer pairs. In the present study, eight primer pairs were detected to compare the different V regions for fingerprinting microbial communities in a paddy soil irrigated with petroleum-wastewater, using denaturing gradient gel electrophoresis (DGGE) and amplified ribosomal DNA restriction analysis (ARDRA) techniques. Results reveal the obvious PCR bias produced by different V regions. Both ARDRA analysis of 16S rDNA clone library and DGGE suggest that V₁-V₃ region amplified with primer pair 8f-519r produced the most informative fingerprinting profiles. Additionally, V₃-V₅ region amplified with 341f-907r was another preferable choice for microbial diversity in petroleum-contaminated soil. The V₄-V₅ region and single V region (V₁, V₃, and V₈) were not recommended for the future study of microbial diversity in soil samples. Phylogenetic analysis of 123 sequences from libraries constructed by amplicons generated from six different V regions suggests that different dominant groups were amplified with distinct primer sets. In detail, V₁-V₃ library (amplified with 8f-519r) and V₃-V₅ library were dominated by Actinobacteria (20.4%) (particularly in genus Arthrobacter), V₁-V₃ library (amplified with 63f-518r) was dominated by γ-Proteobacteria (25.0%) and α-Proteobacteria (22.0%) (particularly in genus Brevundimonas), V₃ library was dominated by β-Proteobacteria (22.3%) (particularly in genus Gallionella) and α-Proteobacteria (20.0%), V₆-V₈ library was dominated by Chlamydiae (20.4%) and β-Proteobacteria (20.4%), V₈ library was dominated by γ-Proteobacteria (27.2%) (particularly in genus Acinetobacter) and β-Proteobacteria (14.0%). The present work strongly recommends that primer pairs should be chosen cautiously in community diversity analysis based on PCR amplification of 16S rDNA, and involving at least two different 16S rDNA universal primer pairs would perform better.