Characterization of root-associated bacterial community structures in soybean and corn using locked nucleic acid (LNA) oligonucleotide-PCR clamping and 454 pyrosequencing

Abstract

The community structure and diversity of root-associated bacteria have been tentatively investigated using polymerase chain reaction (PCR) amplification methods in several studies. However, the homology between small submit ribosomal (SSU) rRNA genes of plant plastids and mitochondria and that of bacteria have hindered in these studies. To address this issue, in this paper, we adopted the methods of locked nucleic acid (LNA) oligonucleotide-PCR clamping with 454 pyrosequencing to analysis the root-associated bacterial community compositions in soybean and corn. Results showed that plant chloroplast and mitochondria genes were effectively inhibited from PCR amplification in the root samples with LNA oligonucleotides (LNA (+)), and PCR amplicons with LNA (+) had higher bacterial operational taxonomic unit (OTU) numbers and ACE, Chao1, and Shannon indices, as well as a lower Simpson index than the corresponding samples without LNA oligonucleotides (LNA (–)). Those findings suggested that the methods of this study provide a much more detail description of root-associated bacterial communities. In the soybean LNA (+) sample, Pseudomonas, Bradyrhizobium and Flavobacterium were the three most abundant genera, whereas the top two predominant genera in corn LNA (+) samples were Streptomyces and Niastella. The presence and absence of major genera varied between soybean and corn, suggesting the root-associated bacterial communities differed between two crops. The rare phylotypes and uncultured root-associated bacterial members detected in this study inferred that the root-associated bacterial communities are highly complex and information on their taxonomic affiliates potentially gives the clues for selecting the optimal medium and method to isolate the novel bacteria for further functional analysis.

Keywords

• bacterial diversity
• high-throughput sequencing
• corn
• soybean
• SSU rRNA genes