Taxonomic profiling of individual nematodes isolated from copse soils using deep amplicon sequencing of four distinct regions of the 18S ribosomal RNA gene

Abstract

Nematodes are representative soil metazoans with diverged species that play crucial roles in nutrient recycling in the pedosphere. Qualitative and quantitative information on nematode communities is useful for assessing soil quality, and DNA barcode-mediated taxonomic analysis is a powerful tool to investigate taxonomic compositions and changes in nematode communities. Here, we investigated four regions (regions 1-4) of the 18S small subunit ribosomal RNA (SSU) gene as PCR targets of deep amplicon sequencing for the taxonomic profiling of individual soil nematodes. We determined the sequence variants (SVs) of 4 SSU regions for 96 nematodes (total 384 amplicons) isolated from copse soils and assigned their taxonomy using the QIIME2 software with dada2 or deblur algorithm and the SILVA database. Dada2 detected approximately 2-fold more nematode-derived SVs than deblur, and a larger number of SVs were obtained in regions 1 and 4 than those in other regions. These results and sufficient reference sequence coverage in region 4 indicated that DNA barcoding using a primer set for region 4 followed by dada2-based analysis would be most suitable for soil nematode taxonomic analysis. Eighteen SSU-derived operational taxonomic units (rOTUs) were obtained from 68 isolates, and their orders were determined based on the phylogenetic trees built by four regional sequences of rOTUs and 116 nematode reference species as well as the BLASTN search. The majority of the isolates were derived from three major orders Dorylaimida (6 rOTUs, 51.5% in 68 isolates), Rhabditida (4 rOTUs, 29.4%), and Triplonchida (7 rOTUs, 17.6%). The predicted feeding types of the isolates were fungivores (38.2% in total nematodes), plant feeders (32.4%), and 14.7% for both bacterivores and omnivores/predators. Additionally, we attempted to improve the branch structure of phylogenetic trees by using long nucleotide sequences artificially prepared by connecting regional sequences, but the effect was limited.

Distinct community structures of soil nematodes from three ecologically different sites revealed by high-throughput amplicon sequencing of four 18S ribosomal RNA gene regions

“…Soil samples were collected from a copse on Toyohashi University of Technology campus, as in a prior study [ 28 ], and from a zucchini-cultivated house garden where chemical fertilizers and water were periodically supplied in June and July 2016 under clear climatic conditions (temperature: 28°C–35°C) in Toyohashi, Japan (137° 24’E, 34° 42’N), which is located in the temperate zone ( Fig 1A ). A field soil sample was collected in November 2013 from an uncultivated field managed by the Research Center for Agrotechnology and Biotechnology of the Toyohashi University of Technology, as detailed in the prior work [ 26 ] ( Fig 1A ).…”

“… (A) Soil sampling sites. Photographs of uncultivated field and copse are reprinted from prior PLoS One papers [ 26 , 28 ]. (B) Relative abundance and phylum of SVs obtained from regions 1 (R1), 2 (R2), 3 (R3), and 4 (R4) of the 18S ribosomal RNA (SSU) gene in each sample.…”

“…PCR primers without tail sequences were only used for the two-step PCR as described below. PCR primers with tail sequences were used for amplification of the 18S SSU gene fragments in the corresponding regions 1–4 (see S1 Fig ), as detailed by Kenmotsu et al [ 28 ]. The PCR reaction mixture (20 μL) contained 10 μL of 2 × Buffer for KOD FX Neo, 4 μL of 2 mM dNTPs, 0.4 units of the KOD FX Neo DNA polymerase (Toyobo, Tokyo, Japan), 2 μL of nematode DNA, as described above, and 0.3 μM each of the tailed forward and reverse primers.…”

“… a These regions are detailed by Kenmotsu et al [ 28 ] and in S1 Fig . b The corresponding primers with tail sequence were also used by Kenmotsu et al [ 28 ].…”

“…To speed up the process, high-throughput sequencing-assisted DNA barcoding has been applied to the taxonomic analysis of soil nematodes. To find suitable regions for DNA barcoding among four SSU gene regions (regions 1–4, 337–388 bp in length), the present authors [ 28 ] clarified the taxa of 68 out of 96 individual nematodes isolated from copse soil using massive amplicon sequencing via the Illumina MiSeq platform. Region 4, located at the 3’-region of the SSU gene, was suggested as the most suitable among these four regions due to the identification of the most abundant nematode-derived sequence variants (SVs) and sufficient reference sequence coverage in DNA barcoding of individual nematodes; however, it is unclear whether these advantages are applicable for DNA barcoding in nematode communities (i.e., involving complex mixed nematodes).…”