Fig 1.
Experimental scheme 1 for the copse-derived nematodes and PCR target regions of the 18S ribosomal RNA gene.
The experimental scheme of the high-throughput sequencing of 12 amplicons prepared from six regions of the 18S ribosomal RNA (SSU) gene using the copse-derived nematode genomic DNA and whole soil DNA is shown (A). Red colored codes in parentheses for sample DNA (e.g., Z03) represent the nematode genomic DNA with the soil sample code (Z: copse) and the experimental ID (03), and the code Z03S indicates the soil DNA purified from the copse soil in the same experiment (S: soil DNA). R1–R4, U1 and U2 in red are the abbreviations of PCR target regions in this study (A). Six PCR target regions with their abbreviations and sizes of amplicons are shown by bold double-headed arrows in gray for nematode-specific primers and in dark red for universal primers (B). The numbers indicating the nucleotide positions of the 5ʹ-end of forward primers are shown on the entire SSU gene prepared from the nucleotide sequence of C. elegans ribosomal RNA gene cluster (X03680). Teal colored boxes correspond to the hypervariable regions of eukaryotic SSU genes reported by Hugerth et al. [33] and Hadziavdic et al. [32]. The regions amplified by two published nematode-specific primer sets (NF1/18Sr2b and NemF/18Sr2b) [35, 60] and four eukaryotic universal primer sets (3NDf/1132rmod [34], 566-F/915-R [37], Ek-NS573/Ek-NSR951[58, 64], F-1183/R-1631 [58, 65], Euk1391f/EukBr [36, 66, 67]) are also indicated by double-headed arrows in gray and dark red with sizes of amplicons, respectively. Fig 1B has been modified and prepared from the Fig 1B appearing in our previous paper [29].
Fig 2.
Experimental scheme 2 for the field-derived nematodes by amplicon sequencing using the universal primers and soil DNA.
The experimental scheme of the high-throughput sequencing of amplicons prepared using the agricultural field-derived soil DNA (H03S) and the universal SSU primers for region U2 is shown (A). Soil samples were isolated from the surface (0–10 cm in depth) and deep (20–30 cm) layers (D) of the distal (control: without plants) and proximal (plants: with plants) points to the growing sweet potato (B, C) as indicated by red/white squares, at two different sites in the field (B: site 1 and C: site 2). Growth of the plants at site 1 (B) was apparently dominated compared with that at site 2 (C). Regarding the code H03S for template DNA, see the legend for Fig 1.
Table 1.
Polymerase chain reaction (PCR) primers used for amplifying the small subunit ribosomal RNA (SSU) gene regions.
Table 2.
Numbers of total and nematode-derived SVs from the copse-derived nematode and soil DNAs in each SSU region.
Table 3.
Numbers of regional nematode SVs derived from nematode genomic and whole soil DNAs.
Fig 3.
Relative read abundances and phylum of sequence variants (SVs) in each region identified by high-throughput sequencing of the copse-derived amplicons.
Relative read abundance and phylum of the SVs obtained from the SSU gene regions 1 (R1), 2 (R2), 3 (R3), and 4 (R4) amplified by the nematode-specific primers, and regions U1 and U2 amplified by the universal primers from the copse-derived nematode DNA (left panel) and soil DNA (right panel) as templates. Boxes in the histogram indicate the read abundances; phyla are indicated by color. Taxonomic classification of SVs was based on the SILVA database, and phyla with less than 1% of relative abundance are omitted. NA: Not assigned.
Fig 4.
Numbers of regional nematode SVs from six SSU regions in each order.
The number of regional nematode SVs identified from the nematode genomic DNA (blue bars) and soil DNA (red bars) from the copse soils is indicated in each order by bar chart for regions 1–4, U1, and U2 (A–F), respectively. NA: not assigned to a single order (i.e., assigned to multiple orders). Target SSU regions are indicated at the top right side of the bar charts.
Fig 5.
Relative abundance of nematode-derived sequence reads at the order level in each region.
The percentage of the abundance of sequence reads in each nematode order obtained from regions 1 (A), 2 (B), 3 (C), 4 (D), U1 (E), and U2 (F) is indicated by colored horizontal histograms in each template DNA: copse-derived nematode genomic DNA (Nema) and soil DNA (Soil). The colors corresponding to orders are indicated at the bottom of (F). Target SSU regions are indicated at the right side of the corresponding horizontal bar charts.
Fig 6.
Beta diversity plots of 12 samples derived from two template DNAs and six SSU regions based on the relative abundance of nematode-derived reads in each order and feeding type.
The beta diversity of the sample was calculated based on the relative abundances of nematode-derived reads in each order (A) and feeding type (B) using non-metric multidimensional scaling (NMDS) with the Bray–Curtis distance matrices in the corresponding SSU region and template DNA indicated in the right of figures. The nematode-derived reads in region 1 (R1, purple), 2 (R2, green), 3 (R3, pink), 4 (R4, red), U1 (orange), and U2 (blue) are derived from the nematode genomic DNA (circles) and soil DNA (triangles). The target SSU region of each sample is indicated by the corresponding symbol. The samples with close relations described in the text are surrounded by broken circles.
Table 4.
Maturity indices calculated by the families identified from the copse-derived nematode DNA and soil DNA in six SSU regions.
Table 5.
Numbers of total and nematode-derived SVs from eight soil samples at sites 1 and 2.
Fig 7.
Relative read abundances and phylum of SVs obtained from high-throughput amplicon sequencing using the agricultural field-derived soil DNAs.
Relative read abundance and phylum of SVs obtained from the amplicon sequencing of region U2 of the SSU gene using soil DNAs derived from the surface (0/10) and deep (20/30) layers at the sampling points without (control) and with (plants) plants at sites 1 and 2, respectively, as shown at the top and bottom of the histograms. Boxes in the histogram indicate the read abundances; phylum is indicated by color as shown in the right legend box. Taxonomic classification of SVs was based on the SILVA database. Phyla with less than 1% of relative abundance were omitted. NA: Not assigned.
Fig 8.
Relative abundances of nematode-derived reads from the field-derived samples in each order and their beta diversity plots.
The percentage of the abundance of sequence reads derived from the soil samples in each order is shown by colored horizontal bar charts (A). Soil samples were isolated from the surface (0–10) and deep (20–30) layers of the sampling points without (control) and with (plants) plants at sites 1 (#1) and 2 (#2), respectively, as shown in the left of the corresponding histogram. Colors corresponding to orders are indicated at the bottom of figure. The principal coordinate analysis (PCoA) plot with the weighted unifrac distances of eight samples derived from the surface and deep layer at the point without (orange-colored circles and squares) and with (green-colored circles and squares) plants from site 1 (circles) and 2 (squares) in the agricultural field (B).
Table 6.
Maturity indices for nematode families from the field-derived soil samples.