Use of universal primers for the 18S ribosomal RNA gene and whole soil DNAs to reveal the taxonomic structures of soil nematodes by high-throughput amplicon sequencing

Abstract

Nematodes are abundant metazoans that play crucial roles in nutrient recycle in the pedosphere. Although high-throughput amplicon sequencing is a powerful tool for the taxonomic profiling of soil nematodes, polymerase chain reaction (PCR) primers for amplification of the 18S ribosomal RNA (SSU) gene and preparation of template DNAs have not been sufficiently evaluated. We investigated nematode community structure in copse soil using four nematode-specific (regions 1–4) and two universal (regions U1 and U2) primer sets for the SSU gene regions with two DNAs prepared from copse-derived mixed nematodes and whole soil. The major nematode-derived sequence variants (SVs) identified in each region was detected in both template DNAs. Order level taxonomy and feeding type of identified nematode-derived SVs were distantly related between the two DNA preparations, and the region U2 was closely related to region 4 in the non-metric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarity. Thus, the universal primers for region U2 could be used to analyze soil nematode communities. We further applied this method to analyze the nematodes living in two sampling sites of a sweet potato-cultivated field, where the plants were differently growing. The structure of nematode-derived SVs from the two sites was distantly related in the principal coordinate analysis (PCoA) with weighted unifrac distances, suggesting their distinct soil environments. The resultant ecophysiological status of the nematode communities in the copse and field on the basis of feeding behavior and maturity indices was fairly consistent with those of the copse- and the cultivated house garden-derived nematodes in prior studies. These findings will be useful for the DNA metabarcoding of soil eukaryotes, including nematodes, using soil DNAs.

In silico assessment of 18S rDNA metabarcoding markers for the characterization of nematode communities

“…For instance, our results are in agreement with previous studies that used mock communities to compare the performance of MMSF-MMSR, NemF-18Sr2b, NF1_18Sr2b and SSU_F04-SSU_R22, and observed a limited coverage of SSU_F04-S-SU_R22 [18,49]. Similarly, Kenmotsu et al [50] confirmed that F_1183-R_1631 and NF1_18Sr2b show a similar, very good performance; Geisen et al [51] confirmed the excellent performance of 3NDf-1132rmod; and Guardiola et al [31] suggested an excellent coverage for Euka02 [see also ref. 36 for in vitro tests confirming the excellent taxonomic coverage of this marker for all the tested invertebrate phyla].…”

Distinct prokaryotic and eukaryotic communities and networks in two agricultural fields of central Japan with different histories of maize–cabbage rotation

“…More than four soil samples were isolated from near the plant and mixed together at each sampling point, where the neighbored sampling point was located 35 cm apart. A total of 36 sample soils were isolated from 3 independent sampling points at 2 fields cultivated with 2 crops at 3 growth stages and pretreated before DNA purification, as described previously 34 . This study was carried out in accordance with relevant guidelines.…”

“…Purified soil DNA was stored at − 20 °C. The 16S (V3–V4 region) and 18S (V7–V8 region) rRNA genes were amplified from soil DNA with the universal primers 16S_Amplicon_MiseqF and 16S_Amplicon_MiseqR (341F and 805R with tail sequences 73 ) and F1183-18S_V7-V8_MiseqF and R1631a-18S_V7-V8_MiseqR 34 , respectively. The PCR mixture (20 μL) contained 10 μL of 2 × Buffer for KOD FX Neo, 4 μL of 2 mM dNTPs, 0.4 units of KOD FX Neo DNA polymerase (Toyobo, Tokyo, Japan), 2 μL of template DNA, and 0.3 mM each of the forward and reverse primers.…”

“…We previously applied 18S rRNA gene-derived amplicon sequencing using Illumina MiSeq to analyze soil nematodes and successfully clarified the nematode communities in sweet potato-cultivated fields 34 . Herein, we applied DNA metabarcoding to investigate both prokaryotes and eukaryotes living in two fields (i.e., field_1 and field_2) cropping maize and cabbage by rotation in central Japan in 2019.…”

Effects of zokor mounds on the stability of soil micro-foodweb in lightly disturbed alpine meadows on the Qinghai-Tibet Plateau

“…The ITS region of the fungi was ampli ed with the primers TS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2 (5'-GCTGCGTTCTTCATCGATGC-3') (Martin and Rygiewicz 2005). For nematodes, PCR reactions used the primers NF1 (5'-GGTGGTGCATGGCCGTTCTTAGTT-3') and 18S r2bR (5'-TACAAAGGGCAGGGACGTAAT-3') to amplify the V4 region of 18S rRNA gene (Kenmotsu et al 2021). Polymerase Chain Reaction (PCR) analysis was performed in triplicate according to the following thermal program: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 40 s, and nal extension at 72°C for 7 min, 25 cycles.…”