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

Abstract

Quantitative taxonomic compositions of nematode communities help to assess soil environments due to their rich abundance and various feeding habitats. DNA metabarcoding by the 18S ribosomal RNA gene (SSU) regions were preferentially used for analyses of soil nematode communities, but the optimal regions for high-throughput amplicon sequencing have not previously been well investigated. In this work, we performed Illumina-based amplicon sequencing of four SSU regions (regions 1–4) to identify suitable regions for nematode metabarcoding using the taxonomic structures of nematodes from uncultivated field, copse, and cultivated house garden soils. The fewest nematode-derived sequence variants (SVs) were detected in region 3, and the total nematode-derived SVs were comparable in regions 1 and 4. The relative abundances of reads in regions 1 and 4 were consistent in both orders and feeding groups with prior studies, thus suggesting that region 4 is a suitable target for the DNA barcoding of nematode communities. Distinct community structures of nematodes were detected in the taxon, feeding habitat, and life-history strategy of each sample; i.e., Dorylamida- and Rhabditida-derived plant feeders were most abundant in the copse soil, Rhabditida-derived bacteria feeders in the house garden soil, and Mononchida- and Dorylamida-derived omnivores and predators and Rhabditida-derived bacteria feeders in the field soil. Additionally, low- and high-colonizer–persister (cp) groups of nematodes dominated in the house garden and copse soils, respectively, whereas both groups were found in the field soil, suggesting bacteria-rich garden soil, undisturbed and plant-rich copse soil, and a transient status of nematode communities in the field soil. These results were also supported by the maturity indices of the three sampling sites. Finally, the influence of the primer tail sequences was demonstrated to be insignificant on amplification. These findings will be useful for DNA metabarcoding of soil nematode communities by amplicon sequencing.

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

“…Two experiments were performed in this study (Figs 1 and 2 ): 1) testing of the suitable universal primers of the 18S ribosomal RNA (SSU) gene and soil DNA used as template DNA for high-throughput amplicon sequencing to analyze the soil nematode communities and 2) application of the developed method for taxonomic profiling of nematodes in the agricultural soils with different depth and plantation. In the experiment 1, we tested six primer sets amplifying each SSU region by PCR: four nematode-specific primer sets for regions 1–4 [ 29 , 30 ] and two universal eukaryotic primer sets for regions U1 and U2 ( Fig 1B ). In parallel, we compared the taxonomic profiles of the nematodes living in the copse soils using the DNA prepared from whole soils and complex nematodes isolated by flotation sieving method [ 28 ].…”

“…Copse soil samples for the experiment 1 were collected from the copse on the campus of Toyohashi University of Technology [ 29 , 30 ] in July 2017 under clear climatic conditions (temperature 32°C and humidity 77%) in Toyohashi, Japan (137° 24’E, 34° 42’N). The copse soil was sampled to a depth of 20–30 cm using a soil sampling auger (2.5 cm in diameter, Fujiwara Scientific Co., Tokyo, Japan).…”

“…The copse soil was sampled to a depth of 20–30 cm using a soil sampling auger (2.5 cm in diameter, Fujiwara Scientific Co., Tokyo, Japan). Field soil samples for the experiment 2 were collected in July 2018 under cloudy conditions (temperature 32ºC and humidity 55%) from two sampling sites (i.e., sites 1 and 2) in the sweet potato-cultivated field managed by the Research Center for Agrotechnology and Biotechnology of the Toyohashi University of Technology [ 30 ]. Growth of plants at site 1 ( Fig 2B ) was apparently dominated compared with that at site 2 ( Fig 2C ).…”

“…In this study, region 4, located at the 3ʹ-region of the SSU gene, was suggested as the most suitable barcode among the four regions because of the identification of the large number of nematode-derived sequence variants (SVs) and sufficient reference sequence coverage in the DNA barcoding of individual nematodes. Recently, Kenmotsu et al have applied high-throughput amplicon sequencing using the genomic DNAs of complex nematodes isolated from three sites (i.e., copse, uncultivated field, and zucchini-cultivated house garden) and successfully clarified distinct proportions of nematode taxa and the ecophysiological status of nematode families in each site [ 30 ]. This study has also suggested that region 4 is the most suitable for the DNA barcoding of complex mixed nematodes among the four regions.…”

“…Currently, DNA barcoding of soil nematodes is performed using the high-throughput sequencing of SSU gene-derived amplicons. Nematode-specific SSU primers are usually used for amplifications from genomic DNAs purified from complex soil nematodes [ 11 , 20 , 22 , 30 , 31 ]. Moreover, universal SSU primers have been recently developed for the DNA barcoding of eukaryotes through high-throughput amplicon sequencing [ 32 , 33 ].…”