ena-STUDY-UPPSALA UNIVERISTY-03-06-2020-07:21:18:871-326
Environmental short read amplicons of soil fungi across Podzol soil profile
Soil samples were collected in mid-October 2013 from Ivantjärnsheden field station in Jädraås (60°49'N, 16°30'E, altitude 185 m), a well-documented field site in central Sweden (Persson, 1980) with Pinus sylvestris L. overstory and an understory of ericaceous dwarf shrubs (Calluna vulgaris (L.) Hull and Vaccinium vitis-ideae L.) and mosses (Dicranum majus Turner and Pleurozium schreberi (Bridel) Mitten). To account for small-scale variability in soil fungal communities we collected 5 soil cores (5 cm diameter and 15 cm deep) in each of 12 plots in the since terminated experiment IhII (9802) (Axelsson & Bråkenhielm, 1980). After visually dividing the plot into four quadrats one core were taken from the middle of each quadrat and from the middle of the plot after peeling back the top shrub and moss layer (incl. most of the litter layer). Soil cores were separated into visually distinct podzol soil layers: organic soil (O), mineral elluvial soil (E) and mineral illuvial soil (B) before pooling the layers for each plot. From each composite soil sample two sub-samples of approximately 0.5 g wet weight were collected for DNA extraction. The ITS2 region of the rRNA genes was amplified using primers gITS7 forward (Ihrmark et al., 2012) and modified ITS4m reverse (5'- TCCTC[C/G][G/C]CTTATTGATATGC-3') (Rosling et al., 2016), with both primers containing adequate barcode sequences for single-ended sequencing (Table S1). Modifications on the reverse primer ITS4 (White et al., 1990) were included to reduce its known bias against the soil-inhabiting fungal class Archaeorhizomycetes (Schadt & Rosling, 2015). A sequencing library was prepared by pooling 35 ng PCR products from each sample, loaded onto a 318 chip for PGM Ion Torrent sequencing technology (Life Technologies Corporation, Carlsbad, CA, US) and sequenced at SciLifeLab/NGI (Uppsala, Sweden).References:Axelsson B, Bråkenhielm S. 1980. Investigation sites of the Swedish coniferous forest project - biological and physiological features. In: Persson T ed. Structure and Function of Northern Coniferous Forests - An Ecosystem Study. Arlöv, Sweden: Berlings, 25 - 64.Ihrmark, K., Bödeker, I. T., Cruz-Martinez, K., Friberg, H., Kubartova, A., Schenck, J., Strid, Y., Stenlid, J., Brandström-Durling, M., & Clemmensen, K. E. (2012). New primers to amplify the fungal ITS2 region–evaluation by 454-sequencing of artificial and natural communities. FEMS microbiology ecology, 82(3), 666-677.Persson, T, ed . 1980. Structure and function of northern coniferous forests – an ecosystem study. Ecological Bulletins 32. Stockholm, Sweden.Rosling, A., Midgley, M. G., Cheeke, T., Urbina, H., Fransson, P., & Phillips, R. P. (2016, FEB 2016). Phosphorus cycling in deciduous forest soil differs between stands dominated by ecto- and arbuscular mycorrhizal trees. New Phytologist, 209(3), 1184-1195. https://doi.org/10.1111/nph.13720Schadt, C. W., & Rosling, A. (2015). Comment on “Global diversity and geography of soil fungi”. Science, 348(6242), 1438-1438. http://www.sciencemag.org/content/348/6242/1438.1.full.pdf White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications, 18(1), 315-322.
Soil samples were collected in mid-October 2013 from Ivantjärnsheden field station in Jädraås (60°49'N, 16°30'E, altitude 185 m), a well-documented field site in central Sweden (Persson, 1980) with Pinus sylvestris L. overstory and an understory of ericaceous dwarf shrubs (Calluna vulgaris (L.) Hull and Vaccinium vitis-ideae L.) and mosses (Dicranum majus Turner and Pleurozium schreberi (Bridel) Mitten). To account for small-scale variability in soil fungal communities we collected 5 soil cores (5 cm diameter and 15 cm deep) in each of 12 plots in the since terminated experiment IhII (9802) (Axelsson & Bråkenhielm, 1980). After visually dividing the plot into four quadrats one core were taken from the middle of each quadrat and from the middle of the plot after peeling back the top shrub and moss layer (incl. most of the litter layer). Soil cores were separated into visually distinct podzol soil layers: organic soil (O), mineral elluvial soil (E) and mineral illuvial soil (B) before pooling the layers for each plot. From each composite soil sample two sub-samples of approximately 0.5 g wet weight were collected for DNA extraction. The ITS2 region of the rRNA genes was amplified using primers gITS7 forward (Ihrmark et al., 2012) and modified ITS4m reverse (5'- TCCTC[C/G][G/C]CTTATTGATATGC-3') (Rosling et al., 2016), with both primers containing adequate barcode sequences for single-ended sequencing (Table S1). Modifications on the reverse primer ITS4 (White et al., 1990) were included to reduce its known bias against the soil-inhabiting fungal class Archaeorhizomycetes (Schadt & Rosling, 2015). A sequencing library was prepared by pooling 35 ng PCR products from each sample, loaded onto a 318 chip for PGM Ion Torrent sequencing technology (Life Technologies Corporation, Carlsbad, CA, US) and sequenced at SciLifeLab/NGI (Uppsala, Sweden).References:Axelsson B, Bråkenhielm S. 1980. Investigation sites of the Swedish coniferous forest project - biological and physiological features. In: Persson T ed. Structure and Function of Northern Coniferous Forests - An Ecosystem Study. Arlöv, Sweden: Berlings, 25 - 64.Ihrmark, K., Bödeker, I. T., Cruz-Martinez, K., Friberg, H., Kubartova, A., Schenck, J., Strid, Y., Stenlid, J., Brandström-Durling, M., & Clemmensen, K. E. (2012). New primers to amplify the fungal ITS2 region–evaluation by 454-sequencing of artificial and natural communities. FEMS microbiology ecology, 82(3), 666-677.Persson, T, ed . 1980. Structure and function of northern coniferous forests – an ecosystem study. Ecological Bulletins 32. Stockholm, Sweden.Rosling, A., Midgley, M. G., Cheeke, T., Urbina, H., Fransson, P., & Phillips, R. P. (2016, FEB 2016). Phosphorus cycling in deciduous forest soil differs between stands dominated by ecto- and arbuscular mycorrhizal trees. New Phytologist, 209(3), 1184-1195. https://doi.org/10.1111/nph.13720Schadt, C. W., & Rosling, A. (2015). Comment on “Global diversity and geography of soil fungi”. Science, 348(6242), 1438-1438. http://www.sciencemag.org/content/348/6242/1438.1.full.pdf White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications, 18(1), 315-322.