DOI: 10.25829/bexis.31401-12
title: Frankia sp. is governing the diazotrophic community composition in deadwood of 13 tree species (raw sequences, total and active fraction)

description: Deadwood is a nitrogen (N) poor environment and its decomposition rate is limited by N-bioavailability. Diazotrophs can reduce such N-limitations as they are capable to fix N2, which employ the functional gene nifH that encode for dinitrogenase reductase. The present study investigates the diazotrophic community composition and activity, and their relationship to environmental factors and wood parameters of 13 different tree species stored as logs in temperate forests. Directly after sampling, each deadwood sample was incubated with water (total fraction) or bromodeoxyuridine (BrdU), which labeled the metabolically active fraction. Thereafter, nucleic acids extract from both total and active fraction were used to assess the nifH gene-based diversity by amplicon sequencing.<br>Here, the dataset contain the Illumina generated demultiplexed paired-end raw fastq sequences (merged).The obtained combined sequences were initially undergone for quality filtering employing DADA2 algorithm.<br>The data collection and analysis was part of the project Woodstock (subproject of the BELongDead project) within the framework of the DFG-funded joint project Biodiversity Exploratories (https://www.biodiversity-exploratories.de).
description: Detailed description of study design: In winter 2008/09, freshly cut logs of 13 different tree species were placed on the forest soil in random order and within 1m distance to each other. The logs were 4m long and around 31cm in diameter. Various parameters have been measured throughout the years. For further details on the study design see: Kahl et al. 2017.<br>In June 2020, after 11 years of decomposition, deadwood samples of the outer 7 cm of the BELongDead logs in the Hainich-Dün exploratory were taken on 9 experimental plots (EP). Bark and leaves were removed. The samples were further processed in the laboratory.<br>Sampling design: The outer 7 cm of the logs were cut laterally from the stem with an electric rip saw and chiseled out (about 2cm wide). For each of the 13 tree species per plot, one sample was taken.<br>Sample preparation: In June 2020, deadwood samples were cut laterally with an electric saw from the outer 7 cm of each log. Deadwood samples consisted almost exclusively of sapwood, but mixtures of sapwood and heartwood were taken from Quercus logs due to the decay and loss of sapwood. One part of the sample was stored in a plastic bag in the field and kept cool (~5 °C) until further treatment. A second part of the sample (sawdust) was transferred into a 15 mL falcon tube in the field. After that, 10 mM bromodeoxyuridine (BrdU) was added sufficiently until the sawdust was completely soaked with solution. The falcon tubes were placed in an upright position and covered with aluminum foil, followed by incubation at room temperature for 48 h. The samples were then frozen at −80 °C in the laboratory before the characterization of the diversity of metabolically active diazotrophs.<br>Sample analysis: The PCR reactions were performed in 10-µL assays containing 5 µL SYBR® Green Supermix (Bio-Rad, Feldkirchen, Germany), 0.5 µL (2.5 µM) of each primer (PolF and PolR), 3 µL sterile and nuclease-free water (Carl Roth GmbH) and 1 µL of either 1:10 diluted DNA-extract, ten-fold diluted standard DNA or 1 µL water as no template control. Cycling conditions were as follows: initial denaturation at 94°C for 5 min followed by 40 amplification cycles (regardless of active or total fraction) of 95ºC for 1 min (denaturation), 55ºC for 1 min (annealing), 72ºC for 1 min 30 s (extension), and a final elongation step at 72°C for 5 min. For checking the product specificity and potential primer dimer formation, runs were completed with a melting analysis starting from 65°C to 95°C with temperature increments of 0.5°C and a transition rate of 5 s. The purity of the amplified products was checked by electrophoresis on a 1.5% agarose gel.<br>Equipment: CFX96™ Real-Time System (Bio-Rad, Feldkirchen, Germany),Petroff counting chamber (Paul Marienfeld GmbH, Germany),Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo, California, USA) and SYBR® Green Supermix (Bio-Rad, Feldkirchen, Germany),Illumina Miseq<br> Data analysis: Illumina generated demultiplexed paired-end sequences were merged using the BBMerge v34.48 (Bushnellet al. 2017). The obtained combined sequences were initially undergone for quality filtering employing DADA2 algorithm , Version1.24.0 (Callahan et al. 2016) that resolves amplicon-sequencing errors to generate amplicon sequence variants (ASVs). Non-target-length sequences, which could potentially be introduced as a result of non-specific priming, were removed from the sequence table. Only sequences with a length between 250 and 400 bp were kept.In the present study, we used the customize nifH gene reference database collated by TaxADivA (Gaby et al. 2018) to analyze nifH gene community in deadwood.<br>Literature references: Kahl T, Arnstadt T, Baber K (2017) Wood decay rates of 13 temperate tree species in relation to wood properties, enzyme activities and organismic diversities. For Ecol Manag 391:86–95. https://doi.org/10.1016/j.foreco.2017.02.012<br>Bushnell B, Rood J, Singer E (2017) BBMerge – Accurate paired shotgun read merging via overlap. PLOS ONE 12:0185056. https://doi.org/10.1371/journal.pone.01850<br>Callahan BJ, McMurdie PJ, Rosen MJ (2016) DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods 13:581–583. https://doi.org/10.1038/nmeth.3869<br>Gaby JC, Rishishwar L, Valderrama-Aguirre LC (2018) Diazotroph community characterization via a high-throughput nifH amplicon sequencing and analysis pipeline. Appl Environ Microbiol 84:01512–17. https://doi.org/10.1128/AEM.01512-17<br>Data references: DADA2 (https://benjjneb.github.io/dada2/tutorial_1_2.html, Version1.24.0)<br>BBMerge (https://sourceforge.net/projects/bbmap/)<br>TaxADivA https://github.com/lavanyarishishwar/taxadiva