THE RHIZOSPHERE BACTERIA DIFFER BETWEEN DOMESTICATED MAIZE LANDRACES – AN EXPERIMENTAL CONFIRMATION
Creators: name: Agerbo Rasmussen, Jacob
Publisher: name: University of Copenhagen
Year: 2021
Subjects: subject: 16S
subject: Metabarcoding
subject: Bacteria
subject: Maize

description: The dataset is a collection of 16S rRNA gene metabarcoding for bacteria, related to rhizopheres in maize. The dataset has been published in Lund &amp; Rasmussen et al. 202? - "THE RHIZOSPHERE BACTERIA DIFFER BETWEEN DOMESTICATED MAIZE LANDRACES – AN EXPERIMENTAL CONFIRMATION". • The plant-associated microbiome has been shown to vary considerably between species and across environmental gradients. The effects of genomic variation on the microbiome within single species are less clearly understood, with results often confounded by the much larger effects of climatic and environmental variation. • In this study, the effect of genomic variation on the rhizosphere bacterial communities of maize was investigated by comparing different landraces grown within controlled environments. Rhizosphere bacterial communities were profiled by metabarcoding the universal bacterial 16S rRNA v3-v4 region. Initially, plants from the B73 and C223 landraces were grown for 12-weeks and compared. The experiment was then repeated with an additional four landraces (C6789, C794, C1850 and C2604) that were grown alongside the B73 and C223 landraces. • In both experiments there was significant differences in the rhizosphere bacterial communities between landraces. Additionally, the bacterial communities were significantly correlated with phylogenetic relatedness, with the more closely related landraces having more similar in rhizosphere bacterial communities. • Despite limited sampling numbers, here we confirm that genomic variation in maize landraces is associated with differences in the rhizosphere bacterial communities. Further studies that go beyond correlations to identify the mechanisms that determine the genotypic variation of the rhizosphere microbiome are urgently required.
description: Sequencing preparation and sequencing All DNA extractions were performed in dedicated pre-PCR laboratories within the GLOBE Institute of the University of Copenhagen, Denmark. Between 200-500 mg of roots or soil were extracted from using a soil fast DNA kit (MPBIO). For GH2, single seeds of individual landraces were also extracted (three per landrace), while an extraction negative was included as part of every batch of extractions. Samples initially underwent a mechanical lysis step by adding samples to a Lysing Matrix E tube and placed within a Tissuelyzer II (QIAGEN), which were lysed for 1 minute at 30 Hz. Extractions were then carried out as per manufacturer’s instructions. DNA extracts underwent quantification and quality checking using Qubit Fluorometric Quantification (Thermo Fisher Scientific). As part of library preparation, DNA extracts initially underwent PCR using the 341F (5'-CCTAYGGGRBGCASCAG-3') and reverse 806R: 5'-GGACTACNNGGGTATCTAAT-3') primers. They target the v3-v4 region of the 16S bacterial gene. They produce an approximate 487 bp amplicon [84]. Each PCR consisted of a 20 µL total reaction volume containing 2 µL of 1x Gold PCR buffer, 2.5mM MgCl2, 0.2mM dNTP, 0.1 U Taq Gold polymerase, 0.5 mg mL-1 BSA ( all of which were from Applied Biosystems, Denmark)) and 2 μL of DNA extract. DNA amplification was carried out in a thermal cycler using the following conditions: 95° C for 10 min, followed by 15 s at 95 °C, 20 s at 53 °C, and 40 s at 72 °C. There was a final extension of 7 minutes at 72 °C. In GH1, PCR reactions ran for 23 cycles for the soil samples and 28 cycles for the rhizosphere samples. However, the first PCR amplification of for the rhizosphere extraction in GH1 failed and the PCR amplification was reperformed with 33 cycles using the same thermal cycler settings. In the following GH2 experiment, the PCR reaction ran for 23 cycles for the soil samples, 28 cycles for the seed samples and 28 cycles for the rhizosphere samples. All samples from both GH1 and GH2 were ran in triplicate to provide technical replicates, while extraction negatives and PCR negatives (sterile water used as a substitute for DNA extract) were included for all batches of PCR amplifications. PCR products were pooled into four separate libraries, with 100 ng of DNA suspended in a total volume of 32 µL. Pools then underwent library preparation for sequencing using the Blunt-End Single-Tube (BEST) approach that was specifically modified for metabarcoding. Library products were purified with magnetic beads (SPRI) using a 1.2:1 ratio of beads to library volumes to remove unbound adapters. Samples were eluted into 35 µL of EB-buffer (QIAGEN, Germany). Samples were then sequenced on the MiSeq v3 sequencing platform (Illumina, CA, USA) at the National High-Throughput Sequencing Centre of Denmark (University of Copenhagen, Denmark). Bioinformatic analysis of metabarcoding data Adapters were removed using AdaptorRemoval (v2.3.1) and Cutadapt (v2.3.1) was used to check and correct the orientation of reads. Technical triplicates (PCR replicates of the same biological samples) were summed together to ensure most comprehensive collection of bacterial species.