DNA extractions and PCR
DNA extractions of 20 foliar samples collected in May, as well as 40
foliar and 40 root samples representing the two different sampling
strategies in roots and leaves in September (100 total extractions),
were performed using the MoBio PowerPlant®Pro-htp DNA isolation kit following the manufacturer’s recommendations.
For five of the plants collected in September, we extracted DNA from all
the remaining foliar tissue of the six leaves collected (1241-2467 mg
dry weight per plant, compared to the 15-30 mg commonly used to infer
differences in microbial community composition). This increased the
amount of tissue analyzed per plant by >40x. The number of
additional extractions depended on the size of the leaves and ranged
from 5 to 10 extractions per plant. These additional bulk material
extractions on larger volumes were performed using the DNeasy Plant Maxi
Kit® Qiagen (Qiagen, Hilden, Germany), also
per manufacturer recommendations. To monitor potential background or
cross contamination among samples, we also included control extractions
and PCRs for all microbial groups and extraction kits used.
After extracting DNA, all samples were prepared for Illumina sequencing
through a two-step PCR amplification. For root tissue we amplified the
18S, internal transcribed spacer 2 (ITS2), and 16S rRNA regions for AMF,
general fungi, and bacteria respectively. In leaf tissue only the ITS2
general fungal region was targeted. Primer pairs for PCR1 included AMF
specific primers WANDA (Dumbrell et al. , 2011) and AML2 (Leeet al. , 2008). For the ITS2 region we used a mixture of the
fungal specific forward primers fITS7 and ITS7o (Ihrmark et al. ,
2012; Kohout et al. , 2014) and the general eukaryotic reverse
primer ITS4 (White et al. 1990). AMF specific primers were used in
addition to general fungal primers because ITS2 primers may not allow
for a thorough characterization of AMF communities due to poor
amplification of AMF when other fungi co-occur (Lekberg et al .,
2018). For 16S we targeted the V4 region using primers 515F and 806R
(Caporaso et al. 2011). More detailed descriptions of PCR reactions are
in Bullington et al ., (2018) and Lekberg et al ., (2018).
Briefly in PCR 1, each primer was flanked by 22 bp Fluidigm universal
tags CS1 or CS2 (Fluidigm Inc. San Francisco, CA, USA). Each reaction
contained a total volume of 12.5 μL which included 1.0 μL of DNA extract
as template, 2.5 pmol of each primer in 1X GoTaq® Green Master Mix
[(Green GoTaq® Reaction Buffer, 200μM dATP, 200μM dGTP, 200μM dCTP,
200μMdTTP and 1.5mM MgCl2) Promega, USA]. Reactions
were performed in duplicate or triplicate, depending on concentration of
amplified product (number of reactions was kept consistent within each
microbial group), in a Techne TC-4000 thermocycler (Bibby Scientific,
Burlington, USA) under the following conditions: initial denaturation at
95 o C for 2 minutes followed by 35 cycles at 95o C for 1 min, 54 o C (18S), 57o C (ITS) or 50 o C (16S) for 1 min,
and 72 o C for one min, with a final elongation for 10
min at 72 o C. To confirm the presence of our target
amplicons, all reactions were analyzed by 1.5% agarose gel
electrophoresis using a 100 bp ladder (O’GeneRuler DNA Ladder, Thermo
Scientific, USA) as a size standard. As a precaution, control samples
were sequenced even if they did not produce a band during gel
electrophoresis.
All amplicons generated during PCR1 were diluted 1:10 for use as
template in PCR2. PCR2 primer complexes consisted of the same Fluidigm
tags (CS1 or CS2) as PCR1 primers, 8 bp Illumina Nextera barcodes
(Illumina Inc., San Diego, CA, USA), and Illumina adapters. To minimize
index hopping, we used unique dual indexing pooling combinations
(Kircher et al., 2012), stored libraries individually, pooled
only immediately before sequencing, and removed free adaptors from our
libraries. PCR2 amplicons were purified using AMPure XP beads (Beckman
Coulter Genomics, USA), quantified by Qubit® 2.0
fluorometer (Invitrigen, USA) and pooled in equimolar concentration
prior to sequencing. Sequencing was done at the Institute for
Bioinformatics and Evolutionary Studies (IBEST) genomics resources core
at the University of Idaho (http://www.ibest.uidaho.edu; Moscow, ID,
USA). Amplicon libraries were sequenced using ¼ of a 2 x 300 paired-end
(PE) run on an Illumina MiSeq sequencing platform (Illumina Inc., San
Diego, CA, USA).