Microbial DNA extraction and 16S rRNA gene sequencing
To characterize the microbial community composition of native locations,
we collected samples of rhizosphere and root from eight haphazardly
selected hallii individuals growing at Austin (WFC) and ninefilipes individuals growing at Corpus Christi (CCBG) – we note
that in this natural sampling scheme location and ecotype are
confounded. Additionally, five bulk soil samples (all plant material
removed) from each site were collected in areas adjacent to livinghallii plants (44 samples total) and DNA was obtained with the
DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany). 16S ribosomal RNA
gene regions were amplified using the 515F-806R primer pair, barcoded
and sequenced on the Illumina Novaseq platform on the SP flowcell using
250x250 reads. To characterize treatments in the greenhouse experiment,
this procedure was repeated on rhizosphere, root and soil samples taken
at harvest from seven replicates of each parent in each treatment (four
treatments x 14 parents x three compartments = 168 samples).