16s-Seq: Wash23
Following boll maturation, we destructively sampled five plants per genotype per treatment (n = 60), excavating entire plants to preserve root structure. Lateral roots were excised, chilled, and stored at 20C alongside rhizosphere soils for later analysis. For DNA extraction, lateral roots were clipped (~1 cm), vortexed in sterile water to obtain rhizoplane, and surface-sterilized with ethanol and bleach. Soils were homogenized and 20 mg aliquots collected. Genomic DNA was extracted using the Qiagen DNeasy 96 Plant Kit with modifications. Extraction blanks were included to monitor contamination. A bacterial mock community was used as a positive control, and template-free reactions served as negative controls. The V4 region of the 16S rRNA gene was amplified using primers 515F/806R, and sequencing was performed on an Illumina MiSeq (2  300 bp) by the University of Arizona Microbiome Center.