Harvest and Phenotyping
Plants were harvested prior to panicle emergence after six weeks of growth over a five-day period. Individual plants were extracted from pots by pulling the plastic bag to prevent root damage and plants with attached roots were removed from the soil by shaking over wire mesh. Soil rhizosphere samples for parents were collected by dipping root systems into sterilized 50 ml tubes filled with 1X phosphate buffered saline. Plants were then hung by the shoot base on a clamping apparatus and soil particles were removed from the root system with a spray of UV-sterilized water and roots were separated from shoots and preserved in 90% ethanol for future phenotyping. Tiller number and flag leaf area of the main tiller was measured. Shoot and leaf tissue were dried at 55°C and weighed separately to obtain aboveground biomass and to calculate specific leaf area (SLA; fresh leaf area / dry mass of the leaf (cm2 g-1)).
Root systems were phenotyped by scanning the entire intact root system and one representative nodal root with attached lateral roots on an EPSON 12000XL flatbed scanner (Epson America, Inc., San Jose, CA, USA) calibrated for use with WinRhizo Pro 2019 root image analysis software (Regent Instruments Inc., Canada) (full description in Appendix S1). For parents, a small portion of the central root system was frozen for DNA extraction and PCR amplification to determine root endosphere microbial community composition. The remaining roots were collected and dried for 96 hours at 55°C, and weighed.