*4.1.3 Growth in rhizotrons*

Fungicide-coated seeds of 30 selected genotypes (**Table 1**) were sown in sheets of moist white filter paper, placed in a square petri dish and wrapped with parafilm. After two days a single germinated seed was placed into a rhizotron, four rhizotrons per genotype. The rhizotrons were 30x60 cm large and filled with 3 kg soil substrate (dried & sieved field soil 50:50 (v/v) mixed with dried organic soil low in nutrients "Nullerde"). Groups of six rhizotrons were grouped into a larger container, inclined to ~45° and covered to reduce light falling onto the transparent plexiglas side used for root imaging. The 30 genotypes in each replicate were randomized and grown in a greenhouse compartment at 24°C during the day (16 h, 70% humidity) and 20°C during nights (8 h, 90% humidity). After 7, 14, 18, and 21 DAS non-invasive phenotyping, and after 21 DAS invasive measurements were performed.

#### **4.2 Non-invasive root and shoot measurements**

Seedlings grown in filter paper were unwrapped gently without removing them from the paper. Roots were photographed with a digital camera and primary and their branched lateral roots imaged under a stereomicroscope (MX12.5, Leica) to evaluate sorgoleone production, root diameter, and root hair formation (length and density).

For rhizotron-grown plants, at the given time points, roots and shoots of the 30 genotypes were non-invasively measured. Shoot length was measured until the tip of the youngest elongated leaf. The root systems of rhizotron-grown plants were imaged using a photo-station equipped with a digital camera. The PaintRhizo software (FZJ) was used to mark and track primary, lateral, and nodal roots separately over time.

#### **4.3 Invasive root and shoot measurements**

#### *4.3.1 Shoot and root dry weight*

At harvest, 21 DAS and following the last non-invasive measurements, shoots were cut off, dried for seven days in a 60°C oven and then weighted to determine total shoot dry weight. After shoot removal, rhizotrons were opened and the soil was gently removed by washing using running tap water. The primary root as well as the longest crown root were gently separated from the remaining root system. From these roots several 1 cm segments (at 5, 10, 20, 30, 40 cm from the root tip) were cut and transferred to 50% ethanol (p.a.) for subsequent root morphological analyses. The remaining root system was dried for seven days in 60°C and then weighted for root dry weight determination.

#### *4.3.2 Root morphological analyses*

All root segments were imaged using a stereomicroscope (MX12.5, Leica) followed by analysis using the image J software (Fiji). Per root segment four images were taken; per image root diameter and ten root hairs were measured in length, while sorgoleone production was noted as presence or absence. Root hair density was scored following the procedure described in [63]. Distinguished were the primary root and the longest nodal root as well as their daughter roots, separated as 'thick' and 'thin' lateral roots.

#### **4.4 Statistical analyses**

The experimental data were analyzed with Excel (version 2019, Microsoft) and R (Rstudio, version 4.0.3). Genotypic variation per time point was analyzed by a one-way ANOVA followed by Tukey's Honest Significant Difference (HSD) and the LSD (Least Significant Difference). Linear correlations over all plants was calculated as Pearson's correlation.

#### **Acknowledgements**

We thank Andreas Neuwohner for technical support. We thank Professor Dr. Mitchell R. Tuinstra for selection and generous provision of seeds of the 30 diverse sorghum genotypes. JK was funded by a Feodor-Lynen-Return Fellowship of the Alexander-von-Humboldt Foundation (DEU/1151987). The authors acknowledge

institutional funding by the Helmholtz Association (POF III Program – Key Technologies for the Bioeconomy).
