*2.1. Allelopathic Assay*

Allelopathic potential was assessed by using the equal compartment agar method (ECAM) [12], with minor modifications. A total of 98 F8 RILs were produced by single-seed descent from a cross between 'Sathi', an indica cultivar with high allelopathic potential, and 'Nong-an', a non-allelopathic Tong-il cultivar [13,14]. Genetically uniform cultivated lettuce 'Yeolpungjeokchima' (Lactuca sativa, cv.)—lettuce exhibits high sensitivity to low concentrations of allelopathic chemicals [4]—was used as a receiver species. For ECAM, dehulled rice seeds were sterilized for 15 min, using 2% sodium hypochlorite to prevent fungal and bacterial contamination. Seeds were then rinsed seven times with sterilized distilled water before placing on Whatman No. 5 filter paper (Whatman, Maidstone, England) in a Petri dish (SPL life Sciences, Pocheon, Korea) with 7 mL of sterilized distilled water. Rice and lettuce seeds were germinated in a controlled environment chamber for 3 days, at 28 ◦C, in the dark. Six germinated rice seeds were transplanted into one side of a Magenta box (SPL Life Sciences, Pocheon, Korea) filled with 30 mL of 0.3% nutrient-free water agar. After 1 week, 6 lettuce seeds were transplanted into the other side of the magenta box (Figure 1). The allelopathic

potential of parent rice lines 'Nong-an' and 'Sathi' and 98 derived F8 RILs were evaluated with ECAM, with lettuce as a receiver plant. Lettuce parameters (root length, root weight, shoot length, shoot weight, total length from base of root to apex of shoot, and total weight) were determined after 1-week co-incubation with rice seedlings. Control lettuce plants were grown in the absence of rice, under the same cultivation conditions. Inhibition rates in lettuce (%) were calculated as follows: [(control − treatment)/control] × 100. Two independent experiments were conducted for each RIL, and thus the average from the total of 12 lettuce plants was used to evaluate each RIL.

**Figure 1.** Evacuating method for allelopathic potentials of rice against lettuces seedlings: (**A**) side view of the magenta box where rice seedlings and lettuce seedlings are grown together; (**B**) top view of the magenta box, where rice seedlings and lettuce seedlings are grown together.

### *2.2. Rice DNA Extraction and High-Throughput SNP Genotyping*

DNA was extracted from the parent rice cultivars 'Nong-an' and 'Sathi' and the 98 F8 RILs, using the CTAB method [15]. The extracted DNA was assessed, using a NanoDrop 2000c spectrophotometer with 230, 260, and 280 nm (Thermo Fisher Scientific, Waltham, MA, USA).

All equipment and resources required for the Axiom 2.0 Assay (Thermo Fisher Scientific, Waltham, MA, USA) with automated target preparation were from the Axiom® 2.0 Assay Automated Workflow (Thermo Fisher Scientific, Waltham, MA, USA). Using the Axiom® 2.0 Reagent Kit, ~200 ng of genomic DNA was amplified and randomly fragmented into 25 to 125 bp fragments and then purified and resuspended. The fragments whose size was confirmed from 25 to 125 bp were denatured and transferred to the hybridization tray in the part of GeneTitan® MC Instrument (Thermo Fisher Scientific, Waltham, MA, USA). The hybridization step followed the GeneTitan® Multichannel Instrument User's Manual, using the KNU Axiom Oryza 580K Genotyping Array [16] After ligation, the arrays were stained and imaged on the GeneTitan MC Instrument. The image was then analyzed by using <sup>A</sup>ffymetrix® GeneChip® Command Console® Software (Thermo Fisher Scientific, Waltham, MA, USA). Genotype calls were conducted, using <sup>A</sup>ffymetrix-power-tools. BRLMM-P algorithm was applied, which is a model-based approach which performs 1-dimensional clustering by fitting a Gaussian mixture model (BRLMM-P: a Genotype Calling Method for the SNP 5.0 Array [17]). The part of the KNU Axiom Oryza 580K Genotyping Array, the PolyHighResolution chip, which is comprised of 247,578 SNP markers, was used for high-throughput SNP genotyping. The 247,578 SNP markers were designed from the genomic data of 3494 accessions including the Korean rice core set version 2 (KRICE\_CORE v2), including wild rice and 3K IRRI world collections [18].
