Phenotypic analysis

The experiment was conducted in a greenhouse at Thailand Science Park, Khlong Luang, Pathum Thani, Thailand. Relative humidity and temperature were controlled at 80 ± 5% and 32 ± 2°C at the daytime/28 ± 2°C at the nighttime, respectively. Seeds of 239 rice accessions were divided into three groups for phenotypic evaluation experiment at seedling, vegetative, and reproductive stages. For the seedling stage, four replicates of rice seeds were germinated on a moist paper towel in Petri dishes. After 3 days, the germinated seeds were sown in a commercial soil until a few leaves appeared. Rice seedlings were then transferred individually into plastic plates floating in a plastic container filled with distilled water. The 14-day-old rice seedlings were then transferred to water containing 0 or 100 mM FeSO₄ solution, pH 4.5, as provided for control and iron toxicity, respectively. For vegetative and reproductive stages, rice seeds were sown with four replicates per accession and grown in commercial soil with chemical properties of electrical conductivity = 2.7 dS m⁻¹, pH = 5.7, total organic carbon = 12.3%, available N = 0.3 mg kg⁻¹, available p = 578 mg kg⁻¹, available K = 3,073 mg kg⁻¹, available Ca = 7,020 mg kg⁻¹, available Mg = 1,034 mg kg⁻¹. The 14-day-old seedlings were then planted in the plastic tray consisted of 80 holes with a diameter of 3.5 cm and a depth of 4.5 cm. The plastic trays were placed in a rubber tank (80 × 80 × 20 cm of width × length × height), and tap water was maintained at the soil surface. The 16-16-16 (N-P-K) fertilizer (5 g per tank) was added at 14 and 45 days after transplanting. At the vegetative stage of the plant (60 days after sowing), 0 or 100 mM FeSO₄ solution with pH 4.5 was added to the tank of the first set of rice plants. On the other hand, the second set of rice plants was grown continuously until the booting stage. One hundred and sixty-nine accessions from the entire rice panel that were capable of flowering were added with FeSO₄ solution of 0 or 100 mM, pH 4.5, before phenotypic traits were collected.

Agro-morphological shoot traits, i.e., shoot height (SH), shoot fresh weight (SFW), shoot dry weight (SDW), and root traits, i.e., root fresh weight (RFW) and root dry weight (RDW), were measured 14 days after treatment for the seedling stage, whereas these traits were measured 7 days after treatment for the vegetative and reproductive stages. All traits were measured and recorded according to IRRI Standard Evaluation System for rice (SES, 2013). SH was measured from the base of rice plants to the tip of the uppermost leaf. SDW and RDW were measured on the harvested shoots and roots after 72-h drying in a hot air oven at 80°C. Physiological traits, i.e., maximum quantum yield (F_v/F_m), photon yield of PSII (Φ_PSII), leaf greenness (Soil Plant Analysis Development or SPAD value), and leaf bronzing score (LBS) were measured. Chlorophyll fluorescence emission was measured from the adaxial surface of second-fully expanded leaf at the vegetative stage or flag leaf at the reproductive stage using a fluorescence monitoring system (model FMS 2; Hansatech Instruments Ltd., Norfolk, United Kingdom) according to Loggini et al. (1999) and Maxwell and Johnson (2000). Leaf greenness was measured using SPAD (SPAD-520Plus, Konica Minolta, Osaka Japan) at the same leaf position (Hussain et al., 2000). LBS was visually scored on a scale of 1 (no symptom on leaf) to 9 (dead leaf) according to the IRRI Standard Evaluation System for rice (SES, 2013; Supplementary Figure S2).