Supplementary Material Figure S1. Mass spectra of major peaks from 'Yuikogane' and lutein and zeaxanthin standards. Liquid chromatography-tandem mass spectrometry of yellow pigments in millet and lutein and zeaxanthin standards. The major yellow pigments were fractionated by HPLC and subjected to LC-MS/MS. Each spectrum corresponds to the HPLC-PAD peaks labeled in Figure 1.

Figure S2. Seed ripening of millet cultivar 'Yuikogane’. Single panicle weight (a), 1,000-seed weight (b), and seed water content (c) are plotted against accumulated temperature.

Supplemental Methods To identify pigments in millet samples, the methanol extracts were separated by HPLC on an InertSustain C18 column (20.0 × 250 mm, 5 µm particle, preparative column; GL Sciences Inc., Tokyo, Japan). HPLC was performed on a Waters HPLC system (Waters 600 Quad Pump and Controller, Waters 717 Plus Automatic Sampler and Waters 996 Photodiode Array Detector; Waters Corp., Milford, MA, USA). Data were recorded and analyzed using Millennium 32 software (Waters). LC-MS/MS analysis was performed on an Agilent 1100 HPLC and AB Sciex MS/MS system consisting of a binary pump (G1312A, CA pump; Agilent Technologies, Santa Clara, CA, USA), an automatic sampler (G1367A, Agilent Technologies), and a triple-quad tandem mass spectrometry system (API4000, AB Sciex, Framingham, MA, USA). The MS/MS parameters were as follows: curtain gas, 15 ml/min; exit potential, 10 V; and drying and nebulizing gas, 50 ml/min. Mass spectra were acquired in atmospheric-pressure chemical ionization (APCI) mode. The analytical column was Develosil XG C30-UG (3 µm particle, 2.0 × 250 mm; Nomura Chemical Co., Ltd, Seto, Aichi, Japan). The method used isocratic flow of 50% acetonitrile, 45% methanol, and 5% distilled water at 0.35 ml/min. Samples (10 μl) of 1 ppm lutein and zeaxanthin standards and 10 ppm extracts were injected into the column, which was maintained at ambient temperature. Analyst version 1.5.2 (Applied Biosystems, Life Technologies Japan, Tokyo, Japan) was used for data acquisition and processing.