Quantifying Rice Grain Quality Traits

Total protein and amylose contents in matured grains were evaluated in each year from 2013 to 2018 using the methods of Hori et al. (2021b (link)). Apparent amylose content was determined using an Auto Analyzer II (Bran + Luebbe, Norderstedt, Germany). Crude protein content was determined by the combustion method with an induction furnace at 900 °C (American Association of Cereal Chemists International, Approved Method 46-30.01). Measurement of low-molecular-weight compounds was carried out in 2015 and 2016, using the methods of Human Metabolome Technologies (Tsuruoka, Japan). Briefly, rice flours from each line were homogenized in 600 µL methanol containing 10 µM internal standards, mixed with 600 µL chloroform and 240 µL water, then centrifuged at 2300 × g for 5 min. The aqueous supernatant fraction was filtered and recovered into 50 µL of MilliQ water prior to metabolite analysis using capillary electrophoresis time of flight-mass spectrometry (CE-TOFMS) (Agilent Technologies, CA, USA) (Soga et al. 2004 (link)). Peaks detected in the CE-TOFMS analysis were extracted using automatic integration software (MasterHands ver. 2.17.1.11) and annotated with putative metabolites based on their migration in CE and m/z values.