*2.1. Plant Materials and DNA Extraction*

The Namil(SA)-flo1 floury endosperm rice mutant was produced via sodium azide mutagenesis of Namil (*O. sativa* L. ssp. japonica), an early maturing, high-yield, non-waxy Korean elite rice cultivar [12]. To evaluate the major agronomic traits and the grain/flour physicochemical properties, Namil(SA)-flo1 was cultivated with Namil, Seolgaeng (non-waxy opaque endosperm japonica cultivar), and Hwaseong (non-waxy japonica cultivar) in the experimental plot of the National Institute of Crop Science (NICS), Rural Development Administration (RDA), Suwon, Korea.

### *2.2. Evaluation of Agronomic Traits and Grain Physicochemical Properties*

Replicated yield trials were conducted to evaluate the major agronomic traits, as well as the grain/flour physicochemical properties, in the field at the NICS, RDA, Suwon, Korea [35]. The seeds of each rice line were sown on April 25, and were then transplanted on May 25 under a randomized complete block design (RCBD) with three replication plots. Each plot, consisting of eight rows with 30 hills per row and three plants per hill, was planted with 30 × 15 cm spacing. The amount of fertilizer application was 90–45–57 Kg/ha for N–P2O5–K2O, and the 10 hills in the middle rows were used to determine days-to-heading (HD), culm length (CL), panicle length (PL), tiller number (TN), spikelet number per panicle (SN), and ripened grains percentage (RGP). The 1000-grain weight (TGW) was measured in grams as the average weight of 1000 fully filled brown rice grains from each plot.

The grain hardness of the brown rice was assessed by determining the pressure at the grain breakage point using a 5 mm probe attachment of a TA.XT Plus instrument (Stable Micro Systems, Godalming, Surrey, UK), using parameters of 0.4 mm/s and 40.0 g for test speed and trigger force, respectively.

The damaged starch content was evaluated using a starch damage assay kit (Megazyme International Ireland, Wicklow, Ireland) following the manufacturer's instructions. The lightness of rice flour was measured with a JS-555 instrument (Color Techno System, Tokyo, Japan). The moisture, protein, lipid, and ash contents of the rice flour were determined using methods 44-15A, 46-30, and 08-01 of the American Association of Cereal Chemists (AACC) 2000. The amylose content of the rice flour was estimated as described previously [36].

Grain samples for scanning electron microscopy (SEM) analyses were prepared as described previously [15]. Cleaved endosperm surfaces were observed under an S-550 scanning electron microscope (Hitachi Hi-Tech, Tokyo, Japan) at an accelerating voltage of 20 kV.
