## 3 Materials and Methods
### Rice Whole Genome DNA Microarray Analysis  
Dry mature seeds (12 of each grade of Y1-Y3) were used for preparing fine powders in liquid nitrogen. Briefly, the seeds were placed in a pre-chilled mortar and pestle containing liquid nitrogen, ground completely to a very fine powder with the chilled pestle in liquid nitrogen and stored at \(-80\)\({}^{\circ}\)C till used for RNA extraction. For total RNA extraction, the stored sample powder (\(\sim\)100 mg) was transferred to a 2 mL sterile microfuge tube, followed by addition of 0.9 mL of CTAB buffer [a 10 mL volume of buffer contains 0.5 mL (50 mM) of 1 M stock Tris-HCl solution (pH 8.0), 1.0 mL (5 mM) of 500 mM ethylenediaminetetraacetic acid (EDTA, pH 8.0), 0.2 g (2%, w/v) of CTAB, 1.68 mL (0.84 M) of 5 M NaCl, and 0.1 M \(\beta\)-mercaptoethanol, which is added just before use of the solution]. The contents were mixed by vortexing for 30 s and incubated for 5 min at RT. After an addition of 0.8 mL of phenol-chloroform-isoamylalcohol (PCIA; 25:24:1), the homogenate was mixed well (by gentle shaking) for 5 min at RT. After centrifugation at 15,000 x g for 5 min at 4 \({}^{\circ}\)C, an aliquot (0.6 to 0.7 mL) of the upper phase was transferred to a 1.5-mL sterile microfuge tube, followed by addition of 1 volume of chloroform, and the mixture was centrifuged at 15,000 x g for 5 min at 4 \({}^{\circ}\)C. The resulting supernatant was transferred to another 1.5 mL microfuge tube and 0.033 volume of 3 M sodium acetate, pH 5.5 and 1 volume of 2-isopropanol were added. The mixture was incubated for 15 min on ice and then centrifuged at 15,000 x g for 5 to 10 min at 4 \({}^{\circ}\)C to collect the RNA. The supernatant was completely removed and the pellet was dissolved in 0.1 mL of RNase-free water (SDW; double sterilized distilled water) followed by using the RNeasy mini protocol for RNA cleanup exactly as described by the manufacturer (QIAGEN, Gaithersburg, MD, USA). To verify the quality of this RNA, the yield and purity were determined spectrophotometrically (NanoDrop, Wilmington, DE, USA) and visually confirmed using formaldehyde-agarose gel electrophoresis. After extraction of high-quality total RNA from dehusked seeds using a modified CTAB extraction protocol, a rice 4 \(\times\) 44K custom (eARRAY, AMAdid-017845) oligo DNA microarray chip (G2514F: Agilent Technologies, Palo Alto, CA, USA) was used for genome-wide gene profiling as described previously [83]. The flip labeling (dye-swap or reverse labeling with Cy3 and Cy5 dyes) procedure was used to nullify the dye bias associated with unequal incorporation of the two Cy dyes into cRNA [84; 85; 86]. The dye-swap approach which is well established in our laboratories and researchprovides a more stringent selection condition for profiling differentially expressed genes rather than simply doing only 2 or 3 replicates, which overlook the dye bias [86; 87; 88; 89; 90; 91; 92]. The experimental design for the DNA microarray analysis of rice seed transcriptome using a two-color dye-swap approach (Figure 7A) is shown along with the extracted seed total RNA quality (Figure 7B). Total RNA (800 ng) for each Y1, Y2, and Y3 sample were labeled with either Cy3 or Cy5 dye using an Agilent Low RNA Input Fluorescent Linear Amplification Kit. Hybridization and wash processes were performed according to the manufacturer's instructions. Hybridized microarray chips were scanned using the Agilent Microarray Scanner G2565 BA. To detect differentially expressed significant genes between control and treated samples, each slide image was processed by Agilent Feature Extraction software (version 9.5.3.1). Normalization of Cy3 and Cy5 signals was performed by LOWESS (locally weighted linear regression), which calculates the log ratio of dye-normalized Cy3- and Cy5-signals. The significance (P) value is based on the propagate error and universal error models. In this analysis, the threshold of significantly expressed differential genes was set to \(<\)0.01 (for the confidence that the feature was not differentially expressed).