*2.3. Fine Mapping of FLO4-5*

In a previous study, the F2 population of Namil(SA)-flo1/Milyang 23 (non-waxy Tongil cultivar) was used for genetic analysis [33]. To investigate the candidate genomic region for *FLO4-5*, F2 lines heterozygous for the genomic region between RM18624 and RM18639 were grown to the F3 generation. The F3 plants were cultivated to generate F3:4 seeds at the Pusan National University (PNU) experimental farm. Total genomic DNA was extracted from fresh leaves using a NucleoSpin ®Plant II kit (MACHEREY-NAGEL GmbH & Co.KG, North Rhine-Westphalia, Germany), according to the manufacturer's instructions.

Parental lines and 96 randomly selected F3:4 recombinants were dehulled for visual inspection of the endosperm, and then genotyped with six cleaved amplified polymorphic sequence (CAPS) markers developed based on the whole-genome resequencing data [34] of Namil(SA)-flo1 and Milyang 23. The whole genomes of Namil(SA)-flo1 and Milyang 23 were re-sequenced with a 75-fold average coverage using the Illumina HiSeq 2500 Sequencing Systems Platform (Illumina Inc., San Diego, CA, USA). Raw sequence reads were aligned against the rice reference genome (IRGSP 1.0) [37], and the predetermined CAPS marker orders were judged by e-landing of each marker on the reference rice genome [38]. ORFs and their functional products were annotated according to the MSU Rice Genome Annotation Project Database [39] based on the defined physical locations. PCRs were performed in a total volume of 20 μL containing 10 ng of DNA template, 10 pmol of each primer, 1× PCR bu ffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, and 1.5 mM MgCl2), 0.2 mM dNTPs, and 1 unit of Taq DNA polymerase (Nurotics, Daejeon, Korea). PCRs were performed with an MJ Research PTC-100 thermocycler (Waltham, MA, USA) using the following conditions: initial denaturation at 94 ◦C for 5 min, followed by 36 cycles of denaturation at 94 ◦C for 30 s, annealing at 58 ◦C for 30 s, and extension at 72 ◦C for 1 min, with a final extension at 72 ◦C for 10 min. The PCR products were digested using restriction enzymes (New England Biolabs, Ipswich, MA, USA), according to the manufacturer's instructions. The digestion of PCR products encompassing CAPS markers was detected using the WatCut program [40]. Digestion products were separated on 3% polyacrylamide gels using 6 M urea

and 1X TAE at 80 volts and visualized using the Molecular Imager ®Gel Doc ™ XR System (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

### *2.4. Cloning of FLO4-5 and Identification of the Mutation Site*

The coding sequence of cy*OsPPDK* in Namil(SA)-flo1 and Namil was compared using the CLC Sequence Viewer 7.0 (QIAGEN, Hilden, Germany). To verify the mutation site, a CAPS marker containing two *MboII* restriction sites in the floury endosperm mutant Namil(SA)-flo1 and one *MboII* restriction site in Namil was developed using the WatCut program. A 177 bp fragment containing the mutation site was PCR amplified from Namil(SA)-flo1 and Namil using the primers flo4-5\_F: CTCCAGTGGGTGGAGGAGTA and flo4-5\_R: GATCGATCAGCAACGGAGAT. The PCR products were digested with *MboII* (New England Biolabs) in a volume of 15 μL containing 5 μL of PCR product, 1.5 μL of 10× NEBu ffer, 0.5 μL of *MboII*, and 8 μL of ultrapure water, and then incubated at 37 ◦C for 2 h. The digestion products were separated using the Fragment Analyzer ™ (Agilent, Santa Clara, CA, USA). The CAPS marker was also used for co-segregation analysis of F3:4 families and 44 Korean rice cultivars with the endosperm phenotype. The complete genomic DNA of the cy*OsPPDK* gene was cloned in three overlapping segments using primers designed on the basis of the cy*OsPPDK* gene sequence of Nipponbare (Table S1).

### *2.5. Predicting the Functional E*ff*ect of Amino Acid Substitutions and Real-Time qRT-PCR Analysis*

The PROVEAN (Protein Variation E ffect Analyzer, v1.0) tool [41] was used to predict amino acid changes that a ffected protein function. Total RNA was isolated from the rice grains of Namil and Namil(SA)-flo1 at 12 days after flowering (DAF) using an RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturers' instructions. Genomic DNA was removed with DNase I (QIAGEN), and reverse transcription was performed using an RNA to cDNA EcoDry Premix Kit (Clontech, Mountain View, CA, USA). A QuantiNova SYBR Green RT-PCR kit (QIAGEN) and the Rotor-Gene Q instrument (QIAGEN) were used for qRT-PCR with the following conditions: 95 ◦C for 10 min followed by 40 cycles of 95 ◦C for 10 s and 60 ◦C for 20 s. Fold-change was calculated relative to Namil. Rice Actin 1 (OsACT1; Os03g0718150) was used as an internal control. The primer sequences used for qRT-PCR are listed in Table S1.
