*3.2. Dry Milling Properties of Namil(SA)-flo1*

The suitability of Namil(SA)-flo1 as a raw material for high-quality dry milled rice flour was assessed. The grain hardness of Namil(SA)-flo1 was 0.45 times that of the wild type, and was softer than that of Seolgaeng, a Korean rice cultivar with an opaque endosperm. The mean particle size of Namil(SA)-flo1 was 86.1 μm, smaller than that of the wild type (109.1 μm), Seolgaeng (97.6 μm), and Hwaseong (112.2 μm). The damaged starch content of dry milled flour in Namil(SA)-flo1 (4.9%) was significantly lower than that in Namil (9.2%), Seolgaeng (7.1%), and Hwaseong (10.3%) (Table 2). Overall, the low grain hardness, low starch damage, and fine particle size indicate that Namil(SA)-flo1 is suitable for dry milling to produce fine flour.

### *3.3. Fine Mapping of the Floury Endosperm Locus*

The Namil(SA)-flo1 locus was previously refined to a region on chromosome 5 and, alongside *FLO4-4* (identified from Namil(SA)-flo2, also known as Suweon 542), was linked to chromosome 5 markers RM18624 and RM18639 [33,35]. To map the Namil(SA)-flo1 target gene more precisely, the whole genomes of Namil(SA)-flo1 and Milyang 23 were re-sequenced and aligned against one another to select homologous SNPs for developing CAPS markers. Six CAPS markers were developed (Table S2) and used to genotype 60 floury and 36 normal individuals derived from F3:4 recombinant families. Multiple comparisons were conducted between the genotypes of these recombinants and the phenotypes of their offspring. Finally, the target region was mapped to BAC clones OJ1174\_H11 and BAC OSJNBb0006J12 in a 33 kb region flanked by the markers CAPS6 and RM18639. Based on the Rice Annotation Project Database [42], four predicted genes were found in the critical 33 kb region (Figure 2a). Of these, *Os05g0404500* was annotated as encoding a hypothetical protein, Os05g0404700 was a functional gene similar to the methyl-binding protein gene MBD1, Os05g0404901 encoded a conserved hypothetical protein, and Os05g0405000 was annotated as a PPDK gene. Sequencing of all four candidates in Namil(SA)-flo1 and the Namil wild type revealed a C to T SNP in exon 2 of PPDK (Figure 2b and Table S3). In rice, PPDK, which has two promoter sites, encodes both a cytosolicand a chloroplast-targeted PPDK protein (cy*OsPPDK* and ch*OsPPDK*, respectively). To analyze the transcript type of PPDK in developing grains of rice, the PPDK of Namil(SA)-flo1 and Namil was sequenced using the primer combinations PF2/PR3 (cy*OsPPDK*) and PF3/PR3 (ch*OsPPDK*) [15] and three additional primer pairs (Table S1). The full-length sequence of cy*OsPPDK* was identified as 7556 bp with a cDNA sequence of 2649 bp containing 19 exons and encoding an 882 amino acid protein. The SNP in exon 2 was a missense mutation that resulted in a serine to phenylalanine change at amino acid position 101 in cy*OsPPDK* (Figure 2b). Comparison of the cy*OsPPDK* sequences of Namil(SA)-flo1 and Namil with that of Nipponbare revealed nine SNPs and six InDels (insertion/deletion mutations) in the intron region and two SNPs in the coding region that were synonymous mutations (Table S3). Sequence analysis of Suweon 542 (*flo4-4*) did not reveal the same mutation site (the Suweon 542 phenotype was caused by a G→A SNP in exon 8 of cy*OsPPDK*) [34]. The novel recessive floury gene was named *floury4-5* (*flo4-5*). The full-length coding sequences of *FLO4-5* from Namil(SA)-flo1 and Namil were deposited in GenBank under accession numbers MG267057 and MG267056, respectively.



**Figure 2.** Map-based cloning of the *flo4-5* mutant. (**a**) Fine mapping of the *flo4-5* locus. The molecular markers and number of recombinants are shown. The 33kb virtual contig, composed of overlapping 2 BAC clones, was delimited by e-Landings of two significant markers on the reference rice genome, 'Os-Nipponbare-Reference-IRGSP-1.0'; (**b**) *flo4-5* gene structure and cDNA sequence comparison showing a nucleotide mutant (C to T) within exon 2 where Ser-101 of the wild was induced to Phe-101 of the *flo4-5*. White boxes represent untranslated regions, black boxes represent coding regions, and solid lines represent introns. 1,2,3 are the PCR results of wild type Namil, *flo4-5*(Namil (SA)–flo1) and *flo4-4*(Namil (SA)–flo2), respectively. 1', 2', 3' are the digested results.
