*3.4. Co-Segregation and Expression Analyses*

The mutation site in *flo4-5* was confirmed using a CAPS marker with a *MboII* restriction site. *MboII* digested the mutant Namil(SA)-flo1 allele at two restriction sites (Figure 3a,b, lane 3) and the wild-type Namil allele at a single site (Figure 3a,b, lane 4). For screening F3:4 family populations using the CAPS marker, 23 plants with the Namil(SA)-flo1 phenotype and 22 with the Namil phenotype were selected. As shown in Figure 3a, sequences from all plants with the Namil(SA)-flo1 phenotype were digested at two restriction sites with *MboII*, whereas those with the Namil phenotype were digested at a single site. Fifteen F3:4 plants were heterozygous and exhibited the Namil phenotype, suggesting that the floury endosperm phenotype was controlled by a recessive gene. The genotypes of the currently cultivated Korean japonica rice were evaluated with this marker. Of the 44 tested varieties, none of them were the mutant genotype, whereas two varieties were the Namil genotype and the others were the Nipponbare genotype (Figure 3b). Overall, the co-segregation analysis indicates that *FLO4-5* is responsible for the floury endosperm in rice.

The PROVEAN tool was used to predict the effect of a single nucleotide mutation (C to T) on the *FLO4-5* gene, resulting in a score of –5.802 and a rating of "deleterious". Next, qRT-PCR was used to investigate the transcript type of *FLO4-5* in rice during the grain filling stage. The result that relative expression of *FLO4-5* was much higher in Namil(SA)-flo1 than in Namil (Figure 4) was unexpected, because the mutation was in exon 2, not in the promotor region. Even though the higher expression level in the mutant was not clearly explained based on our experimental design, it is highly possible that the increased level of *FLO4-5* was caused by the mutation.

**Figure 3.** Co-segregation analysis of the *flo4-5* genotype with floury endosperm phenotype. (**a**) Verification of the CAPS marker (digestion by *MboII*) and tagging the *flo4-5* locus using a part of F3 individuals; (**b**) Verification of the CAPS marker (digestion by *MboII*) and tagging the *flo4-5* locus using Korean rice cultivars. 'A' and 'B' are homogeneous of Namil(SA)-flo1 and Milyang 23, respectively. 'H' is heterozygote. M (100bp ladder), F1 (Namil(SA)-flo1), M23 (Milyang 23), Na (Namil). 1. Namil(SA)-flo2(Suweon542), 2. Aranghyangchal, 3. Baegjinju1, 4. Baekogchal, 5. Boramchal, 6. Boramchan, 7. Borami, 8. Boseog, 9. Boseogchal, 10. Boseogheugchal, 11. Cheongnam, 12. Chindeul, 13. Chucheong, 14. Dabo, 15. Danmi, 16. Danpyeong, 17. Deuraechan, 18. Dodamssal, 19. Dongjin, 20. Dongjin1, 21. Dongjinchal, 22. Geonganghongmi, 23. Geonyang 2, 24. Goami, 25. Goami 2, 26. Goami 4, 27. Haepum, 28. Haiami, 29. Hanam, 30. Hanmaeum, 31. Heugjinmi, 32. Heughyangchal, 33. Heugjinju, 34. Heugnam, 35. Heugseol, 36. Hongjinju, 37. Hopum, 38. Hwanggeumnuri, 39. Hwaseong, 40. Hwawang, 41. Hwayeong, 42. Hyangnam, 43. Hyeonpum, 44. Ilmi.

### *3.5. Mutation of FLO4-5 Changed the Expression Levels of Major Starch Synthesis Enzymes*

Because the starch content and the starch granules were altered in the *flo4-5* mutant, the expression levels of the starch synthesis-related genes in the developing grains were then analyzed using qRT-PCR. Compared with the wild-type Namil, the transcription of several genes decreased in the mutant, including *GBSSI*, *GBSSII*, *AGPL1*, *AGPL2*, *AGPL3*, *AGPL4*, *AGPLS1*, *SSI, SSIIa, SSIIIa, SSIIIb, SSIVa, SSIVb, BEI, BEIIa, BEIIb,* and *PUL*. The expression of *AGPS2a*, *AGPS2b*, *SSIIb*, and *SSIIc* increased in the mutant compared to the wild type, with substantial increases seen for *AGPS2a* and *AGPS2b* (Figure 5a). The expression analysis of Suweon 542 (*flo4-4*) revealed similar expression patterns (Figure 5b).

**Figure 4.** Expression analysis of PPDK between Namil and *flo4-5* at 12 DAF. Values shown are mean ± SD (n = 3).

**Figure 5.** Expression profiles of the genes involved in production of storage starch in endosperm from the flo4-5 mutant (**a**) and Suweon542 (**b**) with the wild type Namil (bar begin with 1). Reference gene was Actin, and the expression levels of these genes are shown relative to the wild type Namil, which is set as 1. Each gene name is indicated by a simplified representation. *GBSSI* and *GBSSII*, granule bound starch synthase I and II; AGPL (*AGPL1*, *AGPL2*, *AGPL3* and *AGPL4*) and AGPS (*AGPS1*, *AGPS2a* and *AGPS2b*), ADP-glucose pyrophosphorylase large subunit and small subunite, respectively; *SSIIa* and *SSIIIa*, soluble starch synthase *IIa* and *IIIa*, respectively; *BEI* and *BEIIb*, branching enzyme *I* and *IIb*, respectively; *PUL*, pullulanase.
