**5. Conclusions**

Our data revealed that a novel SNP (C to T) in the coding region (exon 2) of the gene encoding cytosolic PPDK (cy*OsPPDK*) was responsible for the floury endosperm characteristics of low grain hardness, low starch damage, and fine particle size, and this mutation may be valuable in rice breeding programs. Co-segregation analysis with the developed CAPS marker (flo4-5\_F/flo4-5\_R) revealed co-segregation between the floury phenotype and the *flo4-5* using the segregation population and 44 japonica varieties. This CAPS marker could be applied directly to MAS. Real-time RT-PCR experiments revealed that PPDK was expressed at considerably higher levels in the *flo4-5* mutant than in the wild type during the grain filling stage. Plastids *AGPS2a*, *AGPS2b*, *SSIIb*, and *SSIIc* also exhibited enhanced expression in the *flo4-5* mutant. Although more studies are needed to fully understand the functions of PPDK, our data indicate that PPDK is involved in the endosperm development function through directly or indirectly regulating *AGPS2a*, *AGPS2b*, *SSIIb*, and *SSIIc*.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4425/11/4/465/s1, Table S1: Primers used for sequencing the whole genome sequence of *flo4-5* and qRT-PCR in this study, Table S2: Primer sequences of CAPS markers that developed for fine mapping, Table S3: Compare the whole genome sequences of *cyOsPPDK* gene among Nipponbare, *flo4-5* and its wild type Namil. InDel (Insertion or Deletion).

**Author Contributions:** H.W., T.-H.H. and S.-W.K.; developed ideas, designed and performed all experiments, and wrote the manuscript. Y.M. and J.-U.J.; developed the mutant lines and mapping population. D.-E.I. and J.L.; analyzed the whole genome resequencing dates. S.M.L. and S.-G.J.; produced rice plants for experiments. S.T.K.; support expression analysis. All authors read and approved the final manuscript.

**Funding:** This work was supported by the gran<sup>t</sup> from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology and the research gran<sup>t</sup> from Rural Development Administration, Republic of Korea.

**Acknowledgments:** This work was supported by the gran<sup>t</sup> from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2018R1A4A1025158) and the research gran<sup>t</sup> from the Rural Development Administration, Republic of Korea (PJ012890032020).

**Conflicts of Interest:** The authors declare no conflict of interest.
