*4.7. RNA Isolation and qRT–PCR Analysis*

RNA isolation: RNAPrep Pure Plant Plus Kit (TIANGEN, catalog number DP441, Beijing, China) was used to extract total RNA from embryos of germinated seeds, according to the manufacturer's instructions. IScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) was used for cDNA synthesis for quantitative real-time PCR (qRT-PCR), according to the manufacturer's instructions. qRT-PCR was performed using a QuantStudio (TM) 6 Flex Real-Time PCR instrument (Applied Biosystems, Waltham, MA, USA) with iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). Three biological replicates were used for each sample. The data were normalized to the amplification of a rice ACTIN gene (LOC\_Os03g50885). The mean value was then plotted with its standard error. Primers for real-time PCR are included in Table S2, Supplementary Materials.

### **5. Conclusions**

Rice seed dormancy is an important agronomic trait that is crucial to the quality of rice. Currently, rice breeding programs have been focused on cultivars with a balance between pre-harvest sprouting and deep dormancy [2]. Cultivars with moderate dormancy levels have multiple advantages, including decreasing pre-harvest sprouting, improving the survival rate of direct seedling rice, increasing seedling growth uniformity, and further improving the storage quality of rice. The QTL *qDOM3.1* controlling seed dormancy identified in the present study will accelerate the breeding of new rice varieties with suitable seed dormancy. Besides *qDOM3.1*, many of the QTLs identified in the present study may also be useful in an agricultural context, providing new genes that can be used to improve crop performance under fluctuating environments.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/4/1344/ s1.

**Author Contributions:** Conceptualization, H.H. and S.Y. (Sibin Yu); methodology, S.Y. (Shaowen Yuan), Y.W., and C.Z.; software, Y.W. and C.Z.; writing—original draft preparation, S.Y. (Shaowen Yuan) and Y.W.; writing—review and editing, H.H. and S.Y. (Sibin Yu) All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (31971864), Natural Science Foundation of Hubei Province of China, grant number 2019CFC851, and Fundamental Research Funds for the Central Universities, grant numbers 2662016QD040 and 2662018YJ025.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
