*4.1. Plant Materials*

#### 4.1.1. Experimental Design

The plant materials were planted at the experimental field of Huazhong Agricultural University at Wuhan (29.58◦N, 113.41◦E). The temperature during the late stage of maturity ranged from 25 ◦C to 30 ◦C, which was normal for rice growth and seed maturation. The first flowering date of each plant was recorded by the emergence of the first panicle from the leaf sheath [13].

Seeds were harvested from the individual plants at 35 days after flowering, which was defined as freshly harvested seeds and then equilibrated about 5–6 days at 15% relative humidity (called freshly harvested seeds), and then stored at −20 ◦C for subsequent analyses. A three-day heat treatment at 43 ◦C for dry seeds was used to break seed dormancy, and the seeds were called after-ripened seeds. All the analyses were performed with three biological replicates.

### 4.1.2. CSSL Population

The CSSL population was developed and genotyped in a previous study [32,33]. The details are as follows. A set of chromosomal segment substitution lines (CSSLs) comprising 146 lines was developed in a previous study [32], in which the donor parent was *japonica* variety Nipponbare (NIP) and the recurrent parent *indica* was variety Zhenshan97 (ZS97). The CSSL population was genotyped previously, of which 518 bins were defined with a median size of 400 kb [33].

#### 4.1.3. CSSL Line-Derived Population

One of the CSSLs (NQ96), which contains the target QTL, was selected to backcross with ZS97 to generate an F2 population (called CSSL-derived population) for QTL validation. A total of 338 F2 plants and the F2:3 families of several recombinants were screened with polymorphic markers to identify the respective genotype.
