**2. Results**

#### *2.1. Seed Dormancy Variation in Parents*

The germination assay for the freshly harvested seed of the parental lines, which were *indica* variety Zhenshan97 (ZS97) and *japonica* variety Nipponbare (NIP), was performed. The number of germinated seeds was counted daily for seven consecutive days and the germination percentage was calculated each day to construct cumulative germination curves. ZS97 and NIP exhibited significant difference in seed dormancy, in which ZS97 had significantly higher germination percentage (100%) than NIP (37%) at seven days (168 h of germination) (Figure 1A). Dry seeds were treated at 43 ◦C for three days to break seed dormancy (which was called after-ripened seeds) and then underwent a seed germination experiment. After seed dormancy was broken, the germination percentage of seven days (168 h) of the parental lines was almost the same (around 90%) (Figure 1B). However, the germination rate of NIP was still lower as compared to ZS97. At two days of germination (48 h), the radicle protrusion of ZS97 was 86%, whereas no radicle protrusion occurred for NIP (Figure 1B).

**Figure 1.** (**A**) Germination behavior of freshly harvested *japonica* variety Nipponbare (NIP) and *indica* variety Zhenshan97 (ZS97) seeds; (**B**) germination behavior of after-ripened NIP and ZS97 seeds.

#### *2.2. Seed Dormancy Variation in the CSSL Population*

The CSSL population, which comprised 146 lines, was developed and genotyped in a previous study [32,33] and the lines were used to dissect the genetic architecture of seed dormancy underlying this population.

Seed germination was analyzed using the six germination parameters from the Germinator package [34], which were Gmax (maximum germination percentage at seven days); G3d (germination percentage atthree3 days), T50 (germination speed: time to reach 50% germination of the total number of germinated seeds), U8416 (germination uniformity: time interval between 16% and 84% of viable seed to germinate), AUC (area under the curve), and GI (germination index). The mean performance of the CSSL population is presented in Table 1. The frequency distribution for the six germination parameters of the CSSL population showed large variation (Figure S1, Supplementary Materials). These results indicated that there might be seed dormancy QTLs in this CSSL population.


**Table 1.** Germination parameters of the two parents and the chromosomal segment substitution line (CSSL) population.

Gmax, maximum germination percentage of seven days of germination; G3d, germination percentage at three days; T50, time to reach 50% germination of the total number of germinated seeds; U84-16, germination uniformity, which is time interval between 84% and 16% of viable seed to germinate; AUC; area under the germination curve until 168 h; GI, germination index. SD, standard deviation; CV, coefficient variation; Min to Max, the minimum and maximum value in the CSSL population. Asterisks \*\*, indicate significant difference between the parents at the level of 0.01.
