4.1.4. Plant Materials for ABA Content Measurement, ABA Sensitivity, and qRT-PCR Analysis

A pair of near-isogenic lines (NILs) containing the NIP alleles (NIL-NIP) and ZS97 alleles (NIL-ZS97), respectively, at the target QTL (*qDOM3.1*) in a common background of ZS97 were developed based on polymorphic markers. NIL-NIP and NIL-ZS97 were used for ABA content measurement, ABA sensitivity, and qRT-PCR analysis.

#### *4.2. Seed Trait Measurement*

In all, 50 seeds of each sample were spread on moistened filter paper in Petri dishes in a 25 ◦C growth chamber for germination experiments. The number of germinated seeds was counted daily for seven consecutive days to construct cumulative germination curves. Germination was defined as the length of the protruded radicle by 3–5 mm. Germination tests for the after-ripened seeds were also conducted as described in the above method.

Germination was scored using the Germinator package [34]. We calculated the six relevant parameters from the germination curve. The parameters included maximum germination percentage of seven days germination (Gmax); germination percentage at three days (G3d); germination speed, which is the time to reach 50% germination of the total number of germinated seeds (T50); germination uniformity, which is the time interval between 84% and 16% of viable seed to germinate (U8416) (16% and 84% stands for −1SD and +1SD, respectively); and area under the germination curve (AUC). Germination index (GI) was calculated by the method of Cao et al. [48]: (GI = Σ(Gt/Tt), where Gt is the number of the germinated seeds on Day t, and Tt is the time corresponding to Gt in days.
