*4.3. Seed Setting Rate*

After maturation, the marked spikes from ten plants were harvested separately for each genotype within each treatment. The number of empty and filled grains was recorded, with the seed set rate represented as the percentage of filled grains in all grains. The effect of heat stress on seed set was indicated by the relative seed setting rate, being seed setting rate at high temperature/seed setting rate at normal temperature × 100%.

#### *4.4. Sample Collection for RNA-Seq and Real-Time PCR*

A total of 75 plants per genotype were used for sample collection. When three or more panicles extended from flag leaves by approximately 2 cm, the panicles were labeled, and the plants transferred to the growth chamber for the HS treatment. The labeled spikes were harvested before HS treatment (0 h) and 1, 2, 6, and 12 h after the initiation of the HS treatment, respectively, and placed on ice before anther extraction. The treatments were designated WD\_0 and 9311\_0, WD\_1 and 9311\_1, WD\_2 and 9311\_2, WD\_6 and 9311\_6, and WD\_12 and 9311\_12 for SDWG005 and 9311, respectively. One hour and 2 h were considered as short-term, 6 and 12 h were considered as intermediate and long-term heat treatments, respectively. For each treatment, 15 independent plants were sampled, and mature anthers which ascending to the top of glume from five independent plants pooled as one biological replicate, such that there were three biological replicates per treatment. The labeled panicles from each time point in each treatment were collected on ice, with the mature anthers from the panicles isolated, immediately suspended in liquid nitrogen, and stored at −80 ◦C until RNA extraction.
