**2. Results**

#### *2.1. Thermotolerance Assay of SDWG005 and 9311 in Growth Chamber*

Our previous field work has identified SDWG005 as thermotolerant but 9311 as thermosensitive. In this study, a growth chamber was used to mimic high temperature treatment on SDWG005 and 9311. The results showed that 9311 had more than 60% sterile spikelets after 5 d of heat treatment (relative seed setting rate 43.8%). In contrast, the spikelets of SDWG005 did not significantly differ between the control and heat treatment (relative seed setting rate 96.4%) (Figure 1). These findings suggested that SDWG005 was heat tolerant and 9311 was heat susceptible.

**Figure 1.** Mature spikes of 9311 and SDWG005 in the control (28 ◦C for 12 h per day) and heat treatment (38 ◦C for 12 h per day, for 5 days) conditions. (**a**,**b**) Panicles of 9311 plants under normal and heat stress conditions, (**c**,**d**) Panicles of SDWG005 plants under control and heat stress conditions.

#### *2.2. Microstructure of Anthers in Thermotolerant SDWG005 under Heat Stress*

To examine the status of rice anthers under heat stress, morphology and microstructure of SDWG005 and 9311 anthers under normal conditions and after 12 h of heat treatment were observed under a microscope (Figure 2). As shown in Figure 2, there were no open anthers in either genotype under normal conditions (Figure 2a,c). After 12 h of heat treatment, the anthers of SDWG005 opened slightly (Figure 2d). In contrast to SDWG005, 9311, as a heat-sensitive variety at anthesis, showed widely opened anthers after the same treatment, but there was no pollen released from these opened anthers (Figure 2b).

**Figure 2.** Anther morphology of SDWG005 and 9311 in the control and heat (38 ◦C for 12 h) treatments. Anthers of 9311 plants in the (**a**) control and (**b**) heat treatments, and anthers of SDWG005 plants in the (**c**) control and (**d**) heat treatments. Scale bar = 22 μm.

In addition, the walls of the anther sacs of 9311 had wilted significantly after 12 h of heat treatment, which damaged the anther structure due to water loss (Figure 3a,b). In contrast, the anthers of the

tolerant genotype SDWG005 remained intact under heat stress (Figure 3c,d) and did not differ from those in the control.

**Figure 3.** Microstructure of anthers of SDWG005 and 9311 in the control and after 12 h of heat treatment. Anthers of 9311 plants in the (**a**) control and (**b**) heat treatments, and anthers of SDWG005 plants in the (**c**) control and (**d**) heat treatments. Scale bar = 50 μm.
