Plant materials
By using a rice 6K SNP chip, a BC5F2 plant with minimal N22 introgression harboring qHTSF4.1 in the genetic background of IR64 was selected as a heat-tolerant NIL (Ye et al. 2015b). This NIL is designated as IR64HT4. As a heat escape NIL, The BC3-derived IR64EMF3 were used (Hirabayashi et al. 2015). These two NILs were crossed, and the F1 plants were self-pollinated to generate F2 seeds. Markers linked to qHTSF4.1 and qEMF3 were used to select the plants with homozygotes at both loci, and then the selected plants were genotyped using the rice 6K SNP chip (Thomson et al. 2017). The F2 plant with minimal donor introgression was selected for seeds multiplying, and the F3 seeds were used for the following experiments (Figure 1). The pyramided NIL carrying both qHTSF4.1 and qEMF3 in the IR64 background was designated as IR64HT4EMF3. N22, the donor of qHTSF4.1, and a heat tolerant rice variety GIZA178 were used as heat-tolerant check varieties.
Evaluation of flower open time and spikelet fertility in the glasshouse and indoor growth chamber (IGC)
Seeds of IR64 and the NILs were germinated and sown in soil-filled plastic trays, and the 21-day-old seedlings were transplanted into plastic pots filled with 1 Kg natural clay loam soil and 1g of complete fertilizer (14N-14P-14K). Since the heading date of N22 is earlier than IR64, the seeds of N22 were sown and transplanted 10 days later than IR64 and the NILs. A total of 50 pots were transplanted for each genotype. Only one seedling was maintained in each pot, and the plants were grown inside a net-house under natural temperature and sunlight till booting stage.
At the booting stage, all plants were moved into a glasshouse under controlled temperature condition (30°C/24°C Day/Night, ± 2°C). When the first panicle started heading, the heading date was recorded, the earliest 3-5 panicles of each plant were marked, then the pot was moved into an indoor growth chamber for high temperature treatment.
The temperature inside the IGC was programmed to simulate diurnal temperature changes, and to make sure air temperature reaches up to 38°C during the heat stress period. Four temperature regimes varying in the start time and duration of high temperature were used in subjecting treatments (Table 1). The high temperature treatment at 38 oC started (i) from 08:00 to 14:00 for 6 hours in the first IGC, (ii) from 9:00 in the second IGC for 5 hours, (iii) from 10:00 in the third IGC for 4 hours, and (iv) from 11:00 in the fourth IGC for 3 hours. Relative humidity and number of lights turned on inside the chamber were also programmed to simulate actual conditions. Relative humidity was kept at 70% when the temperature is 38 oC, and at 75% when temperature is below 38 oC. The night (dark) time was from 18:00 to 6:00.
Table 1
Environmental setting for the indoor growth chambers (IGC)
Chamber | Time duration | Temperature (°C) | Relative humidity (%) | Light intensity (µmol m-2s-1) |
| 6: 00-8:00 | 24-38 | 75 | 0-580 |
IGC1 (Treatment 08) | 8: 00-14:00 | 38 | 70 | 580 |
| 14:00-18:00 | 38-24 | 75 | 580-0 |
| 18: 00-6: 00 | 24 | 75 | 0 |
| 6: 00-9:00 | 24-38 | 75 | 0-580 |
IGC2 (Treatment 09) | 9: 00-14:00 | 38 | 70 | 580 |
| 14:00-18:00 | 38-24 | 75 | 580-0 |
| 18: 00-6: 00 | 24 | 75 | 0 |
| 6: 00-10:00 | 24-38 | 75 | 0-580 |
IGC3 (Treatment 10) | 10: 00-14:00 | 38 | 70 | 580 |
| 14:00-18:00 | 38-24 | 75 | 580-0 |
| 18: 00-6: 00 | 24 | 75 | 0 |
| 6: 00-11:00 | 24-38 | 75 | 0-580 |
IGC4 (Treatment 11) | 11:00-14:00 | 38 | 70 | 580 |
| 14:00-18:00 | 38-24 | 75 | 580-0 |
| 18: 00-6: 00 | 24 | 75 | 0 |
Ten plants of each genotype were subjected to each treatment chamber. Beginning of flower opening time (FOT) was recorded and observed by “plot” basis (or for one treatment group as a whole, not individual plants), and observed only from the glass window of the IGC to minimize microenvironment variations that may affect flowering pattern and spikelet fertility. FOT was observed for 3-5 consecutive days. After about 10 days of high temperature treatment, when the marked 3-5 panicles have completed flowering, the plants were moved back to the glasshouse and maintained until physiological maturity.
The plants of control group were remained inside the glasshouse (30°C/24°C Day/Night, ± 2°C). The heading date of each plant was recorded when it started heading, and one panicle per plant was selected and tagged for FOT observation based on a previous method (Hirabayashi et al. 2015). The number of spikelets that flowered (spikelet was open and anthers protruded from the glume) was recorded at 30-minute intervals, starting from 6:00 for the NILs carrying qEMF3 and 8:00 for other genotypes, until the end of anthesis. Observation was conducted for 3 consecutive sunny days.
At physiological maturity, three uniform panicles were collected from each plant for measuring spikelet fertility. The number of filled (full and partially filled) spikelets and empty spikelets were counted for each individual panicle. Spikelet fertility, the ratio of filled grains to the total number of spikelets in the panicle expressed as percentage, was computed for each panicle. The average spikelet fertility of the three panicles was computed.
Evaluation of spikelet fertility and agronomic traits in the field
The five genotypes (IR64, IR64HT4, IR64EMF3, and IR64HT4EMF3, and N22) were grown under field conditions in two sites in the Philippines in 2015: the International Rice Research Institute (IRRI) at Los Baños, Laguna (14° 10' 7.8"N, 121° 15' 18.8" E); and the Cagayan State University (CSU) at Piat, Cagayan (17° 47' 30.7"N, 121° 30' 58.2"E). Based on the past 30 years local temperature records, the seeding dates of the genotypes were adjusted to ensure the plants flowering during the hottest period. Seeds were germinated and sown in the seeding bed, and transplanted at a density of 20 cm x 20 cm, one seedling per hill. The size of each plot was 12 m2 (2 m x 6 m). The plots were arranged randomly with three replications. Heading date of each genotype was recorded when 50% of the panicles in the plot emerged from the flag leaf sheath.
At maturity, fifteen plants were randomly selected from the inner rows of each plot for measuring the plant height, flag leaf length, panicle length, panicle exertion, number of tillers, and three uniform panicles of each plant were harvested for counting the filled and empty grains. The spikelet fertility was calculated as the ratio of filled grains on each panicle. For grain yield computation, all panicles from an 1 m x 1 m (25 hills) area were harvested from the inner rows of each plot. The panicles were threshed, and the grains were dried. The moisture content of the grains were measured, and the potential yield of each plot was computed and converted to kilogram per hectare.
The maximum and minimum temperature data at IRRI and CSU were abstained from local weather station. The high temperature stress (maximum temperature over 35 oC lasted for at least 3 days) at IRRI started from 19 April till 4 June. While the high temperature at CSU started from 2 May till 3 July (Supplemental Figure 1).
Evaluation of flower open time in the field
The five genotypes (IR64, IR64HT4, IR64EMF3, and IR64HT4EMF3, and GIZA178) were grown in the experimental field of Yezin Agricultural University, Nay Pyi Taw, Myanmar (19° 49' 59.6"N, 96° 16' 30.4"E) in the dry season of 2015. Seeds were sown on 22 December 2014, and 21-day-old seedlings were transplanted in the field at a density of 20 cm x 20 cm. The size of each plot was 1.44 m2 (0.6 m x 2.4 m) with randomized block design for four replications per genotype. A total amount of fertilizer application is 74-20-20 kg 10a−1 for N-P2O5-K2O, respectively. Flowering pattern was observed for four days (3-6 April 2015) during heading period using four panicles per plot every day.