qEMF3, a novel QTL for the early-morning flowering trait from wild rice, Oryza officinalis, to mitigate heat stress damage at flowering in rice, O. sativa
Journal of Experimental Botany
qEMF3, a novel QTL for the early-morning flowering trait from wild rice, Oryza officinalis, to mitigate heat stress damage at flowering in rice, O.sativa
KazuhiroSasaki 1 2 7
TakashiKambe 3 8
Ritchel B.Gannaban 1
Monaliza A.Miras 6
Merlyn S.Mendioro 6
Eliza V.Simon 1
Patrick D.Lumanglas 1
DaisukeFujita 0 1
NobuyaKobayashi 1 2 3
Krishna S.V.Jagadish 1
TsutomuIshimaru 1 2 3
0 Present address: Kyushu University, faculty of agriculture , Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581 , Japan
1 International Rice Research Institute (IRRI) , DAPO Box 7777, Metro Manila , The Philippines
2 Japan International Research Centre for Agricultural Sciences (JIRCAS) , 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686 , Japan
3 NARO Institute of Crop Science , NARO, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8518 , Japan
4 Faculty of Agriculture, University of Miyazaki , 1-1 Gakuen kihanadai nishi, Miyazaki, Miyazaki 889-2192 , Japan
5 NARO Tohoku Agricultural Research Centre (TARC) , NARO, Shimo Furumichi, Daisen, Akita 014-0102 , Japan
6 University of the Philippines , Los Banos, Laguna , The Philippines
7 The University of Tokyo, Graduate School of Agricultural and Life Sciences, Institute of Sustainable Agro-ecosystem Services (ISAS) , 1-1-1 Midoricho, Nishitokyo, Tokyo 188-0002 , Japan
8 Present address: Niigata Crop Research Centre , Nagakura-machi 857, Nagaoka, Niigata, 940-0826 , Japan
A decline in rice (Oryza sativa L.) production caused by heat stress is one of the biggest concerns resulting from future climate change. Rice spikelets are most susceptible to heat stress at flowering. The early-morning flowering (EMF) trait mitigates heat-induced spikelet sterility at the flowering stage by escaping heat stress during the daytime. We attempted to develop near-isogenic lines (NILs) for EMF in the indica-type genetic background by exploiting the EMF locus from wild rice, O.officinalis (CC genome). Astable quantitative trait locus (QTL) for flower opening time (FOT) was detected on chromosome 3.AQTL was designated as qEMF3 and it shifted FOT by 1.5-2.0 h earlier for cv. Nanjing 11 in temperate Japan and cv. IR64 in the Philippine tropics. NILs for EMF mitigated heat-induced spikelet sterility under elevated temperature conditions completing flower opening before reaching 35C, a general threshold value leading to spikelet sterility. Quantification of FOT of cultivars popular in the tropics and subtropics did not reveal the EMF trait in any of the cultivars tested, suggesting that qEMF3 has the potential to advance FOT of currently popular cultivars to escape heat stress at flowering under future hotter climates. This is the first report to examine rice with the EMF trait through marker-assisted breeding using wild rice as a genetic resource.
Early-morning flowering (EMF); flower opening time (FOT); global warming; heat stress; quantitative trait locus (QTL); rice (Oryza sativa L; ); spikelet sterility
Rice (Oryza sativa L.) is one of the most important staple food
crops for nearly half the worlds population (Carriger and
Vallee, 2007). Predicted future climate change is expected to
have a negative impact on global rice production. Maximum
and minimum daily temperatures, and the number of hot
days and warm nights in a year, are estimated to increase over
most land areas (IPCC, 2013). In addition, climate
variability is predicted to increase, leading to frequent episodes of
heat stress, often coinciding with key developmental stages in
crops, such as flowering. Seasonal temperatures in the tropics
and subtropics by the end of the 21st century are predicted
to exceed the most extreme seasonal temperatures recorded
in the past (Battisti and Naylor, 2009). Breeding crops able
to cope with future climate change is a pressing requirement
to feed the growing population in the era of global warming.
High temperatures (>35C) at flowering cause spikelet
sterility in rice (Satake and Yoshida, 1978; Matsui etal., 1997).
Heat stress for 1 h induces spikelet sterility if it coincides with
flowering (Jagadish etal., 2007). Exposure of flowering
spikelets to heat stress results in the failure of anther dehiscence
and the reduced number of germinating pollen grains on
the stigma (Satake and Yoshida 1978; Matsui et al., 2001a;
Matsui and Omasa, 2002; Jagadish et al., 2010). Spikelet
sterility positively correlates with a yield reduction in
experimental paddy fields (Kim et al., 1996; Oh-e et al., 2007), in
line with the yield reduction caused by heat-induced
spikelet sterility observed in tropical Asia (Osada et al., 1973)
and Africa (Matsushima et al., 1982). In Bangladesh,
eastern India, southern Myanmar, and northern Thailand, the
flowering and early grain-filling stages of rice are predicted
to coincide with high-temperature conditions, according to
the local cropping patterns and crop calendars (Maclean
et al., 2002; Wassmann et al., 2009). In the Jianghan Basin
in China, significant losses in seed set were observed in local
hybrid rice because of hot and humid conditions during
flowering (Tian etal., 2010). Damage to rice productivity because
of heat stress at flowering has also been reported in temperate
regions of Japan: in comparison with normal summers,
spikelet sterility was significantly higher by 1520% in the summer
of 2007, when maximum temperatures reached a record level
of 40C during the flowering stage (Hasegawa et al., 2011).
Thus, heat stress at flowering poses a real threat to sustained
rice production not only in tropical and subtropical regions,
but also in temperate regions. Existing breeding programmes
are challenged to develop rice that would be heat-resilient at
flowering and could be grown across temperate, tropical, and
To mitigate heat-induced spikelet sterility, two strategies
have been proposed. One is to develop cultivars that shed
larger numbers of pollen grains or produce pollen grains able
to germinate at high temperatures. At the flowering stage,
rice cultivars have different tolerance to high temperatures
(Satake and Yoshida, 1978; Matsui et al., 2001b; Prasad
etal., 2006; Jagadish etal., 2008). Ahighly heat-tolerant
cultivar, N22 (Satake and Yoshida, 1978; Mackill et al., 1982;
Jagadish et al., 2010), has the tolerance allele qHTSF4.1, a
QTL on chromosome 4, as a major genetic factor (Ye etal.,
2012). Another strategy, which is less explored, is to breed
cultivars that escape heat at flowering because of their
earlymorning flowering (EMF) trait (Satake and Yoshida, 1978).
Spikelets are highly susceptible to heat stress at flowering;
however, they remain fertile when flowering occurs 1h prior
to heat stress, because fertilization is completed within 1 h
after the onset of flowering (Satake and Yoshida, 1978).
The EMF strategy has been used to produce an
introgression line, EMF20, with the EMF trait transferred from wild
rice, O.officinalis, into the genetic background of O.sativa cv.
Koshihikari (Ishimaru et al., 2010). EMF20 flowered a few
hours earlier than Koshihikari, which reduced heat-induced
sterility at flowering at elevated temperatures in a greenhouse
test (Ishimaru etal., 2010).
Within the genus Oryza, the flower opening time (FOT)
ranges widely from early morning to midnight. There are
many accessions in wild rice with the EMF trait (Sheehy
et al., 2005). CG14 (O. glaberrima) has been reported as an
EMF cultivar (Jagadish et al., 2008). By using backcrossed
inbred lines derived from a cross between O. rufipogon and
O.sativa, QTLs for FOT have been detected on chromosomes
4, 5, and 10 (Thanh etal., 2010). The O.rufipogon alleles of
all QTLs theoretically contributed to the 30 min advancement
of FOT in O.sativa (Thanh etal., 2010). However, the effect
of the QTLs detected is yet to be proven to be materialized
as a near-isogenic line (NIL) for EMF. Currently, there is no
material available from ongoing rice breeding programmes
that could be used to introduce a heat-escape trait to mitigate
heat-induced spikelet sterility at flowering. In addition, there
has been no systematic analysis of flowering dynamics of a
large number of popular local cultivars occupying large areas
in rice-growing regions, which would help to evaluate their
Hence, our work had three major objectives: (i) to
identify QTLs for EMF and to develop NILs for EMF by using
molecular markers and exploiting the EMF trait of the wild
rice O. officinalis; (ii) to characterize the flowering pattern
conferred by QTLs identified for EMF under various
hightemperature regimes and at different geographical locations;
and (iii) to quantify the FOT of popular cultivars grown
across tropical and subtropical regions by systematic
observation of their daily flowering patterns.
Materials and methods
Plant materials for QTL analysis
Rice plants were grown at the NARO Institute of Crop Science
(NICS), Tsukuba (36 02N, 140 10E), Ibaraki, Japan. The
introgression line, EMF20, was crossed with Nanjing 11 (Supplementary
Figure S1A) because of the similar heading date in that location.
The F2 population and self-pollinated F3 lines were derived from
a cross between EMF20 and Nanjing 11 (Supplementary Figure
S1A). The F2 seeds (in 2007) and F3 seeds (in 2008) were sown in
seedling trays and 20-day-old seedlings were transplanted (146 F2
plants and 10 F3 plants into 250-cm2 pots, one plant per pot). As
a basal dressing, a controlled-release fertilizer was applied in both
experiments, which contained N (0.5 g), P2O5 (2.3 g), and K2O (2.2 g)
in each pot. Plants were kept free from pests and diseases by
preventive application of chemicals.
FOT of F2 and F3plants
To reduce the phenological variation, a short-day treatment (light
conditions for 9 h) was imposed on Nanjing 11, EMF20, F2 plants,
and F3 lines for 2 weeks before the panicle-initiation stage. The
heading date among all the above entries was narrowed down to just
9 days (from 1 to 9 August 2007). In addition, solar radiation and
air temperature are known to strongly affect the daily flowering
pattern (Kobayashi etal., 2010; Julia and Dingkuhn, 2012), and hence
to reduce the variation induced by environmental factors, FOT was
recorded only on sunny days. Opened spikelets were visually counted
every 20 min using two panicles per plant per day and in three plants
each from EMF20, Nanjing 11, and 146 F2 populations. The
beginning of FOT (BFOT, the time when the first spikelet opened on a
given day) and the peak of FOT (PFOT, the time when the largest
percentage of spikelets were open on a given day) were recorded for
at least 2days in 2007 for each individual F2 plant; BFOT of 10 F3
progenies of each F2 plant was recorded daily in 2008 for 4 days.
EMF20 and Nanjing 11 were stagger-sown every 3days to make their
heading dates cover the entire period of heading dates of the F2 and
F3 plants. The BFOT and PFOT data were used for QTL analysis.
DNA extraction, map construction, and QTL analyses
Plant DNA was extracted from young leaves of the F2 plants and
their parents using the CTAB method (Murray and Thompson,
1980). A genetic map was developed using 154 simple sequence
repeat (SSR) markers (McCouch et al., 2002). DNA amplification
was performed for 35 cycles of 94C (1 min), 55C (2 min), and 72C
(3 min), and a final extension at 72C for 7min (PTC100, BioRad)
using Taq DNA Polymerase (Thermo Fisher Scientific, Inc.).
Amplified DNA products were electrophoresed in a 3.0% agarose
gel in 0.5TBE buffer. Analysis of linkage between SSR markers
and linkage map construction for QTL analysis were performed
with MAPMAKER/EXP 3.0 software (Lander et al., 1987). QTL
analysis was performed with composite interval mapping analysis
in the WinQTL Cartographer 2.5 software (Wang etal., 2007). The
optimal log of odds (LOD) threshold values obtained with the
permutation value set at 1000 by WinQTL Cartographer were used to
determine the presence of a putative QTL, the percentages of
variation explained by the QTL, and the additive effect.
Segregation analysis and development of NILs for the
To confirm the effect of the QTL detected (qEMF3), segregation
analysis for BFOT and PFOT was performed by using the BC3F2
population. By using SSR markers in the qEMF3 region (Fig.2A),
BC3F2 plants were classified into EMF20-homozygous, Nanjing
11-homozygous, and heterozygous. BFOT and PFOT were recorded
from 10 plants of each classified group mentioned above and
Nanjing 11 (as described in the section FOT of F2 and F3 plants)
from late July to mid-August 2009 in Japan. To develop NILs for
qEMF3, EMF20-homozygous plants for qEMF3 were selected from
BC3F2 plants by using the same SSR markers used in the
segregation analysis. From these BC3F2 plants, a plant that was Nanjing
11-homozygous for other chromosome segments was selected and
used as a NIL (Nanjing 11+qEMF3) for the following experiments.
The heading date of BC2F1 plants with heterozygous qEMF8 was
2months later than that of the recurrent parent, Nanjing 11,
resulting in a failure to proceed to the BC3F1 generation.
Nanjing 11 is well adapted to temperate rice-growing regions. To
facilitate field testing of the EMF trait in heat-vulnerable regions
in the tropics and subtropics, qEMF3 was transferred to IR64, a
popular indica-type cultivar adapted for tropical and subtropical
regions (Table3; Khush, 1987). To develop IR64+qEMF3, Nanjing
11+qEMF3 and IR64 were used as the donor and recurrent parent,
respectively (Supplementary Figure S1B). A foreground screening
of the BC1F1, BC2F1, and BC3F1 populations was conducted with
the flanking SSR markers RM 14360, RM 14374, and RM 14394 to
confirm the qEMF3 genotype (Fig.2A). Atotal of 82 SSR markers
showing polymorphism between IR64 and Nanjing 11 were used for
a background survey of 98 BC3F1 plants. An individual plant with
a clear IR64 background (except in the qEMF3 region) was selected
and self-pollinated to proceed to the BC3F2 generation. BC3F2
plants with the EMF20 alleles at flanking markers for qEMF3 were
selected and advanced to BC3F3 to obtain seeds of NILs for qEMF3
in the IR64 genetic background (IR64+qEMF3). The flowering
patterns of IR64 and IR64+qEMF3 were evaluated under flooded-field
conditions in the wet season of 2013 (for 4 days in the middle of
September) and in the dry season of 2014 (for 3days in the middle
of February) at the International Rice Research Institute (IRRI),
Los Baos, the Philippines (14 11N, 121 15E). Plants were grown
as described by Fujita et al. (2013). Spikelets that opened during
the experiment were marked using fine-tipped pens every 30min,
from 07.00 until 13.30. Four panicles from four individual plants
were used for each genotype per day. The time from dawn to 50% of
the FOT (T50) was calculated as described below to compare FOTs
between IR64 and IR64+qEMF3.
Greenhouse experiment with elevated day temperatures
Nanjing 11 and Nanjing 11+qEMF3 were used to observe
flowering patterns and to test sterility by using the method of Ishimaru
etal. (2010). The experiment was conducted in 2010 in a glasshouse
with clear-paned windows at NICS. Air temperature was manually
increased by 2.03.5C per hour after 08.00 h. Maximum
temperature reached ~40C around noon. Air temperature was monitored
every 15 min with dataloggers (SK-L200TH II, Sato Keiryoki Mfg.,
Tokyo, Japan). Opened spikelets were marked with fine-tipped pens
every hour. All pots were returned to the open-windowed greenhouse
after marking. Sterile spikelets were manually counted at maturity.
Heat tolerance test in an environmentally controlled growth
Nanjing 11, Nanjing 11+qEMF3, IR64, and IR64+qEMF3 were
grown in 10-l pots. Plants were moved into walk-in growth
chambers (3.3 3.2 2.7 m, or 10.6 m2 area), which provided a
photosynthetic photon flux density of 1000mol m2 s1 at panicle height.
For quantifying heat tolerance at flowering, heading panicles were
exposed for 6 h to 30C (control) or 38C (high temperature) at 60%
relative humidity. The temperature treatment was imposed between
07.00 and 13.00 h for Nanjing 11+qEMF3 and IR64+qEMF3, and
between 09.00 and 15.00 h for Nanjing 11 and IR64 because of the
difference in FOT between the recurrent parents and the EMF lines.
Spikelets that flowered in the growth chamber during temperature
treatment were marked, and after the end of the treatment period,
all pots were returned to the non-stress greenhouse conditions. No
spikelet flowering was observed before and after the temperature
treatment. Sterility of the marked spikelets was estimated by
manually counting the filled and unfilled spikelets.
Response of IR64 and IR64+qEMF3 to elevated high
Twelve seeds were regularly and circumferentially sown in 10-l pots
using the method of Satake (1972). Plants were trimmed to the
main stem once a week and grown in a greenhouse until the
flowering stage. The flowering pattern was investigated in the walk-in
chamber under three settings of temperature regime: (1) 25C at
06.00, linearly increasing to 40C by 14.00, then gradually
decreasing to 25C by 19.00; (2) 30C at 06.00, linearly increasing to 40C
The differences of means were analysed using the t-test or Tukey
Kramer test implemented in the R package (ver. 2.15.1).
Identification of QTLs forEMF
by 14.00 h, then gradually decreasing to 30C by 19.00 h; and (3),
as (1) except that the temperature maximum (40C) was reached
at 12.00 h. The lights were on from 06.00 h until 19.00 h; relative
humidity was constant (60%) during the light period. Two or three
panicles with the same heading date were selected per pot. Opened
spikelets were marked manually every 30 min after 06.00 h. Eight
panicles from three or four pots were used per genotype per day.
Actual values of temperature and humidity were monitored every
15 min with data loggers. After each temperature treatment, pots
were returned to the greenhouse. To acclimate the plants to
chamber conditions before the experiments, new pots were moved from
the walk-in chamber maintained at 25C or 30C to the walk-in
chamber used for this experiment at 19.00 h. No flowering was
observed before 06.00 h. The experiment was repeated for 3days for
each temperature regime.
Calculation of the beginning, peak, and end ofFOT
FOT was included in the time (hour) after dawn criterion because
of the wide range of genotype heading dates and difference in
natural day-length between wet and dry seasons. The exact time
of dawn was calculated using the Koyomi Station software on the
website of the Ephemeris Computation Office, Public Relations
Centre, National Astronomical Observatory of Japan (http://eco.
mtk.nao.ac.jp/koyomi/index.html.en). The time of dawn changed
from 06.49 to 06.05 during the experimental period (from 22
February to 3 May). The flowering pattern was presented as
percentage of opened spikelets on each day (y-axis) plotted against
time after dawn (x-axis) (Supplementary Figure S2A). Curves were
fitted by probit analysis using the R program (ver. 2.15.1) based
on the cumulative percentage of opened spikelets (Supplementary
Figure S2B and S2C). The times from dawn to 10% of the FOT,
to 50% of the FOT, and to 90% of the FOT were calculated and
defined as T10, T50, and T90, respectively. T10, T50, and T90 were
also calculated using the R program.
Fig.1. Frequency distribution of (A) BFOT and (B) PFOT for the F2
population derived from a cross between EMF20 and Nanjing 11. (C)
Frequency distribution of the averaged BFOT in F3 lines (n=10/line).
The mean BFOT SD (n=3) for EMF20 and Nanjing 11 (NJ) are shown
in (A) and (C). The PFOTs for EMF20 and Nanjing 11 are shown with
arrowheads in (B).
Chromosome Nearest- Marker LOD score
Table1. QTLs for the beginning and peak of FOT
a Additive effect of the EMF20 allele.
b Variance explained (%).
normally (Fig.1C). QTLs for BFOT were identified on
chromosome 3 (LOD score of 8.4; 19.7% of phenotypic
variation explained), chromosome 6 (LOD score of 2.5; 4.8% of
phenotypic variation), and chromosome 8 (LOD score of 5.6;
12.9% of phenotypic variation) (Table1). In both the F2 and
F3 populations, the EMF20 alleles of QTLs on chromosomes
3 and 8 advanced FOT to early in the morning; these two
QTLs were designated as qEMF3 and qEMF8 (because of the
QTL for Early-Morning Flowering), respectively.
FOT of NILs carrying qEMF3
Segregation analysis for BFOT and PFOT was performed
by using BC3F2 plants that were either EMF20 homozygous,
Nanjing 11 homozygous, or heterozygous in the qEMF3
region (Fig.2A). The BFOT of EMF20-homozygous plants
(06.54) was significantly earlier than that of Nanjing 11 (08.23;
i.e. an 89-min difference), and also earlier than that of
heterozygous plants (07.47), although the latter difference did not
reach statistical significance (Fig.2B). The BFOTs of Nanjing
11-homozygous plants (08.30) and Nanjing 11 were similar.
The PFOTs of the EMF20-homozygous (08.50), heterozygous
(09.21), Nanjing 11-homozygous (10.20), and Nanjing 11
(10.24) followed a pattern similar to that of BFOT (Fig.2C).
The results confirmed that a single QTL for EMF, qEMF3,
significantly advanced the FOT of Nanjing 11 by ~1.5h.
Nanjing 11 vs Nanjing 11+qEMF3: elevated
temperature experiment in a greenhouse and heat
To prove the effectiveness of qEMF3 in escaping heat stress
at flowering, the NIL developed (Fig. 3A) was subjected to
the elevated high temperature from early morning to noon in
the greenhouse. In this experiment, air temperature steadily
increased from ~28.0C to ~34.0C by 10.00, then exceeded
35.0C, which is generally the threshold for induction of
spikelet sterility (Satake and Yoshida, 1978; Matsui et al.,
1997), and reached 38.040.0C at 11.0015.00 (Table2). The
relative humidity steadily decreased (from >80.0% at 06.00
to ~40.0% by 15.00; Table 2). Nanjing 11+qEMF3 clearly
showed peak flowering at 07.0008.00 and completed
flowering by 11.00, whereas Nanjing 11 started flowering after
07.00, showed two peaks (09.0010.00 and 11.0012.00), and
finished flowering by 14.00 (Fig.3B). After 10.00, only 4.1%
Fig.2. (A) Graphical genotype of the short arm of chromosome 3 used
for the selection of lines for segregation analysis. Markers used for
segregation analysis for FOT in (B) and (C) are italicized. The box with
slanted lines indicates the region which showed LOD scores greater than
the threshold. *RM14407 is the nearest marker to qEMF3 (see Table1).
(B, C) Segregation analysis for FOT among selected genotypes of BC3F2
plants. Values shown are mean SE (n=10). Values labelled with the
same letters do not differ significantly. The TukeyKramer test (P<0.05)
was used for multiple comparisons between genotypes.
of spikelets in Nanjing 11+qEMF3 flowered, whereas 43.1%
of spikelets flowered in Nanjing 11. The percentage of
sterile spikelets was significantly lower in Nanjing 11+qEMF3
(1.2%) than in Nanjing 11 (49.8%) (Fig. 3C). In a chamber
experiment for heat tolerance set constantly at 38C for 6 h
on the day of flowering, spikelet sterility was similar between
Nanjing 11 (33.9%) and Nanjing 11+qEMF3 (30.3%)
(Fig. 3D). Thus, Nanjing 11+qEMF3 escaped heat-induced
spikelet sterility by flowering earlier in the morning (i.e. at
IR64 vs IR64+qEMF3: flowering patterns at elevated
We also transferred the EMF20 allele of qEMF3 into IR64
through marker-assisted selection (Supplementary Figure S1B).
Fig.3. (A) Graphical genotype of Nanjing 11+qEMF3 for the whole
genome region. White bars, Nanjing 11, homozygous; grey bars, Nanjing
11, heterozygous; black bars, EMF20, homozygous. (B) and (C) Impact of
qEMF3 on flowering pattern and spikelet sterility in the Nanjing 11 genetic
background. (B) Hourly changes in the percentage of opened spikelets on
a single day. (C) Percentage of sterile spikelets at maturity under elevated
temperature conditions in a greenhouse test (Table2). 144 spikelets from
five panicles were used for Nanjing 11 and 221 spikelets from seven
panicles for Nanjing 11+qEMF3. Error bars indicate SE; ***, significant at
the 0.1% level (t-test). (D) Percentage of sterile spikelets after temperature
treatment at flowering in a growth chamber test. Under control conditions
(30C), 107 spikelets (Nanjing 11)and 131 spikelets (Nanjing 11+qEMF3)
from five panicles were used. Under high-temperature conditions (38C),
264 spikelets (Nanjing 11)and 238 spikelets (Nanjing 11+qEMF3) from
eight panicles were used. Bars indicate SE; ns, not significant at the 5%
The graphical genotype of the NIL developed (IR64+qEMF3)
is shown in Fig.4A. Under field conditions at IRRI, mean T50
of IR64+qEMF3 was 1.9h in the wet season and 2.2h in the dry
season (Fig.4B), also confirming the effectiveness of qEMF3 in
the genetic background of IR64. These T50s were significantly
earlier (by ~2h) than those of IR64. The IR64+qEMF3 plants
were further tested in the environmentally controlled chamber to
understand the response of FOT to the elevated temperature. In
the first temperature regime, T10 of IR64 and IR64+qEMF3 was
~11.00 and ~09.30, respectively, T50 was ~11.30 and ~10.30, and
T90 was ~12.30 and ~11.00 (Fig.5A). Temperature at T90 was
~35.5C (IR64) and ~33.0C (IR64+qEMF3). In the second
temperature regime, T10 of IR64 and IR64+qEMF3 was ~10.00 and
~06.30, respectively, T50 was ~11.00 and ~07.30, and T90 was
~11.30 and ~09.30 (Fig.5B). Temperature at T90 was ~35.5C
(IR64) and ~33.0C (IR64+qEMF3). Spikelet opening started
within 30min after light exposure in IR64+qEMF3. In the third
temperature regime, which was the most severe among the three
regimes, T10 of IR64 and IR64+qEMF3 was ~10.30 and ~07.30,
respectively, T50 was ~11.00 and ~09.00, and T90 was ~12.00
and ~10.00 (Fig. 5C). Temperature at T90 was ~38.5C (IR64)
and ~34.0C (IR64+qEMF3). Spikelet sterility in the third
temperature regime was significantly lower in IR64+qEMF3 (11.2%)
than in IR64 (55.8%) (Fig.5D). Notably, the temperature at T90
for IR64+qEMF3 was below the sterility threshold (35C) in all
three temperature regimes. In the heat tolerance test constantly at
38C for 6h on the day of flowering, sterility was similar in IR64
(50.2%) and IR64+qEMF3 (57.6%) (Fig.5E).
but significantly later than that of Nanjing 11+qEMF3; T90
of Nanjing 11+qEMF3 was similar to T10 of CG14 (Fig.6).
Comparison of FOT between Nanjing 11+qEMF3 and
popular local cultivars in the tropics and subtropics
The mean T50 ranged from 3.4 to 4.8 h after dawn (HAD)
among popular cultivars (Table3) in the dry season (Fig.6).
Swarna, an Indian cultivar, had the earliest T50 among the
tropical and subtropical cultivars. Nanjing 11 and IR64
were placed in the early-T50 group. Caiapo, an upland
cultivar from Latin America, had the latest T50 (3 h later than
Nanjing 11+qEMF3). The mean T50 of Nanjing 11+qEMF3
was 1.8 HAD, which was significantly earlier than those
of all tested cultivars (Fig. 6). The difference between T10
and T90 varied among the accessions: the difference was
>1.5 h for Nanjing 11, TDK1, KDML105, Epagri108, Pusa
Basmati, and Caiapo, and was less for Ciherang and Sahel
329. For Nanjing 11+qEMF3, the mean T10 was 1.1 HAD
and the mean T90 was 2.5 HAD; this T90 was earlier than
T10 of all other cultivars. This result indicates that Nanjing
11+qEMF3 had almost completed flowering prior to the
beginning of FOT of the tested popular cultivars. The T50 of
CG14 was 40 min to 2 h earlier than those of other cultivars
Table3. Cultivars and lines used in this study
We attempted to develop NILs for EMF in indica-type genetic
backgrounds through the detection of QTLs using a wild rice,
O.officinalis, as a donor parent, aiming for genetic improvement
of FOT to the cooler early morning. Heat-induced spikelet
sterility is expected to aggravate rice yield losses in vulnerable
temperate, subtropical, and tropical regions. We also investigated the
FOTs of cultivars popular in tropical and subtropical
rice-growing regions to identify the presence or absence of the EMF trait
for the broader application of QTLs detected to rice breeding.
qEMF3 contributes to heat escape at flowering by
advancing FOT to early morning
To date, there has been only one attempt to detect QTLs
for EMF derived from O. rufipogon (AA genome; Thanh
et al., 2010); this study detected QTLs for BFOT on
chromosomes 5 and 12, and QTLs for PFOT on chromosomes
4 and 5. However, the effects of these QTLs have not been
Main countries of cultivation
Name of cultivar or line
The flowering patterns of CG14 were observed in 2013.
Fig.6. Comparison of time to 10% (T10), 50% (T50) and 90% (T90) after
dawn in Nanjing 11+qEMF3 and popular rice cultivars grown in the tropics
and subtropics. Grey bars indicate time from T10 to T90 for each cultivar.
T10, T50, and T90 calculations are shown in Supplementary Figure
S2. The same letters on the left side of the cultivar names indicate no
significant difference in T50. The TukeyKramer test (P<0.05) was used
for multiple comparisons between groups.
functionally validated through the development of NILs,
which makes it difficult to use these QTLs in breeding
programmes. In our study, QTLs for BFOT and PFOT derived
from O. officinalis were found on chromosomes 3 (qEMF3)
and 8 (qEMF8) (Table1); thus, we consider them to be
different from those identified by Thanh etal. (2010). We
confirmed the allelic effect of EMF20 on qEMF3 for EMF in
the BC3F2 population (Fig. 2B, C) and developed NILs for
EMF in the genetic background of Nanjing 11 (Fig. 3A)
and IR64 (Fig.4A). Comparison of FOT between the
recurrent parents and the NILs revealed that the EMF20 allele of
qEMF3 advanced FOT by 1.52.0 h both in temperate Japan
(Fig.2B, C) and the tropical Philippines across different
seasons (Fig. 4B), indicating that qEMF3 advances the FOT
under various environmental conditions.
Our previous study revealed that EMF20, an introgression
line containing both qEMF3 and qEMF8, avoids heat-induced
spikelet sterility at flowering under an elevated temperature in
the greenhouse (Ishimaru etal., 2010). In this study, we found
two QTLs for the EMF trait, qEMF3 and qEMF8 (Table1).
We tested the hypothesis that a single QTL, qEMF3, is effective
in avoiding heat stress at flowering using Nanjing 11+qEMF3
and IR64+qEMF3. By 10.00, when the temperature exceeded
35C (Table2), over 95% of spikelets had completed flowering
in Nanjing 11+qEMF3 but only 57% in Nanjing 11 (Fig.3B).
As a result, spikelet sterility was significantly reduced in
Nanjing 11+qEMF3 (Fig.3C), although sterility was similar
in Nanjing 11+qEMF3 and Nanjing 11 in the heat tolerance
test (Fig.3D). This result supports our hypothesis that qEMF3
is sufficient to advance the FOT of Nanjing 11, which allows
this cultivar to escape heat stress during the daytime. Using
an environmentally controlled chamber and three different
temperature regimes, we confirmed that almost all spikelets
of IR64+qEMF3 flowered before the temperature reached
35C, whereas the percentage of spikelets that opened at
temperatures below 35C varied in IR64 depending on the
temperature regime (Fig.5AC). This difference in the flowering
pattern significantly reduced spikelet sterility in IR64+qEMF3
(Fig.6D). The results clearly show that qEMF3 has a similar
effect on FOT in the IR64 background. Thus, qEMF3
contributes to heat escape at flowering by advancing FOT. It is
notable that qEMF3 shifted its FOT drastically responding to
the given temperature regimes (Fig.5AC). The NILs
developed could be a novel material for revealing the interaction of
genetic and environmental control of FOT.
qEMF3 has the potential to shift the FOT of cultivars
popular in the tropics and subtropics to earlier in the
The vulnerability of the major rice-producing regions to
heat-induced spikelet sterility at the flowering stage has been
mapped both regionally (Wassmann etal., 2009) and globally
(Teixeira etal., 2013). Although major local cultivars listed in
Table3 occupy a large proportion of rice cultivation areas in
the tropics and subtropics, no assessment of their FOT traits
has been conducted so far. Our investigation of the
flowering patterns revealed that none of them had the EMF trait
(Fig. 6). Among them, Nanjing 11 and IR64 were
categorized in the early-FOT group (Fig. 6). The advancement in
FOT due to qEMF3 in the genetic background of Nanjing 11
(Fig.2B, 2C; Fig.7) and IR64 (Fig.4B) shows that qEMF3
has a significant impact even on the early-FOT cultivars.
Heat-induced spikelet sterility occurs even upon
exposure of a flowering spikelet to a short-term heat stress (1h at
~37C; Jagadish etal., 2007). However, similar heat exposure
1 h after flowering hardly affects fertility (Satake and Yoshida,
1978; Ishimaru etal., 2010). Therefore, advancing FOT by 1 h
results in a significant difference in the proportion of sterile
spikelets in rice under heat stress. Among the cultivars tested,
Caiapo had the latest FOT, 3 h later than Nanjing 11+qEMF3
(Fig.6). Two-thirds of the cultivars tested had their T50 2 h
later than Nanjing 11+qEMF3 (Fig. 6), and these cultivars
should be prioritized as targets for breeding programmes for
EMF through the introgression of qEMF3. The fact that
none of the popular cultivars tested, including CG14, which
is known as an EMF cultivar (Jagadish etal., 2008), had the
same EMF trait as Nanjing 11+qEMF3 shows a pressing
need to transfer qEMF3 to those cultivars by marker-assisted
breeding. The allele of the EMF20 locus from O. officinalis
has a potential to shift the FOT of these cultivars to a time
earlier in the morning when the temperature is lower. Thus,
the wild rice, O.officinalis, could provide an excellent genetic
resource for the EMF trait to mitigate heat-induced spikelet
sterility at flowering, which would help in breeding rice
cultivars able to cope with future hotter climates.
Supplementary Table1. Meteorological data inside the
greenhouse at IRRI in the 2012 and 2013 dry season.
Battisti DS , Naylor RL . 2009 . Historical warnings of future food insecurity with unprecedented seasonal heat . Science 323 , 240 - 244 .
Carriger S , Vallee D. 2007 . More crop per drop . Rice Today 6 , 10 - 13 .
Fujita D , Trijatmiko KR , Tagle AG , etal. 2013 . NAL1 allele from a rice landrace greatly increases yield in modern indica cultivars . Proceedings of the National Academy of Sciences , USA 110 , 20431 - 20436 .
Hasegawa T , Ishimaru T , Kondo M , Kuwagata T , Yoshimoto M , Fukuoka M. 2011 . Spikelet sterility of rice observed in the record hot summer of 2007 and the factors associated with its variation . Journal of Agricultural Meteorology 67 , 225 - 232 .
IPCC. 2013 . Fifth Assessment Report (AR5 ) . WMO/UNEP Ishimaru T, Hirabayashi H , Ida M , Takai T , San-Oh YA , Yoshinaga S , Ando I , Ogawa T , Kondo M. 2010 . A genetic resource for earlymorning flowering trait of wild rice Oryza officinalis to mitigate high temperature-induced spikelet sterility at anthesis . Annals of Botany 106 , 515 - 520 .
Jagadish SVK , Craufurd PQ , Wheeler TR . 2007 . High temperature stress and spikelet fertility in rice ( Oryza sativa L.). Journal of Experimental Botany 58 , 1627 - 1635 .
Jagadish SVK , Craufurd PQ , Wheeler TR . 2008 . Phenotyping parents of mapping populations of rice for heat tolerance during anthesis . Crop Science 48 , 1140 - 1146 .
Jagadish SVK , Muthurajan R , Oane R , Wheeler TR , Heuer S , Bennett J , Craufurd PQ . 2010 . Physiological and proteomic approaches to address heat tolerance during anthesis in rice ( Oryza sativa L.). Journal of Experimental Botany 61 , 143 - 156 .
Julia C , Dingkuhn M. 2012 . Variation in time of day of anthesis in rice in different climatic environments . European Journal of Agronomy 43 , 166 - 174 .
Khush GS . 1987 . Rice breeding: Past, present and future . Journal of Genetics 66 , 195 - 216 .
Kim HY , Horie T , Nakagawa H , Wada K. 1996 . Effects of elevated CO2 concentration and high temperature on growth and yield of rice . II. The effect on yield and its components of Akihikari rice . Japanese Journal of Crop Science 65 , 644 - 651 .
Kobayashi K , Matsui T , Yoshimoto M , Hasegawa T. 2010 . Effect of temperature, solar radiation, and vapor-pressure deficit on flower opening time in rice . Plant Production Science 13 , 21 - 28 .
Lander ES , Green P , Abrahamson J , Barlow A , Daly MJ , Lincoln SE , Newburg L. 1987 . MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations . Genomics 1, 174 - 181 .
Mackill DJ , Coffman WR , Rutger JN . 1982 . Pollen shedding and combining ability for high temperature tolerance in rice . Crop Science 22 , 730 - 733 .
Maclean JL , Dawe DC , Hardy B , Hettel GP . 2002 . Rice almanac: Source book for the most important activity on earth . The Philippines: International Rice Research Institute.
Matsui T , Omasa K. 2002 . Rice (Oryza sativa L.) cultivars tolerant to high temperature at flowering: Anther characteristics . Annals of Botany 89 , 683 - 687 .
Matsui T , Omasa K , Horie T. 1997 . High temperature-induced spikelet sterility of Japonica rice at flowering in relation to air temperature, humidity and wind velocity conditions . Japanese Journal of Crop Science 66 , 449 - 455 .
Matsui T , Omasa K , Horie T. 2001a . Comparison between anthers of two rice (Oryza sativa L.) cultivars with tolerance to high temperature at flowering or susceptibility . Plant Production Science 4 , 36 - 40 .
Matsui T , Omasa K , Horie T. 2001b . The difference in sterility due to high temperatures during the flowering period among japonica-rice cultivars .
Plant Production Science 4, 90 - 93 .
Matsushima S , Ikewada H , Maeda A , Honma S , Niki H. 1982 . Studies on rice cultivation in the tropics . 1. Yielding and ripening responses of the rice plant to the extremely hot and dry climate in Sudan . Japanese Journal of Tropical Agriculture 26 , 19 - 25 .
McCouch SR , Teytelman L , Xu Y , etal. 2002 . Development and mapping of 2240 new SSR markers for rice ( Oryza sativa L.). DNA Research 9 , 199 - 207 .
Murray MG , Thompson WF . 1980 . Rapid isolation of high molecular weight plant DNA . Nucleic Acids Research 8 , 4321 - 4325 .
Oh-e I , Saitoh K , Kuroda T. 2007 . Effects of high temperature on growth, yield and dry-matter production of rice grown in the paddy field .
Plant Production Science 10 , 412 - 422 .
Osada A , Sasiprapa V , Rahong M , Dhammanuvong S , Chakrabandhu H. 1973 . Abnormal occurrence of empty grains of indica rice plants in the dry, hot season in Thailand . Proceedings of the Crop Science Society of Japan 42 , 103 - 109 .
Prasad PVV , Boote KJ , Allen LH , Sheehy JE , Thomas JMG . 2006 .
Species, ecotype and cultivar differences in spikelet fertility and harvest index of rice in response to high temperature stress . Field Crops Research 95 , 398 - 411 .
Satake T. 1972 . Circular dense-culture of rice plants in pots, the purpose of obtaining many uniform panicles of main stems . Proceedings of Crop Science Society of Japan 41 , 361 - 362 .
Satake T , Yoshida S. 1978 . High temperature-induced sterility in indica rices at flowering . Japanese Journal of Crop Science 47 , 6 - 17 .
Sheehy J , Elmido A , Centeno G , Pablico P. 2005 . Searching for new plants for climate change . Journal of Agricultural Meteorology 60 , 463 - 468 .
Teixeira EI , Fischer G , Velthuizen HV , Walter C , Ewert F. 2013 . Global hot-spots of heat stress on agricultural crops due to climate change .
Agricultural and Forest Meteorology 170 , 206 - 215 .
Thanh PT , Phan PDT , Mori N , Ishikawa R , Ishii T. 2010 . QTL analysis for flowering time using backcross population between Oryza sativa Nipponbare and O.rufipogon. Genes and Genetic Systems 85 , 273 -279 Tian X , Matsui T , Li S , Yoshimoto M , Kobayasi K , Hasegawa T. 2010 .
Heat-induced floret sterility of hybrid rice (Oryza sativa L.) cultivars under humid and low wind conditions in the field of Jianghan Basin , China. Plant Production Science 13 , 243 - 251 .
Wang S , Basten CJ , Zeng ZB . 2007 . Windows QTL Cartographer 2 . 5 .
Wassmann R , Jagadish SVK , Heuer S , Ismail A , Redona E , Serraj R , Singh RK , Howell G , Pathak H , Sumfleth K. 2009 . Climate change affecting rice production: The physiological and agronomic basis for possible adaptation strategies . Advances in Agronomy 101 , 59 - 122 .
Ye C , Argayoso MA , Redoa ED , etal. 2012 . Mapping QTL for heat tolerance at flowering stage in rice using SNP markers . Plant Breeding 131 , 33 - 41 .