Plant Science 174 (2008) 618–625

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Plant Science
journal homepage: www.elsevier.com/locate/plantsci

QTLs affecting morph-physiological traits related to drought tolerance detected in

overlapping introgression lines of rice (Oryza sativa L.)
Xiu-Qin Zhao a,b,1, Jian-Long Xu a,b,1, Ming Zhao a, Renee Lafitte b,2, Ling-Hua Zhu a, Bin-Ying Fu a,b,
Yong-Ming Gao a,b, Zhi-Kang Li a,b,*
a
Institute of Crop Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China
b
International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines

A R T I C L E I N F O A B S T R A C T

Article history: Plant photosynthetic traits such as net photosynthetic rate (Pn), stomata conductance (gs), transpiration
Received 28 September 2007 rate (Tr), and intercellular CO2 concentration (Ci), are known to relate to drought tolerance in plants, but
Received in revised form 2 February 2008 the genetic basis of these traits remains largely uncharacterized because of the difficulty in phenotyping
Accepted 19 March 2008
physiological traits in a large mapping population. In this study, a set of 55 overlapping introgression
Available online 26 March 2008
lines (ILs) in the Teqing (indica) background were used to genetically dissect several morph-physiological
traits and their relationship with grain yield under water stress and non-stress conditions. These traits
Keywords: included specific leaf weight (SLW), chlorophyll content (CC), leaf stomata frequency (SF), Pn, gs, Tr, and Ci.
Rice
A total of 40 QTLs affecting the measured traits were identified and mapped to 21 genomic regions in the
Drought tolerance
rice genome. Clustered QTLs affecting Pn, gs, Tr, and Ci in the same genomic regions suggest common
Photosynthesis
QTLs genetic bases for the physiological traits. Low or no phenotypic correlations between leaf morphological
traits and photosynthetic traits and between morph-physiological traits and grain yield (GY) appeared to
be due to inconsistence in QTL effect for clustered QTLs, unlinked QTLs affecting different traits, and to
possible epistasis that could not be adequately addressed in this study. Our results indicate that
improving drought tolerant (DT) of rice by selecting any single secondary traits is not expected to be
effective and the identified QTLs for GY and related morph-physiological traits should be carefully
confirmed before to be used for improving DT in rice by MAS.
ß 2008 Published by Elsevier Ireland Ltd.

1. Introduction
The increasing threat from water shortage and drought in many
rice-growing areas of Asia, particularly the rainfed areas, has posed
a great challenge to rice breeders to develop drought tolerant (DT)
and/or water-saving rice cultivars. As a result, DT and water use
efficiency (WUE) have become two priority target traits in many
breeding programs of Asia [1]. During the past decade, tremendous
efforts have been taken in identifying and mapping genes/QTLs
affecting traits related to DT in order to facilitate marker-assisted
selection (MAS) for genetic improvement of DT in rice [1–3].
However, despite a large number of DT QTLs identified, progress in
* Corresponding author at: Institute of Crop Sciences/National Key Facility for
Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural
Sciences, 12 South Zhong-Guan-Cun Street, Beijing 100081, China.
Tel.: +86 1062136040; fax: +86 1068918559.
E-mail addresses: lizhk@caas.net.cn, zhkli@yahoo.com.cn (Z.-K. Li).
1
These authors contributed equally to this work.
2
Current address: Pioneer Hi-Bred International, 18285 County Road, 96,
Woodland, CA 95616, USA.
breeding for DT by MAS has been slow. Part of the reason is that DT
in rice, when defined as grain yield under drought is very complex
both genetically and physiologically [4,5]. Thus, it was suggested
that deliberate selection for secondary traits related to DT is likely
to achieve better results than direct selection for yield per se under
stress [6–8].
Classical studies have generated significant amounts of
information regarding the physiological traits related to DT in
plants. Of these traits, photosynthesis traits are among the most
important factors influencing biomass and yield in rice and are
known to have high heritability [9–11]. Under drought stress, rice
photosynthetic capacity decreased as a result of stomata closure to
prevent water losses, which is an early and efficient event in plant
response to water deficit [12–14], resulting in reduced assimilation
rate and yield [15]. Identification of QTL conditioning the net
photosynthetic rate (Pn), stomata conductance (gs) and transpira-
tion rate (Tr) is a logic step to facilitate MAS for improving yield of
rice under drought stress. Unfortunately, few studies in this regard
have been reported, partly because measuring leaf instant Pn, gs
and Tr under the field conditions is greatly influenced by sampling
environments and thus requires more replications in order to

0168-9452/$ – see front matter ß 2008 Published by Elsevier Ireland Ltd.

doi:10.1016/j.plantsci.2008.03.009
 X.-Q. Zhao et al. / Plant Science 174 (2008) 618–625
obtain accurate data. This creates a major problem in phenotyping
a typical mapping population of 200 individuals or lines for these
physiological traits.
Three morphological traits, stomata frequency (SF), specific leaf
weight (SLW), and leaf chlorophyll content (CC) are also known to be
associated with Pn, gs and Tr and supposed to be less affected by the
sampling environments. SF is known to be able to influence gs and
thus Pn [16–18]. Under drought, the negative effect of reduced gs on
plant growth can be partially compensated by increased SF [19,20].
The variation of leaf SF is an adaptive feature of plants to ensure an
optimum gs and assimilation rate to maintain the maximum CO2
fixation, which is known as one of the ‘‘gain and loss’’ compensation
mechanics in crop plants [21]. SLW is reportedly another factor
influencing photosynthesis. Genotypes with higher SLW tend to
have greater photosynthesis [22], which may allow plants to better
adapt to water stress conditions. High SLW also enables greater
carbon gain by reducing transpiration losses under drought [12]. For
example, cotton cultivars with high SLW is reportedly associated
with increased yield in the dryland conditions [23]. Moreover, SLW
was highly correlated with WUE in cotton, which appeared to be
responsible for higher yield under water stress [24]. Thus, it is
suggested that selection for increased yield may be achieved by
indirect selection for high SLW [25,26]. CC is reportedly to be
correlated significantly with Pn and is one of the key components
involved in the photosynthetic process [9,27]. Down-regulation of
photosynthesis under stress was also associated with decrease in CC.
A low CC reduces light absorbance and thus the heating effect from
high light intensities, which is always accompanied by drought
stress. Moreover, lower CC in plants is associated with a higher lipid/
protein ratio which can increase lipid fluidity of the thylakoid
membrane, and thus increases DT [28]. Despite the known
influences of above-mentioned morph-physiological traits on DT
and photosynthesis in rice, little is known on the genetic bases of
these traits and their associations with grain yield under water stress
and non-stress conditions in rice.
Here, we report an effort to genetically dissect QTLs affecting
the above-mentioned morph-physiological traits, characterize
their relationships with grain yield under drought stress and
non-stress conditions using a unique set of overlapping introgres-
sion lines (ILs).
2. Materials and methods
2.1. Plant materials
A large set of 254 advanced BC introgression lines (ILs) were
developed by crossing Teqing (the recurrent parent) with Lemont
(the donor) followed by 2–4 times of backcrossing and 3–5 times
of selfing [29,30]. Teqing is a high yielding Chinese indica rice
cultivar with a moderate level of DT, whereas Lemont is a japonica
variety from Southern US which has a higher photosynthetic
capacity [31] and is susceptible to drought [32]. The 254 ILs were
genotyped with 160 well-distributed polymorphic single
sequence repeat (SSR) markers and a complete linkage map
was constructed, as previously described [29]. From the 254 ILs,
we selected a subset of 55 ILs as the materials for this study based
on the following criteria: (1) each IL has minimum donor (Lemont)
genome; (2) each introgressed donor segment was partially
shared by at least two ILs such that the 55 ILs together contain the
whole donor genome in overlapping segments; (3) all ILs had
similar heading date under the normal irrigated conditions so that
environmental influences in phenotyping physiological traits
from the variation in heading date of the ILs were minimized. The
parents were used as control in the experiments to monitor the
severity of drought stress.
619
2.2. Phenotyping experiments and data collection
The ILs were evaluated in two replicated experiments under the
water stress (rainfed upland) and fully irrigated lowland (non-
stress) conditions in the experimental farm of the International
Rice Research Institute (IRRI) during the 2002 wet season (June–
November). In the upland field, seeds were directly sown into two-
row plots on the raised beds with a spacing of 25 cm  20 cm in a
randomized block design with an incomplete block arrangement in
three replications. Water was supplied by the sprinkler irrigation
for the initial 60 days of the season to maintain the normal growth
of rice plants. Irrigation was then completely stopped until the
maturity. During this period of time, a total of 726 mm of rainfall
was recorded and plants suffered severe yield loss because the
uplands could not hold the water. In the irrigated lowland control,
seeds of the ILs were sown in the seedbed and 20-day seedlings
were transplanted into three-row plots (36 plants per plot) at a
spacing of 25 cm  20 cm. The field was managed according to the
standard procedures, with a basal fertilization rate of 30 kg ha 1
for each of N, P, and K, and two additional 30 kg ha 1 N
applications made at the 44 and 66 days after sowing. Weeds in
both the lowland and upland fields were controlled by a
combination of chemical and manual methods. Insects (particu-
larly stem-borers) were controlled chemically.
In the upland field, the middle parts of three fully expanded flag
leaves on the main culms of three middle plants in each plot were
measured at the flowering time for Pn, gs, Tr and Ci from 9:00 a.m.
to 11:30 a.m. under the sunny condition (1500 mol m 2 s 1 of
photosynthetic active radiation, 30 8C of the leaf chamber
temperature, and 350 cm3 m 3 of CO2 concentration) using a
portable Li-COR6400 Photosynthesis System. CC were measured
on the three last fully expanded flag leaves of the main stems from
three different plants in each plot at the booting stage in both the
lowland (non-stress) and upland (water stress) experiments using
a SPAD-502 chlorophyll meter (Spectrum Technologies Inc.). CC
was presented as the SPAD meter reading. SF was measured using
the imprint method [33] in which stomata numbers on the middle
parts (0.5  0.5 = 0.25 cm2 on the right side of the leaf main vein)
of three sampled flag leaves from each IL were counted in two
vision fields under the plan-NEOFLUAR 40*/0.75 lenses of the
microscope (Axioskop, IEISS, West Germany). SF was expressed as
the average stomata number per vision field. In addition, three
fully expanded flag leaves of the main stems of three different
plants in each plot was sampled and measured for leaf area (LA)
using a LI-3000 leaf area meter (LI-COR Inc., Lincoln, NE, USA).
After that, the sampled leaves were dried in an oven at 70 8C for
72 h and the dry weight of the leaves from each IL was recorded.
Specific leaf weight was then calculated as the dry leaf weight (in
mg) per cm2 of LA. Because of the damages of some ILs from the
tongro disease, the data of four photosynthesis traits (Pn, Tr, gs, and
Ci) and grain yield (GY) in the non-stress lowland experiment in
2002 were inaccurate and thus were excluded from the data
analyses.
The 55 ILs were again evaluated in two replicated experiments
under fully irrigated (non-stress) and water stress conditions in
2003 dry season to measure GY, as previously described [29]. Seeds
of the ILs were sown in the seedbed and 30-day seedlings were
transplanted into three-row plots (36 plants per plot or entry) with
a spacing of 25 cm  20 cm in a randomized block design with an
incomplete block arrangement in three replications in 2003. The
field was managed in the same way as the experiments conducted
in 2002. For the stress treatment, the field was drained at 60 days
after transplanting and no further irrigation was applied. This
treatment resulted in leaf rolling by 15 days after the field was
drained. By the date of heading in the control plots, soil moisture in
620 X.-Q. Zhao et al. / Plant Science 174 (2008) 618–625

the stress plots reached 100 kPa at 15-cm depth. Grain yield (g/m2)
was recorded by harvesting all plants in each plot [29].

2.3. Data analyses and QTL identification

Analysis of variance was performed to evaluate differences

among the ILs, between the parents and the conditions (water
irrigation and water stress) using the SAS PROC GLM [34]. Correlation
between the measured traits in each of the conditions and between
lines for the same traits across the conditions was determined using
the SAS PROC CORR [34]. The average values of the measured traits of
the ILs obtained from the non-stress and stress conditions were used
as input data to identify QTL affecting the target traits by one-way
Fig. 1. Distribution of the Lemont genome in the 55 and 254 Teqing introgression
ANOVA using SAS PROC GLM [34]. A probability level of P  0.005 lines (ILs).
was used for claiming a significant QTL. When a QTL was detected by
two or more linked markers, the one with the highest F value was
presented. Since the use of a single arbitrary threshold in QTL all measured traits in the parents, particularly for GY, which
mapping could easily detect a QTL in one environment but not in decreased by 93.8% for Lemont and 89.9% for Teqing, respectively.
another [29], putative QTLs detected by the minimum threshold of The parents differed significantly for Pn, Tr, SLW, CC and GY under
P  0.05 in one environment will also be reported as long as they water stress, but only for CC and GY under the non-stress
reached the selected threshold of P  0.005 in another. condition. Under water stress, Lemont had significantly higher Pn
and Tr, and significantly lower SLW, CC, and GY than Teqing. Under
3. Results
the non-stress condition, Teqing had significantly higher CC and GY
than Lemont. The 55 ILs showed transgressive segregations
3.1. Genetic constitution of the 55 ILs
towards both directions for all measured traits under stress and
non-stress conditions. ANOVA based on the data from the stress
The 55 ILs together carry a total of 695 homozygous segments
conditions indicated that the variation among ILs accounted for the
and 52 heterozygous introgressed segments from Lemont with a
most of the phenotypic variation for Pn (R2 = 82.0%), gs (R2 = 77.7%),
total length of 15569.6 cM, or 9.3 times as much as the Lemont
Tr (R2 = 79.1%), and Ci (R2 = 82.9%). ANOVA results indicated that
genome-equivalent. The proportions of the Lemont genome in the
highly significant differences were detected between the stress
55 ILs were normally distributed with an average 15.2% per IL,
conditions for SF (R2 = 10.6%), SLW (R2 = 17.2%) and CC (R2 = 63.5%),
ranging from 3.5 to 32.1% (Fig. 1). The average number and length
among the ILs for SF (R2 = 50.8%), SLW (R2 = 48.9%) and CC
of homozygous and heterozygous donor segments per line were
(R2 = 18.2%), and the stress by IL interaction for SF (R2 = 29.5%),
13.6 and 37.4 cM, respectively. The overlapping introgressed
SLW (R2 = 26.8%), CC (R2 = 7.9%), and GY (R2 = 10.8%). As compared
segments cover the whole Lemont genome (Fig. 2). The flowering
to the normal condition, SF, SLW, CC and GY of the ILs decreased
time of the 55 ILs under the lowland conditions was very uniform,
significantly under drought, but this reduction, though significant,
ranging from 86 to 89 days.
was much more pronounced for GY than for the morph-
3.2. Trait variation and correlations physiological traits measured. The average reduction in the 55
ILs was similar to that in the parents for all measured traits except
Table 1 shows the summary statistics of the parents and ILs for for SF and SLW, which were affected little by drought, and showed
the measured traits. Water stress caused significant reductions for a very high level of genotype by environment interaction.

Table 1
Phenotypic performance for flag leaf net photosynthetic rate (Pn, mmol CO2 m 2 s 1), stomatal conductance (gs, mol H2O m 2 s 1), transpiration rate (Tr, mmol H2O m 2 s 1),
intercellular CO2 concentration (Ci, mmol CO2 mol 1), stomata frequency (stomata number per vision field), SLW (mg cm 2) of leaf area, CC (SPAD reading) and grain yield
(g m 2) of the 55 introgression lines (ILs) evaluated under the irrigated lowland (control) and/or rainfed upland (stress) conditions

Treatment Traits Lemont Teqing Teqing ILs

Mean  S.D. CV (%) Range

Stress Pn 24.4** 20.2 19.7  3.7 18.9 10.4–6.7

gs 0.17 0.14 0.15  0.04 23.7 0.06–0.21
Tr 5.15* 4.46 4.94  1.05 21.2 2.23–6.65
Ci 112.0 102.6 140.7  41.1 29.2 55.2–232.0
SF 61.5 59.9 70.0  12.3 16.1 47.5–103.5
SLW 4.57 5.84* 5.58  0.73 13.2 3.53–7.24
CC 32.4 38.0* 37.4  3.1 8.3 29.8–44.5
GY 43.4 83.4*** 70.9  56.6 79.9 0.0–236.3

Control SF 72.6 70.1 68.0  13.5 19.8 47.0–111.0

SLW 5.82 6.27 6.48  1.08 16.7 4.14–10.09
CC 39.4 45.9* 46.4  2.5 5.5 38.5–51.7
GY 705.3 822.4* 776.3  280.5 36.1 188.1–1342.4

Differencea SF 11.1*** 10.2*** 1.8  14.7 – 36.5–26.0

SLW 1.25*** 0.43*** 0.89  1.12 – 5.08–2.39
CC 7.0** 7.9** 9.2  3.1 – 16.7–2.1
GY 661.9*** 739.0*** 719.5  290.9 – 1298.8–175.9

*, ** and *** represent significantly different levels at P < 0.05, 0.01 and 0.001, respectively.
a
Difference = Stress control for all measured traits of the individual IL.
 X.-Q. Zhao et al. / Plant Science 174 (2008) 618–625 621

Fig. 2. Overlapping introgressed segments (the thick black lines on the right of each chromosome) in the 55 Teqing introgression lines and QTL for grain yield (GY), net
photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (Tr), intercellular CO2 concentration (Ci), stomatal frequency (SF), specific leaf weight and chlorophyll
content (CC) detected under irrigated and/or water stress conditions.

Under water stress, there was a very high and positive
correlation (r = 0.92, P < 0.0001) between gs and Tr in the 55 ILs
(Table 2). Pn was moderately and positively correlated with gs, Tr
and SLW (r = 0.65, 0.62 and 0.35, P < 0.0001). Ci was moderately
and positively correlated with gs and Tr (r = 0.63 and 0.66,
P < 0.0001). GY was weakly and positively correlated with CC,
but not with any other traits. Interestingly, there was weak and
positive correlation between the trait values of CC (r = 0.40,
P < 0.001), SF (r = 0.29, P < 0.01) and SLW (r = 0.26, P < 0.05)
measured under the stress and non-stress, indicating that the ILs
behaved similarly for the three traits under stress and non-stress
conditions.
3.3. QTL affecting morph-physiological traits
A total of 14 QTLs affecting Pn, gs, Tr and Ci were identified under
the stress condition (Table 3, Fig. 2). These included five QTL (qPn2,
qPn5, qPn10, qPn11 and qPn12) for Pn, which were mapped to
chromosomes 2, 5, 10, 11 and 12 and collectively explained 39.9%
of the total phenotypic variation of the trait. The Lemont alleles at
qPn2 and qPn11 increased Pn, but decreased Pn at the other three
loci. Two QTL (qGs1 and qGs11) for gs were mapped to
chromosomes 1 and 11, which collectively explained 14.9% of
the total trait variation. The Lemont allele at qGs1 decreased gs but
increased gs at qGs11. Three QTL (qTr1, qTr5 and qTr11) affecting Tr
622 X.-Q. Zhao et al. / Plant Science 174 (2008) 618–625

Table 2
Correlation coefficients between specific leaf weight (SLW), chlorophyll content (CC), stomatal frequency (SF), net photosynthetic rate (Pn), stomatal conductance (gs),
transpiration rate (Tr) and intercellular CO2 concentration (Ci) evaluated at the booting/flowering stages under irrigated (control) and/or upland (stress) conditions in the 55
Teqing ILs

Stress Control

GY SLW CC SF Pn gs Tr Ci GY SLW CC

Stress
SLW 0.24
CC 0.26* 0.25*
SF 0.13 0.03 0.04
Pn 0.21 0.35** 0.05 0.05
gs 0.02 0.21 0.23 0.04 0.65**
Tr 0.02 0.22 0.21 0.04 0.62** 0.92***
Ci 0.22 0.06 0.23 0.01 0.16 0.63*** 0.66***

Control
GY 0.18 0.02 0.33** 0.13 0.02 0.19 0.21 0.17
SLW 0.04 0.26* 0 0.11 0.09 0.05 0.05 0.27* 0.01
CC 0.19 0.31** 0.40*** 0.02 0.12 0.03 0.03 0.04 0.22 0.24
SF 0.07 0.18 0.06 0.29** 0.09 0.02 0.04 0.12 0.05 0.23 0.16

*, ** and *** represent the levels of significance at P < 0.05, 0.01 and 0.001, respectively.

were identified on chromosomes 1, 5 and 11, which together
explained 25.7% of the total phenotypic variation. The Lemont
alleles at the two loci (qTr1, qTr5) were associated with decreased
Tr while associated with increased Tr at qTr11. Four QTL for Ci (qCi3,
qCi7, qCi8 and qCi9) were mapped to chromosomes 3, 7, 8 and 9,
which collectively explained 52.8% of the total trait variation. The
Lemont alleles at all these loci increased Ci.
Eight QTLs affecting SF were identified and mapped to
chromosomes 3, 4, 7, 10 and 11, including three QTLs (qSf4, qSf7b
and qSf11) that expressed under both stress and non-stress
conditions, four QTLs (qSf3a, qSf3b, qSf7a, and qSf10a) that
expressed specifically under the control condition, and a single
QTL (qSf10b) which expressed specifically under water stress.
These QTLs collectively explained 66.4% and 40.0% of total
phenotypic variation for SF under stress and non-stress conditions,
respectively (Table 3, Fig. 2). The Lemont alleles at all loci except
qSf7a and qSf10b increased SF.
Six QTLs affecting SLW were identified, including two QTLs
(qSlw7 and qSlw8) that expressed under both stress and non-stress
conditions, two QTLs (qSlw3 and qSlw12) that expressed specifi-
cally under the irrigated condition, and two QTLs under (qSlw5 and
qSlw10) that were induced specifically under water stress.
Together, these QTLs explained 48.8% and 36.6% of total trait
variation under stress and non-stress conditions, respectively
(Table 3, Fig. 2). The Lemont allele at qSlw12 increased SLW while
the Teqing alleles at the remaining five QTLs increased SLW.
Five QTLs affecting CC were identified, including two QTLs (qCc8
and qCc12) that expressed specifically under the non-stress
condition and three QTLs (qCc3, qCc6 and qCc11) that were
induced by water stress (Table 3, Fig. 2). These QTLs collectively
explained only 14.6% and 28.6% of the total trait variation under
the control and stress conditions, respectively. The Lemont alleles
at all loci except qCc12 were associated with decreased CC.
3.4. QTLs affecting GY
A total of seven QTLs affecting GY were detected and mapped to
rice chromosomes 3, 5, 7, 8, 9, 10, and 12, including three QTLs (qGy5,
qGy8 and qGy10) that expressed under both stress and non-stress
conditions, three QTLs (qGy3, qGy9 and qGy12) that expressed only in
the control condition and 1 QTL (qGy7) that expressed specifically
under the stress. Collectively, these QTLs explained 57.0% and 39.2%
of the trait variation under the non-stress and stress conditions,
respectively. The Teqing alleles at qGy3, qGy5, qGy9, qGy10 and
qGy12 increased GY and the Lemont allele at qGy7 increased GY.
Interestingly, the Lemont allele at qGy8 increased GY under stress
but decreased GY under the control condition.
4. Discussion
In this study, we deliberately selected 55 ILs from a larger IL
population based on known marker and phenotypic information
such that the mapping population had a manageable size and
allowed more timely and accurate measurements of the studied
traits in the replicated experiments. The tremendous segregation
for the measured morph-physiological traits in the ILs indicated
that this subset of ILs had sufficient amounts of genetic variation
for mapping QTLs affecting the measured traits. The small
experimental errors from our ANOVA results clearly indicated
that we were successful in controlling the micro-environmental
noises on the measured morph-physiological traits in our field
experiments. The 55 overlapping ILs covers the whole donor
genome, which minimized the chance of the main-effect QTLs
escaping from incomplete coverage of the donor genome, which
was proven to be true according to our mapping results for a highly
heritable trait, 1000-grain weight, in which a total of 7 grain
weight QTLs were identified in the same genomic regions under
water stress condition in both the whole population of 254 ILs and
the subset population of 55 ILs (Table 4). Similarly, 60% of the GY
QTLs detected in the 254 ILs [29] were also identified in the 55 ILs,
further demonstrating that the 55 ILs had sufficient power to
detect most QTLs segregating in the original population. We noted
that the statistic powers associated with the detected grain weight
QTLs were significantly lower in the small subpopulation than that
in the big population. However, our overlapping IL population
should be much more powerful in detecting QTLs than commonly
used overlapping chromosomal single-segment substitution lines
[35–37], because this population had, on average, eight more
replications for any specific genomic regions across the genome
and can even detect some of digenic interactions of large effect.
Evidence for this argument came from the fact that 11 of the 19 QTL
regions (Fig. 2) affecting the morph-photosynthesis traits identi-
fied in this study matched closely to previously identified QTLs for
the same or related traits in different mapping populations[16,38–
40], suggesting our mapping results were quite robust.
As expected, GY and its related morph-physiological traits
changed dramatically under water stress, reflecting important
aspects of plants to adapt to the stress. It is well known that GY in
rice tends to show considerable G  E interactions [41,42]. Some
changes in morph-physiological traits caused by water stress are
 X.-Q. Zhao et al. / Plant Science 174 (2008) 618–625 623

Table 3
Forty QTL affecting flag leaf net photosynthetic rate (Pn, mmol CO2 m 2 s 1), stomatal conductance (gs, mol H2O m 2 s 1), transpiration rate (Tr, mmol H2O m 2 s 1),
intercellular CO2 concentration (Ci, mmol CO2 mol 1), stomata frequency (mean stomata number per vision field), SLW (mg cm 2), chlorophyll content (SPAD meter reading)
and grain yield (g m 2) detected at the booting/flowering stages in 55 Teqing introgression lines under irrigated (control-C) and/or upland (stress-S) conditions

Trait QTL Ch. Marker interval Control Stress QTL and population reporteda
b 2 2
F A R (%) F A R (%)

Pn qPn2 2 RM324-RM145 11.18 1.93 11.2

qPn5 5 OSR35-RM159 10.37 1.53 9.3
qPn10 10 RM258-RM228 10.47 1.71 8.8 GY5 (C-)
qPn11 11 RM209-RM229 12.99 0.52 1.5 GY5 (C-, S-)
qPn12 12 OSR20-RM277 10.51 1.53 9.1 CC4, GY5 (C-)

gs qGs1 1 OSR27-RM212 9.42 0.01 7.2 Tr1, SF3, GY5 (C-)

qGs11 11 RM209-RM229 8.57 0.01 7.7 GY5 (C-, S-)

Tr qTr1 1 OSR27-RM212 10.64 0.38 7.7 Tr1, SF3, GY5 (C-)

qTr5 5 OSR35-RM159 6.68 0.40 8.5
qTr11 11 RM209-RM229 8.11 0.46 9.5 GY5 (C-, S-)

Ci qCi3 3 RM148-RM227 16.87 38.4 19.9 Pn1, Rubisco/CC4

qCi7 7 RM478-RM234 7.81 26.1 9.9
qCi8 8 RM137-RM72 7.22 16.3 9.4 CC1, gs1, Photo capacity4
qCi9 9 RM242-RM278 11.94 22.0 13.6

SF qSf3a 3 RM168-OSR31 7.20 4.15 6.9 SF3, CC4

qSf3b 3 RM148-RM227 17.20 8.98 17.9 Pn1, Rubisco/CC4
qSf4 4 RM252-RM303 9.48 6.60 8.5 13.12 5.25 11.1 Pn 1
qSf7a 7 RM481-RM436 7.77 6.00 4.4 CC1, Tr 2
qSf7b 7 RM118-RM18 12.99 13.65 11.4 12.08 9.48 10.3
qSf10a 10 RM311-RM239 7.60 5.37 7.1 gs1, Ci1
qSf10b 10 RM258-RM228 9.45 3.71 7.8 GY5 (C-)
qSf11 11 RM209-RM229 11.25 5.71 10.2 12.76 4.31 10.8 GY5 (C-, S-)

SLW qSlw3 3 RM148-RM227 12.32 0.53 12.7 Pn2, Rubisco/CC4

qSlw5 5 OSR35-RM159 11.65 0.35 11.7
qSlw7 7 RM118-RM18 9.75 0.80 8.2 9.07 0.31 7.8
qSlw8 8 RM126-RM483 7.51 0.43 7.8 10.24 0.31 8.9
qSlw10 10 RM258-RM228 9.76 0.26 8.2 GY5 (C-)
qSlw12 12 OSR20-RM277 17.73 0.98 20.1 CC4, GY5 (C-)

CC qCc3 3 RM36-RM282 10.88 3.83 11.9 CC2

qCc6 6 RM3-RM141 7.28 1.22 7.3 Pn 2
qCc8 8 RM126-RM483 10.44 1.14 5.7
qCc11 11 RM123-RM224 9.87 1.49 9.4
qCc12 12 OSR20-RM277 8.05 1.18 8.9 CC4, GY5 (C-)

GY qGy3 3 RM148-RM227 9.40 185.1 13.0 Pn2, Rubisco/CC4

qGy5 5 RM509-RM163 7.82 120.0 14.9 10.20 27.9 5.8 GY5 (C-, S-)
qGy7 7 RM478-RM234 12.17 57.0 13.1 GY5 (S+)
qGy8 8 RM137-RM72 13.24 145.1 9.1 10.18 31.6 10.7 CC1, gs1, Photo capacity4
qGy9 9 RM242-RM278 11.00 124.2 4.1 GY5 (C-)
qGy10 10 RM258-RM228 8.78 108.9 8.3 8.79 29.2 9.6 GY5 (C-)
qGy12 12 RM309-RM463 10.48 145.7 7.6
a
1–5 in superscript indicate cases where QTL were previously detected in the populations of Asominori  IR24 CSSL (Hu et al., 2007), JYQ8  JX17 DH [39],
Nipponbare  Kasalath BIL [16], Nipponbare  Kasalath BIL [38] and 254 Teqing ILs [29], respectively. C and S represent the 254 Teqing ILs evaluated under the control and
stress conditions with + and representing the directions of the QTL additive effects identified at the Lemont allele.
b
QTL additive effects were associated with the Lemont allele.

potentially able to contribute to GY, even though genetic evidence measured morph-physiological traits were significantly sup-
for this association has been particularly lacking. Thus, traits pressed by drought, and SF and SLW showed considerable G  E
showing greater G  E interactions across the stress and non-stress interaction across the stress and non-stress conditions, suggesting
conditions are expected to contribute more to GY. In this study, all they were important components of GY. In contrast, the large

Table 4
The comparison of QTLs for grain weight (GW) detected in 55 and 254 ILs from Lemont/Teqing under water stress condition in 2003 dry season

QTL Ch. Marker interval P55 P254

F a F a

qGw3 3 RM85-RM148 10.70 0.85 19.29 0.70

qGw4 4 RM551-RM417 14.28 0.78 15.65 0.47
qGw5 5 RM289-RM249 16.52 0.45 54.26 0.90
qGw6 6 RM30-RM439 8.96 0.70 89.78 1.69
qGw8 8 RM210-OSR7 14.25 1.05 28.96 0.89
qGw9 9 RM278-OSR28 14.64 0.79 43.60 1.03
qGw12 12 RM235-RM17 14.76 1.42 53.62 1.63
624 X.-Q. Zhao et al. / Plant Science 174 (2008) 618–625
change in CC caused by drought appeared to be more responsive
instead of adaptive to the stress because of this trait did not vary
much across the stress and non-stress conditions.
Similar to many previous studies, most QTLs detected in this
study tended to be clustered in certain genomic regions across the
rice genome (Fig. 2). In particular, QTLs affecting Pn, gs, Tr, and Ci
tended to cluster in the same genomic regions, suggesting their
common genetic bases (pleiotropy) for the observed high and
positive phenotypic correlations between the physiological traits.
In addition, the SLW QTLs were mapped three times with the SF
QTLs (qSf3, qSf7, and qSf10), twice with the CC QTLs (qCC8 and
qCC12) and the Pn QTLs (qPn5 and qPn10). The SF QTLs were
located twice in the Pn QTLs (qPn10 and qPn11). In these cases, the
QTL effects on the morphological traits and physiological traits
were often in different directions, suggesting tight linkage instead
of pleiotropy was more likely responsible for the observed
associations, even though the resolution of our genetic mapping
could not clearly distinguish one from the other. The genomic
correspondence and the inconsistent relationship in effect of the
QTL clusters provided a reasonable explanation for the low
correlation between the leaf morphological traits (SLW, SF and
CC) and photosynthesis traits (Pn, gs, Tr and Ci) observed in this
study.
Our results provided direct evidence that GY under stress were
genetically associated with all measured morph-physiological
traits except CC. This was reflected by the high genomic
correspondence between QTLs affecting the morph-physiological
traits and GY (Fig. 2). For example, five (qGy3, qGy7, qGy8, qGy9,
and qGy10) of the seven GY QTLs were mapped to the same
genomic locations as QTLs affecting one or more morpho-
photosynthesis traits. Of these, two QTL regions were particularly
interesting. The first one was flanked by RM148 and RM227 on
chromosome 3, where QTLs affecting Ci, SF, SLW and GY were
mapped together. The Lemont allele at this QTL region was
associated with increased Ci and SF, but decreased SLW and GY.
We further note that QTL for Pn, Rubisco content per chlorophyll
content were also mapped to the same region in different rice
populations previously [38,39], the second one was flanked by
RM258 and RM228 on chromosome 10, where QTLs affecting Pn,
SF, SLW and GY were mapped together. The Lemont allele at this
region was associated with reduced Pn, SF, SLW and GY (Fig. 2 and
Table 3). In three remaining regions on chromosomes 7, 8 and 9,
QTLs for Ci and GY were mapped together. In the former two cases,
the Lemont alleles were associated with increased Ci and GY under
stress. However, in the latter case (chromosome 9), it increased Ci
but reduced GY. This kind of inconsistence between GY QTLs and
component trait QTLs explained at least partially the observed
complex phenotypic relationships between GY and its component
traits. In two additional regions on chromosomes 11 (between
RM209 and RM229) and 12 (between OSR20 and RM277), clusters
of QTL affecting multiple photosynthesis traits were mapped with
GY QTL that were detected in the 254 ILs [29]. In both cases, the
Lemont alleles for increased photosynthesis traits were asso-
ciated with reduced GY under either stress and/or non-stress
conditions. Surprisingly, none of the CC QTLs were mapped with
the GY QTLs detected under stress even though there was a weak
phenotypic correlation between GY and CC. This conflict reflects
the complex genetics underlying GY because epistasis, which is an
important genetic basis of GY in rice [43,44], could not be
adequately addressed in the 55 ILs. In conclusion, our results
indicate that improving DT of rice by selecting any single
secondary traits is not expected to be effective and identified
QTLs for GY and related morph-physiological traits should be
carefully confirmed before to be used for improving DT in rice by
MAS.
Acknowledgements
The authors are grateful to the financial support from Chinese
Ministry of Agriculture (948 # 2006-G51), the National 973 project
of Chinese Ministry of Science and Technology (grant no.
2003CB114308) and from the Rockefeller Foundation (#2005
FS029) to Z.K. Li. Q.X. Zhao was also supported by a Ph.D. fellowship
from the Rockefeller Foundation.
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