Journal of Experimental Botany, Vol. 57, No. 11, pp. 2815–2824, 2006
doi:10.1093/jxb/erl046 Advance Access publication 25 July, 2006
RESEARCH PAPER
Heavy genetic load associated with the subspecific
differentiation of japonica rice (Oryza sativa ssp.
japonica L.)
Jian-Long Xu1,2,*, Jun-Min Wang2,*, Ye-Qing Sun3,*, Li-Jun Wei3, Rong-Ting Luo2, Ming-Xian Zhang2 and
Zhi-Kang Li1,4,†
1
3
4
Harbin Institute of Technology, Harbin 150001, PR China
International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines
Received 13 February 2006; Accepted 18 May 2006
Abstract
Introduction
Genetic load in the genome of the model species, rice,
was genetically dissected by mapping quantitative
trait loci (QTLs) affecting the radiosensitivity of 226 recombinant inbred lines (RILs) to c-ray- and spaceflightinduced radiation. The parents and RILs varied
considerably in their radiosensitivity to c-ray irradiation. A total of 28 QTLs affecting the two index traits,
seedling height (SH) and seed fertility (SF), of radiosensitivity were identified. The japonica parent, Lemont, was much more sensitive to c-ray irradiation than
the indica parent, Teqing, and its alleles at almost all
QTLs were associated with increased radiosensitivity,
suggesting a much higher genetic load in the japonica
genome of rice. Six QTLs (QSh2a, QSh2b, QSh5a,
QSh7, QSf3b, and QSf10b) were located in the genomic
regions particularly sensitive to radiation and thus
might represent possible ‘mutation hot spots’ in the
japonica genome. Detailed characterization of these
genomic regions may shed light on the evolution and
subspecific differentiation of rice.
Genetic load, originally defined as the total amounts of
potentially deleterious mutations in the genome of an organism (Muller, 1950), has been of tremendous interest to
geneticists and evolutionary biologists because of its association with the adaptability of the organism to changing
environments. Most plants contain a significant portion
(more or less) of mutated genes in their genomes that are
potentially harmful or even deleterious. Genetic load is
believed to have played an important role in evolution and
contributed frequently to the extinction of small populations (Kimura et al., 1963; Lynch et al., 1995). Because of
their polyploid nature and the redundancy in gene copy
number and function, most plants may not show reduced
fitness despite the genetic load they carry. In plants, genetic
load tends to be associated with outbreeders as compared
with inbreeders because many deleterious mutations are
masked in the heterozygous state (Wiens et al., 1987). The
number of deleterious mutations and the genetic load in
an organism’s genome are difficult to measure directly
because of the complexity of the genome. However, the
amount of genetic load in a species’ genome can be indirectly measured by its radiosensitivity, which is defined
as the observable damage in fitness-related traits caused by
mutagenic treatments. It is well known that radiosensitivity
differs greatly among different plant species, subspecies,
Key words: Genetic load, mutagenesis, g-rays, radiation
damage, radiosensitivity, spaceflight.
* Jian-Long Xu, Jun-Min Wang, and Ye-Qing Sun contributed equally to this work.
y
To whom correspondence should be sent. E-mail: [email protected] or [email protected]
Abbreviations: SF, seed fertility; SH, seedling height; QTL, quantitative trait locus; RAPD, randomly amplified polymorphic DNA; RFLP, restriction fragment
length polymorphism; SSR simple sequence repeat.
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
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Institute of Crop Sciences/The National Key Facility for Gene Resources and Genetic Improvement,
Chinese Academy of Agricultural Sciences, 12 South Zhong-Guan-Cun Street, Beijing 100081, PR China
2
Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, PR China
2816 Xu et al.
observed in the treated materials, and these mutagenic
effects derived from the treated plant seeds were apparently
heritable (Bayonove et al., 1984; Kostina et al., 1984; Mei
et al., 1994, 1998; Liu, 2000). Stable and promising lines
with improved performance have been identified from the
progeny of rice subjected to spaceflight and wheat seeds
(Cyranoski, 2001; Dennis and Ding, 2002; Liu et al., 2004),
even though the mechanism(s) of mutagenesis of the space
environment on plants remain unclear.
In the present study, the QTL mapping strategy was
adopted to dissect genetically the amounts of genetic load
in the rice genome that are responsible for the differential
responses of two rice subspecies to c-ray irradiation and
space environment treatments. The results provided insights into the genomic distribution and magnitudes of
genetic load associated with the subspecific differentiation
of rice.
Materials and methods
Experimental materials
Lemont (Oryza sativa ssp. japonica), a commercial semi-dwarf rice
variety from southern USA, was used as the female parent to cross
with Teqing (O. sativa ssp. indica), a high-yielding semi-dwarf
variety from China. Two hundred and fifty-five F2 individuals were
generated from the cross (Li et al., 1999) and allowed to self
consecutively until F10, from which 292 recombinant inbred lines
(RILs) were generated by single-seed descent (Xu et al., 2004).
The parents, Lemont and Teqing, together with a random subset of
226 RILs were used as the materials in this study.
Induction treatment and data collection
Three hundred purified air-dried seeds from each of the RILs were
divided into three equal parts. The first part was irradiated with
300 Gy of c-rays at an exposure rate of 1.4 Gy minÿ1 from a 60Co
source located in the Zhejiang Academy of Agricultural Sciences,
China. The second part was carried by the Chinese spacecraft ‘ShenZhou 4’ launched in December of 2002 and travelled in a low-Earth
orbit for 162 h. In the spacecraft, 100 seeds of each RIL were fixed
with two layers of a plastic holder (6 cm35 cm) and sandwiched
between the nuclear track detectors that were used to measure the
high energy cosmic rays around the seeds. The environmental
parameters of the spacecraft included a flight altitude of 200–
400 km, 638 inclination, 15–26 8C inner temperature, 1.5310ÿ4 g
microgravity, and 1.58 mGy of total radiation detected by an
integrating thermal luminous dosimeter (TLD). It was estimated
that all seeds were hit at least once by the moderate energy (Z/b >20)
cosmic ray particles, and ;80% of the seeds were hit at least once
by the high energy (Z/b >50) cosmic ray particles based on the records on the nuclear track detectors. The third part of the seeds was
kept as the untreated control. Here Z/b is an exploration parameter for measuring the level of energy in which Z is the atomic
number of incident particles, and b is the ratio of the velocity of
incident particles to the velocity of light.
To measure the radiosensitivity of the RILs, all the treated and
control RILs were sown in the concrete beds of the greenhouse in
the Zhejiang Academy of Agricultural Sciences, Hangzhou, China
during the summer of 2003 with three replications for each RIL/
treatment. One week after sowing, 20 seedlings from each treatment
were randomly selected to measure seedling height (SH, cm). Then,
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 27, 2014
and even among different genotypes of the same species
(Sparrow and Evans, 1961; Ukai, 1967; Ukai and Yamashita,
1980), indicating a tremendous variability in plants regarding genetic load in their genomes. In applied biology,
particularly in agriculture, mutation breeding, i.e. generating mutations using chemical or physical treatments
followed by selection for heritable changes of specific
phenotypes, has achieved considerable success in genetic
improvement of crop plants (Micke et al., 1985; Liu et al.,
2004). Crop plants have been classified into three types, i.e.
sensitive, intermediately sensitive, and non-sensitive based
on their sensitivity to radiation (Takagi, 1974). Further
studies indicated that radiosensitivity of several higher
plants (tomato, peas, and rice) to c-ray irradiation is
a heritable trait (Davis, 1962; Blixt, 1969; Ukai, 1970),
which is either under the control of Mendelian factors
(Yamashita, 1964; Takaki and Yamashita, 1967) or of
polygenic nature (Davis, 1962; Lawrence, 1963; Al-Rubeal
and Godward, 1981). Thus, genetic load in plants can be
dissected regarding its distribution and effects at individual
genomic regions using the conventional quantitative trait
locus (QTL) mapping approach (Paterson et al., 1988;
Lander and Botstein 1989).
Among many methods used to produce mutagenesis,
c-ray irradiation is the most common way to generate
mutations in plants. It is well known that c-ray treatment
tends to cause random deletions of large genomic segments
and serious phenotypic damage in treated plants, which are
linearly related to the treatment dosage (Soriano, 1961;
Nuclear Institute for Agriculture and Biology, 2003). Thus,
the sensitivity of a plant to c-ray irradiation provides an
indirect measurement of the genetic load in its genome. The
more sensitive to c-ray irradiation a plant is, the larger the
genetic load it has in its genome.
Recently, treating plants in the space environment has
become an increasingly common method of mutagenesis as
part of many countries’ space ambitions since the 1960s
(Halstead and Dutcher, 1987; Horneck, 1992). Although
not fully understood, the space radiation environment
consists of a complex mixture of galactic cosmic radiation,
trapped belt radiation, and solar particles (Badhwar, 1997).
Of these, charged particles are the most important constituent of the radiation in the manned spacecrafts flying in
a near-Earth orbit (;400 km). Various biological effects on
treated plants from exposure of the seed to space radiation,
especially to the high-energy cosmic rays, have been
documented, which include chromosomal aberrations
(Krikorian and O’Connor, 1984; Slocum et al., 1984),
developmental abnormalities (Bayonove et al., 1984;
Kuang et al., 1996), and increased mutation rates (Kostina
et al., 1984; Mei et al., 1994). In China, a total of 16
batches of biological materials (including dry seeds of
different plant species, micro-organisms, etc.) have been
sent into space in retrievable satellites and spacecrafts since
1987. Morphological mutations have been consistently
Characterization of genetic load in rice
30-d-old seedlings were transplanted into three-row plots at a spacing
of 25320 cm in the field (36 plants per plot for each of the treated
and untreated RILs) with three replications for each of the RILs. The
field was managed under standard procedures with three sprays
of insecticides to control brown planthoppers (Nilaparvata lugens
Stål.). At maturity, 15 main panicles were randomly sampled from
15 plants in each plot and the total number of spikelets per panicle
was counted, which included the number of filled grains and that of
unfilled grains. The seed fertility (SF, in %) was measured as the ratio
of filled grains to the total number of spikelets per panicle.
Data analysis and QTL mapping
Analysis of variance (ANOVA) was performed to evaluate differences in the induction treatments, among the RILs, and genotype by
treatment interactions for the measured traits using SAS PROC GLM
(SAS Institute, 1996). Least significant difference (LSD) tests were
performed to compare the differences between the radiation-treated
lines and untreated control for the parents and all RILs. Correlation
between the traits in both treatments and between lines for the same
traits across the treatments was determined using SAS PROC CORR
(SAS Institute, 1996).
Phenotypic data of the RILs, obtained from the control and two
induction treatments, as well as trait differences between the treatments and control (treatment–control) of the RILs were used as input
data to identify QTLs affecting SH, SF, and trait differences using the
software QTLMAPPER v. 1.0 based on the mixed model approach
(Wang et al., 1999). Specifically, significant markers (QTLs)
associated with mean trait values of individual RILs were identified
using stepwise regression with a threshold of P <0.005. Then, QTL
parameters (locations, effects, and test statistics) of all putative QTLs
were estimated using interval mapping and the restricted maximum
likelihood estimation method, with all significant markers identified
in the first step fixed in the model to control the background genetic
variation. The permutation method (Churchill and Doerge, 1994) was
used to obtain empirical thresholds for claiming QTLs of the
experiment based on 1000 runs of randomly shuffling the trait values,
which ranged from 2.50 for SF under the spaceflight treatment to 3.10
for SH in the controls.
The use of a single arbitrary threshold in QTL mapping could
easily detect a QTL in one treatment but not in another (Li et al.,
2003). Thus, while all QTLs detected at the selected thresholds are
presented, any QTLs detected in only one treatment was interpreted
with caution. Also, to examine the extent to which inconsistent QTL
detection across the treatments actually arose from type II errors, all
QTLs identified in one treatment were re-examined using the data
from the other treatments and the trait differences under the minimum
threshold of P <0.05. In other words, when a QTL was identified
using the data from the control experiment, this QTL was also tested
by the data from the spaceflight, c-ray irradiation, and differences
between the treatments and control, and vice versa. The test statistics
and QTL parameters associated with the QTL were also reported as
long as the QTL reached the minimum threshold.
Results
Performance of the parents and RILs after
induced treatments
No significant differences between the parents were
observed for SH and SF after the spaceflight (Table 1). cray irradiation resulted in a significant reduction of 50% in
SH and of 46% in SF for the japonica parent Lemont, but
caused no apparent damage in the indica parent, Teqing.
The RILs showed tremendous segregation for the two
measured traits and differences between the induction
treatments and control, which were approximately normally
distributed (Table 2, Fig. 1), indicating that the responses
of the RILs to the radiation treatments behaved like quantitatively inherited traits. ANOVA indicated highly significant differences among the treatments (R2=34.3 and 48.2%
for SH and SF), among the RILs (R2=20.4 and 18.1%), and
the genotype by treatment (R2=22.2 and 12.5%). c-ray
irradiation caused significantly reduced SH in 167 lines
(74.2%) and significantly decreased SF in 141 lines
(74.2%), resulting in a general reduction of 49.5% in SH
and of 54.4% in SF of the RILs. Only two lines showed
significantly increased SH and one line showed significantly increased SF after the c-ray treatment. By contrast,
spaceflight caused no shift in the mean SH of the RILs
(Table 1; Fig. 1), but 13 of the RILs (5.8%) showed
significantly improved seedling growth and 17 lines (7.5%)
showed significantly reduced seedling growth, as compared
with the controls. Similar to SH, spaceflight caused no
change in the mean SF of the RILs (Table 1; Fig. 1), but 12
of the RILs (5.5%) showed significantly improved SF and
nine lines (4.1%) had significantly reduced SF.
Table 1. Performance of seedling height (SH) and seed fertility (SF) of the parents and the Lemont/Teqing RILs after different
induction treatments
All values given are the means 6SD. Significance level of ***P <0.001 based on t-tests between the treatments and the control.
SH (cm)
Lemont
Teqing
RILs
SF (%)
Control
Spaceflight
c-rays
Control
Spaceflight
c-rays
7.660.6
11.060.5
9.162.4
9.460.6
9.961.0
9.362.0
3.860.5***
9.160.8
4.662.2***
89.262.6
92.961.3
80.1611.7
88.064.8
93.961.8
79.5612.8
48.167.0***
89.167.1
36.5615.6***
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Linkage map construction
Genotyping of the RILs with molecular markers and the construction
of a complete linkage map were carried out in several steps, as
described by Xu et al. (2004). The linkage map which was used for
QTL analysis in this study (Fig. 2) consists of 164 well-distributed
markers [40 restriction fragment length polymorphism (RFLP)
markers, 100 simple sequence repeat (SSR) markers, 21 randomly
amplified polymorphic DNA (RAPD) markers, and three morphological markers]. This map covered the whole rice genome with a
total length of 1921 cM and an average distance of 11.7 cM between
adjacent markers.
2817
2818 Xu et al.
Table 2. Comparison of seedling height (SH) and seed fertility (SF) between spaceflight- and c-ray-treated Lemont/Teqing RILs and
untreated controls based on ANOVA/LSD tests
Treatment
Spaceflight
c-rays
SH
SF
No. of lines
evaluated
No. of lines with
significantly increased
trait value
No. of lines with
significantly decreased
trait value
No. of lines
evaluated
No. of lines with
significantly increased
trait value
No. of lines with
significantly decreased
trait value
226
225
13
2
17
167
219
190
12
1
9
141
P1-γ
P2-γ
70
60
P1-C
50
P1-S
60
P2-S
50
40
40
30
1.0
2.4
-2.7
7.7
3.8
-0.4
-4.6
-44.3
-13.1
-6.0
-54.7
-1.8
-7.4
-65.1
-23.5
-88.8
-75.5
-3.2
-10.2
12.4
11.2
10.0
8.8
7.6
6.4
5.2
4.0
2.8
0
1.6
10
0
0.4
20
10
-85.9
30
20
P1-S P2-S
80
140
70
P1-C
60
P2-γ
50
P2-C
120
100
P1-γ
80
40
18.1
94.9
86.3
77.7
69.1
60.5
51.9
43.3
0
34.7
20
0
26.1
10
17.5
40
8.9
20
-33.9
60
30
control
spaceflight treatment
γ-irradiation treatment
Fig. 1. Frequency distribution of seedling height (SH) and seed fertility (SF) detected under the control, spaceflight, and c-radiation treatments, and
their differences between the treatments and the control in the Lemont (P1)/Teqing (P2) RILs.
Correlation among tested traits after different induction
treatments in the RILs
The space-induced SF was highly and positively correlated
with the SF of the control (r=0.81, P <0.0001), indicating
that spaceflight had minimum effects on SF (Table 3). High
and positive correlation (r=0.83, P <0.0001) was also
observed between c-ray-induced SF and its effects (trait
difference) on SF, indicating that the damaging effect of
c-ray irradiation was quite consistent across all RILs. For
SH, moderate and positive correlation (from 0.38 to 0.58)
was observed between spaceflight or c-ray irradiation
treatment and the control, and between the two treatments,
indicating there was some degree of similarity in the
seedling responses of the RILs to the two radiation treatments (Table 3). Partial and negative correlation (r=ÿ0.46
and ÿ0.52) existed between the controls and the effects of
radiation treatments on SH, indicating that lines with faster
seedling growth tended to suffer less damage from the
radiation treatments.
Identification of QTLs associated with radiosensitivity
For SH, 18 QTLs were identified and mapped to 10 rice
chromosomes except chromosomes 10 and 12, including
eight detected in the untreated RILs, 13 detected in the
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P2-C
Characterization of genetic load in rice
2819
Table 3. Correlation coefficients among seedling height (SH), seed fertility (SF), and differences betwen treatments and control (DSH
and DSF) in Lemont/Teqing RILs after different induction treatments
Significance levels of *P <0.05, **P <0.01, and ***P <0.001, respectively. The upper data are the correlation coefficients, and the lower data are the
mean degrees of freedom for each pair of correlations. Superscripts C, S and c on the right of the trait abbreviations represent control, spaceflight, and
c-ray irradiation treatments, respectively.
SHC
SFC
SHS
SFS
SHc
SFc
DSHS
DSFS
DSHc
SFC
0.14*
223
0.58***
226
0.08
220
0.38***
225
ÿ0.01
192
ÿ0.46***
226
ÿ0.09
219
ÿ0.52***
225
ÿ0.10
191
0.10
223
0.81***
219
0.17*
222
0.13
190
ÿ0.05
223
ÿ0.14*
219
0.02
222
ÿ0.44***
190
0.10
220
0.40***
225
0.09
192
0.46***
226
0.01
219
ÿ0.13*
225
0.06
191
0.19**
219
0.09
187
0.04
220
0.47***
219
0.10
219
ÿ0.38***
187
0.04
192
0.03
225
0.04
218
0.60***
225
0.02
191
0.09
192
ÿ0.08
186
0.04
192
0.83***
191
0.11
219
0.42***
225
0.14
191
0.11
218
0.06
186
0.09
191
SH
S
SFS
SH
c
SFc
DSH
S
DSFS
DSH
c
DSFc
c-ray-treated RILs, nine detected in the spaceflight-treated
RILs, and 12 by the trait differences between the treated and
untreated lines (Table 4; Fig. 2). Based on their differential
behaviours, these QTLs could be classified into seven
types. Type 1 included QSh5a that was only detected in
the control but not under the two treatments. Type 2 included QSh2c, QSh3b, QSh4a, QSh6, QSh8a, and QSh9,
which were induced specifically by c-ray irradiation. Type 3
included QSh8b which was induced specifically by spaceflight. Type 4 included QSh3c and QSh11b that were
detected in both the control and c-ray irradiation-treated
groups. Type 5 included QSh3a, QSh5b, and QSh11a
detected in both the control and spaceflight groups. Type 6
included QSh1and QSh4b that were detected in both the
control and the two treatment groups. Type 7 included
QSh2a, QSh2b, and QSh7 that were apparently induced by
both treatments. Twelve QTLs (QSh1, QSh2a, QSh2b,
QSh2c, QSh3b, QSh4b, QSh6, QSh7, QSh8a, QSh8b, and
QSh9) contributed to SH differences of the RILs between the
two treatments and control. All these QTLs except type
6 were expected to have arisen from the parental allelic
diversity in radiosensitivity. Specifically, types 2, 3, and
7 represented QTLs at which the parental allelic differences were specifically induced by either or both induction
treatments, and types 1, 4, and 5 represented QTLs where the
parental allelic differences were specifically removed
by either or both induction treatments. The Lemont alleles
at all 18 loci resulted in reduced SH or increased SH
sensitivity.
For SF, 10 QTLs were identified and mapped to seven
rice chromosomes, including eight detected in the control,
four by c-ray irradiation, five by spaceflight, and five by the
trait differences between the radiation treatments and
control (Table 4; Fig. 2). These included QSf3b and
QSf10b of type 1 that were detected only in the control,
QSf6 and QSf12a of type 2, which were induced by c-ray
irradiation, QSf9 of type 4 that were expressed in the
control and c-ray irradiation-treated groups, QSf3a, QSf8,
QSf10a, and QSf12b of type 5 that were expressed in the
controls and those undergoing spaceflight, and QSf7 of type
6 that were expressed in the control and after treatments.
The Lemont alleles at all these loci except QSf7 under c-ray
irradiation were associated with reduced fertility or increased SF sensitivity. Five QTLs (QSf6, QSh10a, QSh12a,
QSf7, and QSh9) contributed to SF differences of the RILs
between the radiation treatments and control, and the
Lemont alleles at QSf6, QSh10a, and QSh12a showed a
reduced SF difference, whereas the Teqing alleles at QSf7
and QSh9 were associated with a reduced SF difference.
Discussion
A high genetic load in japonica subspecies and
its implications
Although the amounts of genetic load in plant genomes
are a subject of general interest and have been reported in
many species (Barrett and Charlesworth, 1991; Kirkpatrick
and Jarne, 2000), this study represents the first effort to
understand its magnitude and genomic distribution in the
model plant, rice, using molecular markers. The tremendous differences in radiosensitivity, measured as phenotypic changes or damage in SH and SF between the
radiation-treated seeds and untreated control, between the
parents and among RILs observed in this study were
expected to have resulted from two factors: (i) the direct
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 27, 2014
Trait
2820 Xu et al.
Table 4. Twenty-eight QTLs affecting seedling height (SH) and seed fertility (SF) detected in the Lemont/Teqing RILs after mutationinducing treatments
Marker intervala
Parametersb
Controlc
c-rays
Spaceflight
1
RM129–RM24
2.42
ÿ0.35
QSh2a
2
OSR17–RM154
QSh2b
2
RM27–RM324
2
OSR26–RM208
2.01
ÿ0.34
3.98
ÿ0.47
6.35
ÿ0.49
7.25
ÿ0.54
11.94
ÿ0.57
8.64
ÿ0.52
2.58
ÿ0.36
QSh2c
QSh3a
3
C6363–RG450
QSh3b
3
G06075–RM156
QSh3c
3
RM227–RM85
QSh4a
4
RM252–RM303
QSh4b
4
Q05050–A17130
QSh5a
5
gl1–RM13
QSh5b
5
RM163–RM161
QSh6
6
OSR19–RZ516
QSh7
7
RM11–OSR4
QSh8a
8
CSU754–G104
QSh8b
8
OSR7–RM230
QSh9
9
RM201–RM215
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
LOD
Effect
Trait
QTL
SH
QSh1
QSh11a
11
RM167–RZ53
QSh11b
11
RM229–RM21
QSf3a
3
RM16–RZ403b
QSf3b
3
RM227–RM85
QSf6
6
RM50–RM276
QSf7
7
RM234–CDO405
QSf8
8
CSU754–G104
QSf9
9
J01090–RZ698
QSf10a
10
RM271–RZ400
QSf10b
10
RG1094f–RM228
QSf12a
12
RM260–RG20q
QSf12b
12
RM17–G1106
2.74
ÿ0.37
13.72
ÿ0.61
3.76
ÿ0.44
3.23
ÿ0.40
3.11
ÿ0.39
c-rays–control
2.94
ÿ0.44
2.75
ÿ0.43
8.37
ÿ0.54
4.19
ÿ0.43
5.67
ÿ0.45
7.67
ÿ0.56
3.01
ÿ0.45
8.11
ÿ0.54
2.80
ÿ0.43
7.60
ÿ0.51
3.61
ÿ0.48
2.87
ÿ0.37
2.42
ÿ0.31
3.03
ÿ0.47
3.22
ÿ0.41
3.78
ÿ0.46
8.35
ÿ3.90
5.20
ÿ2.54
2.24
ÿ1.83
2.43
ÿ1.89
3.26
ÿ2.24
2.20
ÿ0.28
3.72
ÿ0.49
2.76
ÿ0.36
3.99
ÿ0.47
8.62
ÿ3.28
3.67
ÿ4.05
3.83
1.27
2.22
ÿ2.89
3.24
ÿ2.54
2.14
ÿ3.3
2.40
4.93
2.74
ÿ3.35
3.16
1.48
3.17
ÿ1.46
6.10
ÿ2.79
2.20
ÿ3.20
3.81
ÿ2.38
5.48
ÿ0.38
2.36
ÿ0.30
5.25
ÿ0.42
6.31
ÿ0.46
3.02
ÿ0.41
3.83
ÿ0.46
5.30
ÿ0.50
6.46
ÿ0.47
3.42
ÿ0.41
5.38
ÿ2.56
3.89
ÿ2.42
Spaceflight–control
3.21
ÿ4.00
2.88
ÿ2.37
a
The underlined markers are those closer to the true QTL positions.
The log-odds ratio (LOD) is the test statistics defined as: LOD=ÿlog (L0/L1). The thresholds of LOD=2.50 and 3.10 for SH and SF are equivalent to
P <0.0007 and 0.00016, respectively (Lander and Botstein, 1989).
c
Underlined data are the parameters of the QTL detected under the threshold of P <0.05. QTL effects were associated with the Lemont allele.
b
effects of radiation on DNA molecules in the seeds and
expression of the affected genes; and (ii) the buffering
abilities in trait expression of individual lines involved. At
the genomewide level, the former were the direct effects
of mutagenesis arising from the c-irradiation treatment,
and was expectedly random in the sense that every gene in
the genomes of individual lines had an equal chance to
be affected. The latter could be considered as the indirect
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 27, 2014
SF
Chromosome
Characterization of genetic load in rice
2821
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 27, 2014
Fig. 2. The molecular linkage map and the location of the main-effect QTLs affecting seedling height (SH) and seed fertility (SF) detected in the RILs
in the controls and in the two treatment groups. Markers starting with RG, RZ, R, CDO, G, C, Y and CSU are RFLP markers. RM (or OSR) and
RD represent SSR and RAPD markers.
measurements of genetic load in the genomes of individual
RILs that varied considerably. The large difference in
radiosensitivity between the parents observed in this study
indicated that the japonica parent, Lemont, has a much
higher genetic load in its genome than the indica parent,
Teqing. Consistent with this result, the Lemont alleles at
almost all identified QTLs were associated with increased
radiosensitivity (Table 4). In a separate study, it was found
2822 Xu et al.
show less damage owing to their better ability to repair
the damage (Inoue, 1980). Thus, it would be of great
interest to distinguish the identified QTLs with regard to the
types of genes they actually represent. Further study is
underway to determine which of these identified QTLs are
involved in heritable mutations.
Comparison between c-rays and spaceflight as
mutagenic treatments
In this study, severe damage in the parents and RILs caused
by c-ray irradiation were consistent with the fact that 11
(39.3%) of the identified QTLs for radiosensitivity (QSh2a,
QSh2b, QSh2c, QSh3b, QSh4a, QSh6, QSh7, QSh8a,
QSh9, QSf6, and QSf12a) were associated with the c-ray
treatment. These QTLs, detected under c-ray irradiation but
not in the control, indicated that the differences between
alleles of Lemont and Teqing were not significant in the
control but were significant under the treatment, which may
result from knock-out of functions of the Lemont alleles at
these loci by c-ray irradiation. By contrast, the spaceflight
treatment appeared to have small effects on the treated
materials and there were only four QTLs (QSh2a, QSh2b,
QSh7, and QSh8b) at which functions of the Lemont alleles
were apparently disrupted by spaceflight. No significant
differences in magnitude of QTL effects were found
between QTLs specifically induced by c-rays and those
induced by spaceflight (Table 4), indicating that more
genomic regions were responsible for the greater damage
caused by c-rays than by spaceflight. Thus, the phenotypic
effects of the space treatment on rice observed in this
study and reported in other plant species (Bayonove et al.,
1984; Kostina et al., 1984; Braam et al., 1997; Hampp
et al., 1997; Mei et al., 1998) appear to resemble very
closely those treated by the chronic low-dose external
ionizing radiation reported previously (Zaka et al., 2002;
Sahr et al., 2005), but continuous efforts should be made
in order to determine if the space treatment is a unique
and cost-effective way for mutation induction as compared with other types of mutagenesis.
Supplementary material
The marker information and the seeds of Lemont/Teqing
RILs are freely available. Interested persons should contact
Dr Zhi-Kang Li or Dr Pinson (USDA-ARS, sr-pinson@
tamu.edu) directly for the materials and related marker
information.
Acknowledgements
We are very grateful for the valuable comments and suggestions
on the earlier versions of the manuscript from the two anonymous reviewers and Dr Luxiang Liu of the Chinese Academy of
Agricultural Sciences. This project was supported by grants of
‘Application of Aerospace Mutation in Rice Breeding’ and ‘Obtaining and Analyzing Techniques of Space Biological Information’
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 27, 2014
that the Teqing alleles at ;50% of the loci across the
genome, particularly chromosome 7, were favoured in both
the indica (Teqing) and japonica (Lemont) genetic backgrounds without apparent selection (unpublished observations), which is generally true for all populations derived
from indica/japonica crosses (Lyttle, 1991; Lin et al., 1992;
Nakagahra, 1996; Xu et al., 1997). Empirical practices in
mutation breeding indicate that the recommended dosage
of c-ray treatment to achieve the desirable level of mutation
is much higher for indica varieties (250–350 Gy) than
that for japonica lines (200–300 Gy) (IAEA, 1977). Consistent with this observation, recent comparative genomic
sequence analyses of rice chromosomes 4 and 10
clearly indicate that there are many more insertions in the
Nipponbare (japonica) genome than in the indica genomes
of Guang-Lu-Ai 4 and 9311, due primarily to activities of
transposable elements (Feng et al., 2002; Goff et al., 2002;
Yu et al., 2002). All these results lead us to the conclusion
that the japonica genome has a much higher genetic load
than the indica genome, and the evolution of the japonica
subspecies was associated with loss of function at many
loci arising primarily from insertions of transposable
elements. Thus, the observed low level of genetic variation
and greater genetic load in almost all japonica varieties
(Glaszmann, 1987; Zhang et al., 1992; Li and Rutger,
2000) suggest that evolution of japonica rice probably went
through severe genetic bottlenecks, which must have
happened during domestication, as suggested by the multilocus structure of isozyme variation (Li and Rutger, 2000).
Given the large difference in radiosensitivity to c-rays
between the parents, all identified QTLs, except for QSh1,
QSh4b, and QSf7 of type 6, represented the genomic
regions where the parental allelic diversity was associated
with the parental difference in radiosensitivity. Of these, six
QTLs are of particular interest, including QSh5a, QSf3b,
and QSf10b of type 1 at which functions of the Teqing
alleles appeared to have been knocked-out by both c-ray
and spaceflight treatments, and QSh2a, QSh2b, and QSh7
of type 7 that were induced by both c-ray and spaceflight
treatments. Because of the random nature of the radiation
treatments, the six QTLs appeared to be in the genomic
regions particularly sensitive to radiation and thus might
represent possible ‘mutation hot spots’ in the japonica
genome.
It is conceivable that at least three types of genes might
have been involved with the identified QTLs, even though
they were indistinguishable by phenotype. The first type
could be those whose functions were interrupted directly by
the radiation treatments. The second type included those
that could compensate in function for the first type. The
third type included those affecting the ability to repair
induced DNA damage. The third type of genes are more
important to determine whether the induced DNA damage
will become heritable mutations. Compared with c-ray
irradiation-sensitive varieties, resistant varieties tend to
Characterization of genetic load in rice
from the 863 Programs of the Ministry of Science and Technology of
China.
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