Euphytica DOI 10.1007/s10681-013-1026-8
Simple sequence repeat markers reveal multiple loci governing grain-size variations in a japonica rice (Oryza sativa L.) mutant induced by cosmic radiation during space flight Junmin Wang • Lijun Wei • Tianqing Zheng Xiuqin Zhao • Jauhar Ali • Jianlong Xu • Zhikang Li
•
Received: 20 May 2013 / Accepted: 6 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Quantitative trait locus (QTL) for grain size traits that include grain length (GL), grain width (GW), grain thickness (GT) as well as thousand grain weight (TGW) were identified using F2 population derived from a cross between a japonica cultivar Nongken58 and its large grain-sized mutant, ‘Dali’, which was selected in SP2 generation of plants from Nongken58 seeds exposed to cosmic radiation upon space-flight, and then advanced it over eight successive generations by bagging the panicles to ensure self pollination. ‘Dali’ had similar GW and GT but 4.8 mm
Junmin Wang, Lijun Wei and Tianqing Zheng have equally contributed to this study. J. Wang Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China L. Wei Harbin Institute of Technology, Harbin 150001, China T. Zheng X. Zhao J. Xu (&) Z. Li Institute of Crop Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, 12 South ZhongGuan-Cun Street, Beijing 100081, China e-mail:
[email protected] J. Ali (&) PBGB Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines e-mail:
[email protected]
longer in GL, and 18.1 g heavier in TGW than those of Nongken58. Seven main-effect QTLs (M-QTLs) were identified for the grain size and weight traits. Among them, three M-QTLs, QGs3a and QGs3b for both GL and TGW, and QGs5 for GW, GT and TGW, which had strong additive effects on grain shape and grain weight, were validated in the two F2 plant-derived F3 populations. The three M-QTLs were found to be nonallelic to the cloned genes GS3, GL3.1, qSW5 and QGs5 by comparative mapping. However, there was only one pair of digenic epistasis involving QGs3b for TGW detected in this population. Interestingly, homozygous ‘Dali’ alleles at the QGs3a, QGs3b and QGs5 showed significant increase in the grain size and weight, suggesting these novel alleles of ‘Dali’ at the above three loci may be a very useful for markerassisted improvement of grain quality for japonica cultivars. Keywords Grain size Grain quality Quantitative trait locus (QTL) Cosmic radiation mutagenesis Japonica rice
Introduction Grain size is determined by grain length (GL), grain width (GW) and grain thickness (GT) and they all affect grain weight. Grain size traits and grain weight, i.e. thousand grain weight (TGW) are key components
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responsible for both grain yield and grain quality. The grain size traits have been found to be controlled by multiple loci (McKenzie and Rutger 1983). In recent years, the quantitative genetic research on rice grainsize related traits has made significant progresses and more than 110 QTLs have been identified so far (Gramene 2013). Amongst them, a larger number of QTLs were found to be located on chromosomes 1, 2, 3 and 5. Some of them have been cloned, such as GW2 for GW and TGW (Song et al. 2007), GS3 mainly for GL and also for GW and GT (Fan et al. 2006), GL3.1 for GL and TGW (Qi et al. 2012), qSW5/GW5 for GW and TGW (Shomura et al. 2008; Weng et al. 2008), GS5 for grain size (Li et al. 2011), GW8 for GW (Wang et al. 2012a, b) and GIF1 for GT (Wang et al. 2008), and some of them have been fine-mapped such as qGL1 and qGW1 for GL and GW (Yu et al. 2008), qGL-3a for GL (Wan et al. 2006), gw3.1 and GW3 for TGW (Li et al. 2004; Guo et al. 2009), qGL7 for GL (Bai et al. 2010), GS7 and qSS7 for GL and GW (Qiu et al. 2012; Shao et al. 2012), and gw8.1 for GL and TGW (Xie et al. 2006). However, most of these identified genes were as a result of natural mutations and they may not be sufficient for carrying out indepth studies pertaining to complete understanding of the complex genetic mechanisms underlying grain size. Likewise, we find very few instances where cloned genes governing grain-shape or size related traits were utilized in improving grain quality in rice breeding programs by marker-assisted selection (MAS) (Ramkumar et al. 2010; Wang et al. 2011; 2012a, b). Broadening the genetic variation is essential through use of classical and innovative breeding methods that includes induced mutation, genetic engineering and use of wild relatives. Mutations induced by physical treatment, e.g. c-ray irradiation and chemical mutagens have been successfully adopted in the genetic improvement of many crops including rice (Ali and Siddiq 1999) and wheat (Liu et al. 2004). In comparing with the artificial gamma irradiation on ground, cosmic radiation exposure during space flight gave relatively lower mutation frequencies but showed higher mutagenic efficiency, which is calculated as the ratio of mutation frequency to the absolute value of physiological damage and is expressed in percentage (Wei et al. 2006a). Exposure of plant materials to cosmic radiation during space flights since 1960s has become an increasingly
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common method of induced mutagenesis in many advanced countries as a part of their ambitious space programs (Halstead and Dutcher 1987). A wide spectrum of mutations and biological effects have been observed in plant materials from seeds treated in the manned spacecrafts flying in a near-earth orbit (* 400 km) that includes chromosomal aberrations (Slocum et al. 1984), developmental abnormalities (Kuang et al. 1996), and increased mutation rates (Mei et al. 1994). In China, a total of 21 batches of biological materials (including dry seeds of different plant species, micro-organisms, etc.) have been sent into space by retrievable satellites and spacecrafts since 1987 (Liu et al. 2009). Morphological mutations have been consistently observed in the treated materials, and these mutagenic effects derived from the treated plant seeds were apparently heritable (Bayonove et al. 1984; Mei et al. 1998; Yu et al. 2007), even though the mechanism(s) of induced cosmic ray mutagenesis on plant materials remains largely unclear. Nongken58, a popular japonica inbred variety was developed in Japan and was introduced into China in the 1960s. Accumulated planting area for this inbred reached 9.46 million hectares in China, and was ever widely used as one of the dominant parents for japonica rice breeding in China. A novel mutant, ‘Dali’, was selected from the progeny of Nongken58 seeds exposed to cosmic radiation during a space flight by the Chinese retrievable satellite ‘8885’ in 1988. Interestingly, this mutant showed favorable grain quality characteristics with slender grain shape and aroma, and is a potential source material for japonica grain quality improvement. Normal distributions for grain size traits were observed in a F2 population of a cross of Nongken58 9 ‘Dali’, strongly indicating them to be governed by a complex genetic mechanism (Xu et al. 2002) despite limited polymorphism between the mutant and the wild type detected by RAPD markers (Xing et al. 1995). In this study, we carried out a polymorphism survey by relatively stable PCR marker, simple sequence repeats (SSRs) and QTL mapping for grain-size related traits of ‘Dali’ mutant. This research activity was carried out with the primary objective to map QTLs and identify the favorable alleles for grain-size related traits of ‘Dali’ mutant that could provide useful information on marker-assisted breeding to enhance grain quality features of japonica rice varieties.
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(A)
(C)
(B)
Dali
NK58
NK58
Dali
NK58
Dali
Fig. 1 Relative comparisons of Nongken58 and its mutant ‘Dali’ for a plant morphology b panicle and c grain size
Materials and methods
‘Dali’ based on its large grain size in comparison to its original variety, Nongken58 (Fig. 1).
Selection of the mutant Dry seeds harvested from single plant of Nongken58 after bagging two cropping seasons were taken to outer space by a Chinese retrievable satellite ‘8885’ in 1988 for exposure to cosmic radiation. The flight characteristics of ‘8885’ spacecraft included a flight altitude of 200–400 km, 638° inclination, inner spacecraft temperature between 15 and 26 °C, 1.5 9 10-4 g microgravity, and a total radiation of 1.58 mGy, which was detected by an integrating thermal luminous dosimeter. It was estimated that all seeds were hit at least once by the moderate energy (Z/b C 20) cosmic ray particles, and *80 % of the seeds were hit at least once by the high energy (Z/b C 50) cosmic ray particles based on the records on the nuclear track detectors. 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. The retrieved Nongken58 seeds that received cosmic radiation were then planted in the field separately. SP1 plants had normal fertility. Bulk harvested seeds from SP1 plants comprised SP2 generation with a plant population size of 15,000 individuals. A large grain mutant with very slender grain shape and aroma was observed and selected by naked eye and rubbing the young leaves and smelling them in SP2. It was found to be genetically stable by pedigree selection for successive eight generations. This mutant was designated as
Mapping population construction and trait investigation Research study was carried out at the Experimental Station of Institute of Crop Sciences, Chinese Academy of Agricultural Sciences in Sanya, Hainan province (18.3°N, 109.3°E, 7 m.a.s.l) during 2009–2010 dry season (from November 2009 to April 2010). Soil type at the study site in the paddy field was determined as sandy yellow clay. Experimental site had favorable paddy growth conditions with an average photoperiod of 11.5/12.5 h (day/night) and a daily mean temperature of 26 °C besides 120 mm of rainfall during dry cropping season. The stable mutant ‘Dali’ was crossed with its original parent, Nongken58 to develop F2 population for QTL mapping of its grain-size related traits. Thirty-day-old seedlings of Nongken58, ‘Dali’ and their derived reciprocal F1s were planted as single plant per hill into two-row plots with 12 plants per row and a spacing of 20 9 17 cm (row to row 9 hill to hill) in three replications. A total of 360 F2 individual plants derived from Nongken58 9 ‘Dali’ were planted with a spacing of 20 9 17 cm (row to row 9 hill to hill) in the experimental field at Sanya. Experimental materials in the field were managed by adopting standard agronomic practices with two sprays of pesticide to control rice stem borers, leaf folders and plant hoppers. Agronomic characteristic features for ‘Dali’ and
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Nongken58 were recorded as follows. Days to heading (in days) were recorded on plot basis when C50 % of the plants in a plot showed complete heading. Plant growth duration was recorded as number of days from sowing to complete maturity. At maturity stage (about 40 days after flowering), eight plants in the middle of each plot separately for the two parents were measured and averaged for plant height, productive panicles per plant, panicle length and number of grains per plant. Plant height (in cm) was measured from the soil surface to the top of the tallest panicle (awns excluded). Productive panicles per plant were counted as number of panicles with more than five filled grains per panicle. Panicle length was measured from panicle base to top of panicle (awns excluded). Grain-size related traits, including grain length (GL), grain width (GW) and grain thickness (GT) were measured and averaged over 20 randomly picked fully filled grains from the panicle of the primary productive tiller belonging to each F2 single plant and representative single plants separately from the middle for both the parents using an electronic digital caliper (Guang-lu Metrical Instrument Co. Ltd, Guilin, China) with a precision of ±0.01 mm. The same set of twenty seeds were weighed on an electronic weighing balance in grams and then multiplied to a factor of 50 to measure the thousand grain weight (TGW). Genotyping and linkage map construction Out of 600 simple sequence repeat (SSR) markers surveyed for polymorphism between Nongken58 and ‘Dali’, only 46 SSR showed polymorphism. Genomic DNA was extracted from separate leaf samples belonging to 281 individual F2 plants that were randomly selected from 360 F2 individuals derived from the cross of Nongken58 9 ‘Dali’. These 281 F2 individual DNA samples were then used for genotyping by assaying the 46 polymorphic SSR markers with clear bands. The genetic map of Cornell SSR (Temnykh et al. 2001) was used as the reference map for marker order. Data analysis and QTL mapping Correlation between the grain-size related traits among plants in F2 mapping population was determined using the SAS PROC CORR (SAS 2002). Phenotypic data of the four grain-size related traits,
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were used as input data to identify main-effect QTLs (M-QTLs) and epistatic QTLs (E-QTLs) affecting GL, GW, GT and TGW by single marker analysis embedded the IciMapping Package Version 3.2 (Li et al. 2007a, b). The LOD threshold was set at 2.9 for claiming a significant M-QTL based on 1,000 runs of randomly shuffling the trait values (Churchill and Doerge 1994). Threshold level to claim a statistically significant E-QTL was set at P B 0.001 and LOD C 3. Differences in mean trait values between the two parental alleles at all significant markers associated with M-QTLs for additive effect were verified by t test. Also, all identified E-QTL pairs were also confirmed by two-way analysis of variance using SAS PROC GLM (SAS 2002). M-QTL verification To verify some important M-QTLs, several F2 individuals with heterozygous genotypes at SSR markers tightly linked with the target M-QTLs were selected to develop F3 segregation populations. The F2-derived F3 populations were genotyped using the markers closest to each M-QTL, and all individuals were then divided into two groups based on marker genotypes, i.e. homozygous Nongken58 and homozygous ‘Dali’. A t-test was performed to find the significant differences in mean data of the related traits between the two homozygous groups. Significant differences indicated that the M-QTL was true.
Results Phenotypic variation and correlation The different traits measured on the semi-dwarf japonica variety Nongken58 in Sanya (Hainan province of China) during the 2009–2010 were as follow: plant height of 85 cm, 11 productive panicles per plant and growth duration of 125 days. Large and significant differences (P \ 0.001) were found between the mutant, ‘Dali’ and its original variety, Nongken58 for GL and TGW (Table 1). ‘Dali’ was 4.8 mm longer in GL and 18.1 g heavier in TGW than those of Nongken58. GL is responsible for the difference in TGW between ‘Dali’ and Nongken58 as the other two traits GW and GT showed non-significant differences between them. The average number of grains per plant
Euphytica Table 1 Evaluation of grain size traits of Nongken58 (P1) and its mutant ‘Dali’ (P2) as well as their reciprocal hybrids Trait
Nongken58 (P1)
Dali (P2)
P1–P2
F1 (P1/P2)
-4.8***
F10 (P2/P1)
F1–F10
GL (mm)
7.1 ± 0.10
11.9 ± 0.29
9.2 ± 0.22
9.3 ± 0.22
-0.1
GW (mm)
3.2 ± 0.10
3.1 ± 0.09
0.1
3.2 ± 0.09
3.2 ± 0.09
0
GT (mm) TGW (g)
2.2 ± 0.04 26.5 ± 0.78
2.4 ± 0.06 44.6 ± 1.02
-0.2 -18.1***
2.3 ± 0.05 36.5 ± 1.58
2.3 ± 0.05 35.9 ± 1.45
0 0.6
GL grain length, GW grain width, GT grain thickness, TGW 1,000-grain weight Significant at *** P \ 0.001
GW (mm) 160 140
60 40
NK 58
Dali
11.79 12.13
11.45
10.43 10.77 11.11
9.07 9.41 9.75 10.09
8.05
0
8.39 8.73
20
120 100 80 60 40 20 0
TGW (g)
GT (mm) Dali
No. of lines
120 100 80 60
NK 58
40
0
2.07 2.12 2.17 2.22 2.27 2.32 2.37 2.42 2.47 2.52 2.57 2.62 2.67 2.72
20
160 140 120 100 80 60 40 20 0
Dali NK58
6.66 10.06
140
40.66 44.06 47.46 50.86
80
NK 58
13.46 16.86 20.26 23.66 27.06 30.46 33.86 37.26
No. of lines
100
Dali
3.02 3.05 3.08 3.11
120
3.14 3.17 3.20 3.23 3.26 3.29 3.32 3.35 3.38 3.41
GL (mm)
Fig. 2 Frequency distribution of grain-size related traits for 281 individuals in the F2 population derived from the cross between the original variety Nongken58 (NK58) and its mutant
‘Dali’. GL grain length, GW grain width, GT grain thickness, TGW 1,000-grain weight
was 608.8 and 880.3 for the mutant ‘Dali’ and Nongken58, respectively under Hainan conditions and showed significant difference. The reciprocal F1 plants had comparable the number of grains per plant of 760.2 and 768.6, respectively. Besides, ‘Dali’ was 10 days shorter in plant growth duration, 30 cm taller in plant height, 8 cm longer in panicle length and has three less productive panicles per plant as compared to Nongken58 (Fig. 1). No significant differences existed for grain-size related traits that included GL, GW, GT and TGW between the reciprocal F1 plants (Table 1), indicating
grain-size traits of the mutant were only governed by nuclear genes and had no relation with cytoplasmic genes. The F2 population of Nongken58 9 ‘Dali’ showed transgressive segregation for all the traits except GL suggesting all these traits are quantitatively inherited (Fig. 2). Positive correlations for each pair of grain-size related traits were highly significant except between GL and GW (Table 2). In the particular population of Nongken58 9 ‘Dali’, GL and GW seemingly showed independent inheritance due to their non-significant association.
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Euphytica Table 2 Correlation coefficient of grain-size related traits in the 281 individuals derived from Nongken58 9 ‘Dali’ F2 population GL GW
GW
GT
-0.045
GT
0.214***
0.467***
TGW
0.587***
0.320***
0.684***
GL grain length, GW grain width, GT grain thickness, TGW 1,000-grain weight Significant at *** P \ 0.001
Chr.3
Chr.1
Chr.5
Genomic differences between Nongken58 and ‘Dali’ Among 600 SSR markers evenly distributed on 12 rice chromosomes, 46 (7.7 %) loci showed polymorphism between Nongken58 and ‘Dali’, including 4, 2, 7, 2, 6, 2, 9, 3, 2, 2, 5 and 2 on chromosomes 1–12, respectively (Fig. 3). It seemed that cosmic radiation had resulted in multiple site mutagenesis across the whole genome of Nongken58, which was in agreement with the differences in many phenotypic traits between Nongken58 and ‘Dali’ as indicated above. Chr.10
Chr.7
Chr.11 RM286
RM159
qGL1/qGW1
RM481
GS5
QGs1a
RM405
RM1 RM283
RM180
GW5/qSW5 qTGW3-1
GW3
RM562
RM21 RM209
GS3/qGL-3a gw3.1 QGs5
RM3351
GL3.1/qGL3
RM432 RM11
RM163 RM440 GS7
RM229 QGs10
RM228 RM333 RM224
RM336
qSS7
RM473C QGs3a
QGs1b
RM426 RM3405 RM3856 RM8277
Qgs7
RM134
qGL7
RM31
RM248 RM420 QTLs for grain size-related traits
RM297 RM7000 QGs3b
RM570
1.0 Mb QTLs cloned or fine-mapped for grain size-related traits
RM7389
Fig. 3 Location of M-QTLs affecting grain-size related traits detected in the F2 population derived from the cross of Nongken58 9 ‘Dali’. Polymorphic markers were shown only
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on the chromosomes on which QTLs were detected. Bold markers by a double-head arrow are associated with digenic epistasis
RM440RM3663
RM473A
1
3
3
5
7
10
QGs1b
QGs3a
QGs3b
QGs5
QGs7
QGs10
22372129
25457233
19912517– 21363398
33795490– 36155173
27588613– 28804886
32099566
4635793– 4885915
Physical Range (bp)
7.7
10.5
11.1
4.0
2.9
0.22***
0.46***
0.47***
0.30***
0.23***
-0.16
-0.01
0.06
0.06
0.06
4.9
15.9
15.7
6.2
4.4
R2d (%)
7.6
LOD
D
LODa A
GW
GL
0.07***
A
10.0
R2 (%)
6.7
4.3
LOD
0.04***
-0.04***
A
0.01
0
D
[1] Previously mapped locus, qGL7-2 (Shao et al. 2012) within 1 Mb to the peak markers
LOD values confirmed by IciMapping package Version 3.2 (Li et al. 2007a, b) for the loci significantly detected by one-way ANOVA
0.03
D
GT
*** Represents significant differences at P B 0.001 based on t-test between the two parental alleles at the significant markers
b
a
A additive effects of substitution of Nongken58 alleles by ‘Dali’ alleles, D dominant effects
Bold represents peak markers
RM333
RM7000RM7389
RM426RM8277
RM297
RM1RM283
1
QGs1a
Marker
Ch
QTL
7.0
6.2
R2 (%)
6.0
16.2
15.1
6.0
LOD
TGW
1.84***
-
3.22***
1.89***
A
0.64
-0.50
-0.16
-0.13
D
4.2
10.9
10.3
4.2
R2 (%)
[1]
Ref.b
Table 3 Detection of main-effect QTLs (M-QTLs) affecting grain-size related traits i.e. grain length (GL), grain width (GW), grain thickness (GT) and 1000-grain weight (TGW) in the Nongken58 9 ‘Dali’ F2 population
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Identification of QTLs associated with grain-size related traits A total of seven M-QTLs on five of 12 rice chromosomes were significantly associated with the four grain-size related traits, including 5 for GL, 1 for GW, 2 for GT and 4 for TGW (Fig. 3 and Table 3). Five M-QTLs (QGs1a, QGs1b, QGs3a, QGs3b and QGs7) affecting GL were identified on chromosomes 1, 3 and 7, and three of them (QGs1a, QGs3a, and QGs3b) were also found to be affecting TGW. The ‘Dali’ alleles at all loci increased GL with an additive effect ranging from 0.23 mm to 0.47 mm. For GW, only one M-QTL (QGs5) was detected on chromosome 5 with an additive effect of 0.07 mm. The ‘Dali’ allele at this M-QTL increased GW and GT as well as TGW. Two M-QTLs (QGs5 and QGs10) were identified for GT on chromosomes 5 and 10. The ‘Dali’ alleles at QGs5 increased GT while it reduced GT at QGs10. A total of four grain shape M-QTLs (QGs1a, QGs3a, QGs3b, and QGs5) were found to be responsible for TGW. Interestingly, ‘Dali’ alleles at all these loci increased TGW with an additive effect ranging from 1.84 g to 3.22 g. Dominant effects at all M-QTLs were much less than the additive effects (Table 3), indicating additive effect being the main component of genetics underlying grain-size related traits. Only one pair of digenic epistasis was detected for TGW that involved the RM7389 (M-QTL QGs3b) locus on chromosome 3 and the RM224 locus on chromosome 11. Epistasis involving additive by additive effects resulted in increased TGW. Validation of the important M-QTLs From QTL mapping results as shown in Table 3 and Fig. 3, the M-QTLs associated with RM8277 and RM7389 on chromosome 3 simultaneously affected GL and TGW, and the M-QTL associated with RM440 on chromosome 5 affected GW, GT and TGW. At all these M-QTLs increased trait values were associated with the ‘Dali’ alleles with mainly gene action showing additive effects. Considering the importance of these M-QTLs for grain-size related traits for crop improvement, there was a further need for their validation in subsequent F2-derived F3 populations. Two F2 individuals (#323 and #192) were selected based on phenotypic and genotypic data, to further verify the M-QTLs on chromosomes 3 and 5 in their
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Grain length or width (mm)
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9.9**
10.03***
10.00 7.34
7.45
8.00 6.00 3.13
4.00
3.22*
2.00 0
QGs3a for GL at RM8277
QGs3b for GL at RM7389
Homozygous Nongken58 allele
QGs5 for GW at RM440
Homozygous Dali allele
Fig. 4 Relative comparisons of GL and GW between the two homozygous genotype classes of Nongken58 and ‘Dali’ alleles at RM8277 and RM7389 loci on chromosome 3 and RM440 locus on chromosome 5 for the two F3 populations derived from the F2 individual plants #323 and #192 from the cross of Nongken58 9 ‘Dali’. Mean significant differences at *P \ 0.05, **P \ 0.01 and ***P \ 0.001 respectively
selffed progenies. The F2 individual plant #323, which was heterozygous at RM8277 locus associated with QGs3a but homozygous for Nongken58 alleles at the other four GL-QTLs was selected for verification of QGs3a. Likewise, the plant #192, which was heterozygous at RM7389 locus associated with QGs3b but homozygous for Nongken58 alleles at the other four GL-QTLs was selected for confirmation of QGs3b. Also, the plant #192 was used for validating QGs5 as it was heterozygous at RM440 locus. Two F3 populations were planted with 410 and 612 plants derived from two individual F2 plants #323 and #192, respectively. Based on genotype at RM8277 and RM7389 on chromosome 3 and RM440 on chromosome 5, individual F3 plants were classified based on two parental homozygous genotypes (Nongken58 homozygous-NN and ‘Dali’ homozygousDD) for phenotypic comparisons, respectively. Significant differences for average GL were detected between plants with NN and those with DD at RM8277 and RM7389 loci (Fig. 4). Likewise, significant difference of average GW was detected between plants with NN and those with DD at RM440 (Fig. 4). Thereby, it was indicated that the three M-QTLs linked to RM8277, RM7389 and RM440 did exist and had effects on GL and GW, respectively.
Discussion Galactic cosmic radiation (GCR) comprising of protons and high charge (Z) and energy (E) (HZE)
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particles in outer space is known to cause genetic mutations in biological organisms upon being exposed to it and reported by several researchers (Kostina et al. 1984; Hagen 1989; Mei et al. 1994; Durante 2009). HZE particles in cosmic radiation hitting the seed materials in outer space is responsible for the mutations that can often cause chromosomal aberrations upon longer duration of exposure (Halstead and Dutcher 1987). Plant seeds when exposed to cosmic radiation under a micro-gravity environment especially during a space flight in a near-earth orbit (*400 km) could support induction of multiple trait mutations such as plant height, heading date, leaf and seed color etc. (Bayonove et al. 1984; Mei et al. 1994, 1998; Wei et al. 2006b; Yu et al. 2007). Space breeding program in China got its boost upon launch of Shijian-8, a specially designed breeding satellite on September 9, 2006. This breeding satellite took a special payload of 2,000 accessions of plant seeds belonging to 133 species. China could utilize space breeding program and successfully released as many as 66 new varieties of crops that included rice, wheat, cotton, rapeseed, sesame, pepper, tomato and alfalfa (Liu et al. 2009). Molecular marker survey for polymorphism percentage between the mutants and their original wild types for rice crop were found to vary from 0.35 to 14.6 % depending upon the kind of molecular markers researchers used and it clearly revealed existence of mutations (Li et al. 2007a, b; Yu et al. 2007; Liu et al. 2009; Lu et al. 2010). Moreover, a six fold increase of SSR mutation rate was observed in wheat crop when exposed to post Chernobyl radioactive contamination (Kovalchuk et al. 2000). In the present study, efforts were made to ensure that all the mutants derived from Nongken58 were strictly maintained in isolation both in space and time from other regular breeding materials to avoid pollen contamination. Comparative analysis using SSR markers with the mutant ‘Dali’ and its original parent Nongken58 revealed polymorphism of 7.7 % at 600 SSR loci across whole genome which is in agreement with the earlier mutation studies (Yu et al. 2007; Lu et al. 2010). We found that as many 46 SSR loci out of 600 markers studied had shown polymorphism without creation of much genetic variation in ‘Dali’ except for the grain size-related traits, panicles and grains per plant, heading date and plant height. These mutations on or near SSR sites must have resulted in the polymorphism in relation to
Nongken58 that allowed us to map the QTLs governing the seed size-related traits. Mutation events being random in nature and much of it occurring in intergenic regions may not be resulting in noticeable change in phenotype which also acts like a buffer to bear the mutations and allowing relatively fewer mutations to occur in the exon regions thereby creating genetic variation. In fact, the SSR sites being repetitive sequence in nature and any mutation on such region or due to replication slippage could be easily detected as in our case that showed 7.7 % to be polymorphic in comparison to its original parent. Our results further demonstrated that seeds exposed to cosmic radiation during space-flight could be useful for screening mutants for crop improvement (Dennis and Ding 2002; Liu et al. 2009), although mechanisms of such cosmic radiation-induced mutagenesis in space remains largely unknown. Many researchers have earlier identified QTLs to be associated with GL and TGW and located them in the peri-centromeric region of rice chromosome 3 especially in several inter-specific crosses in rice (Xiao et al. 1998; Brondani et al. 2002; Li et al. 2004; Xu et al. 2004). Another QTL associated with GW and/or TGW located in a recombination hotspot on chromosome 5 was identified by several researchers (Shomura et al. 2008; Wan et al. 2008; Zheng et al. 2011). M-QTLs affecting grain size-related traits identified in this study were effectively compared with those previously reported using the same SSR markers and comparative linkage maps (Kurata et al. 1994; Temnykh et al. 2001; Ware et al. 2002) as well as a sequence map, Oryza sativa subsp. japonica Gramene annotation sequence map 2009 (Gramene 2013). Some M-QTLs identified in this study were found to be located in the same or adjacent regions with those previously reported. For instance, QGs3b associated with marker RM7389 on chromosome 3 for GL and TGW was mapped in the same region with qGL3c (Bai et al. 2010) and Bin3.7 QTL (Zheng et al. 2011) for GL. QGs5 associated with RM440 on chromosome 5 for both GW, GT and TGW were mapped together with the QTL associated with RM6621 (Kato et al. 2011) for GT. By comparative mapping, the three validated M-QTLs (QGs3a, QGs3b and QGs5) were non-allelic to the cloned genes, GS3 (Fan et al. 2006), GL3.1 (Qi et al. 2012), qSW5/GW5 (Shomura et al. 2008; Weng et al. 2008) and GS5 for grain size (Li et al. 2011) owing to their locations in obvious
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different chromosomal regions (Fig. 3). The identification of multiple loci have largely contributed to our understanding based on the normal distribution of grain size-related traits in the F2 population from ‘Dali’ and Nongken58 (Xu et al. 2002), which has been puzzling us for a long time, ever since the RAPD marker assay that showed only 0.25 % differences between ‘Dali’ and Nongken58 (Xing et al. 1995). Genetically, QTLs for grain-size related traits were found to be independent of those for heading date based on their detection on different chromosomes and a few heading date QTLs had a pleiotropic effect on TGW (Huang et al. 2010, Liang et al. 2013). The weak negative association between grain-size related traits and grain number per panicle appeared to be largely due to linkage, rather than pleiotropy as previously reported by Xu et al. (2004). This suggests that the ‘Dali’ alleles identified at the three validated M-QTLs (QGs3a, QGs3b and QGs5), which had strong additive effects with pleiotropy on grain shape and weight, could be used in improvement of rice grain quality although ‘Dali’ has significant differences for number of grains per plant and flowering time compared with Nongken58. These three genes have not been intensively utilized in many japonica rice breeding programs especially in China, as evident by the high frequency of the round-shaped grain japonica cultivars. Therefore these three genes, derived from the novel japonica mutant, ‘Dali’, need to be considered for improving grain shape and grain yield. For instance, if we just focus on the above three M-QTLs and their eight possible homozygous genotype combinations, the plants fixed with ‘Dali’ alleles at QGs3a and QGs3b and Nongken58 allele at QGs5 would produce grains with a largest GL/GW ratio of 3.28; however, in the reciprocal case, the grains would have a smallest GL/GW ratio of 2.65. Among them eight homozygous genotypes, various ratio of GL/GW, ranging from 2.65 to 3.28, will be providing breeders a wide range of choice to design new varieties with different grain shapes by MAS. It is also expected that any plant from these eight homozygous genotypes at QGs3a, QGs3b and QGs5 would possess longer grain type, thus may result in good grain appearance quality primarily due to reduced chalkiness. In Yangtze valley areas of China, japonica cultivars with round-shaped grains frequently produced chalkiness with poor grain appearance quality especially owing to high temperature during grain-filling stage
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from August to September in the fall season. Larger ratio of GL to GW in japonica cultivars over shorter ones are now becoming popular and have now higher market value in China. In general, GW was found to be closely associated with the occurrence of chalkiness (Luo et al. 2004). Rice breeding efforts to combine higher grain yield potential with premium grain quality features, it will be worthwhile to increase the GL and proportionately reduce the GW thereby simultaneously improve grain weight and quality. Using ‘Dali’ as a donor parent, an attempt was made to cross with round-shaped grain japonica variety Jia59 and the resulting F1 was then repeatedly backcrossed with the recipient parent Jia59 for two backcross generations without losing the grain quality features of ‘Dali’. By this approach, three japonica lines were developed by conventional procedures with improved grain quality features of ‘Dali’ besides aroma and decreased percentage of chalkiness (from 55.5 % in the original Jia59 to 10 % in the new lines) (Xu et al. 2002), thus indicating the long grain mutant, ‘Dali’, is a valuable source for rice breeding for premium grain quality in japonica background. Interestingly, the ‘Dali’ being in japonica background with improved grain features that matches with indica rice grain type that could be easily introgressed into other japonica high yielding varieties without posing any cross incompatibility problems as noticed in indica 9 japonica crosses (Jia et al. 2012). Therefore, marker-assisted introgression and deployment of the ‘Dali’ alleles at the three M-QTLs (QGs3a, QGs3b and QGs5) into round-shaped grain japonica varieties will be facilitating to develop japonica varieties with improved grain shape and size. Further efforts are underway to fine-map the three targeted M-QTLs for precision based MAS and gene cloning.
Conclusion A novel mutant ‘Dali’ with longer GL and heavier TGW was induced by cosmic radiation during a space flight on the original variety Nongken58. Among 7 QTLs for grain size-related traits, 3 (QGs3a, QGs3b and QGs5) were validated and found to be non-allelic to the earlier cloned genes for grain size-related traits on chromosomes 3 and 5. The ‘Dali’ alleles at these three loci had strong additive effects with pleiotropic influence on grain shape and weight. Therefore,
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marker-assisted introgression and deployment of ‘Dali’ alleles especially at the three M-QTLs into round-shaped grain japonica varieties will be facilitating improvement of japonica varieties for grain shape and grain quality. Acknowledgments This research was funded by the ‘‘948’’ Project (2011-G2B) from the Ministry of Agriculture of China, the ‘‘863’’ Project (2012AA101101) from Ministry of Science and Technology of China, and the National Science and Technology Key Project (2008BAD97B02).
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