Published online 23 February 2005
Published in Crop Sci 45:712-716 (2005)
© 2005 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Quantitative Trait Loci Associated with Seed Dormancy in Rice
J. M. Wana,*,
Y. J. Caoa,
C. M. Wanga and
H. Ikehashib
a National Key Lab. for Crop Genetics and Germplasm Enhancement, Nanjing Agricultural Univ., Nanjing 210095 China
b Dep. of Plant and Resources, College of Bioresource Sciences, Nihon Univ., 1866 Kameino, Fujisawa, Knagawa 252-8510, Japan
* Corresponding author (wanjm{at}njau.edu.cn)
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ABSTRACT
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Seed dormancy is a key agronomic trait related to the quality of seed and rice production because it imparts resistance to preharvest sprouting. A population was constructed from a three-way cross, IR50/Tatsumimochi//Miyukimochi, in which IR50 was an indica donor of strong seed dormancy and Tatsumimochi and Miyukimochi were nondormant japonica cultivars. Seed dormancy was tested for 166 F1 plants of the three-way cross and their progeny population (F2). A total of 108 simple sequence repeats (SSR) markers were mapped on 12 rice chromosomes, and quantitative trait locus (QTL) analysis was performed for seed dormancy in the two segregating generations. Three putative QTLs affecting seed dormancy were detected on chromosomes 1, 3, and 7 in the F1 generation. Phenotypic variations explained by each QTL ranged from 4 to 21%, indicating that seed dormancy is affected by a number of minor genetic effects. The putative QTLs on chromosomes 1 and 3 were also detected in the F2 generation at the same map sites. Considering the present and past results there are a number of genetic factors that affect rice seed dormancy; however, the two SSR markers on chromosomes 1 and 3 could be used to develop near-isogenic lines (NILs) for map-based cloning of seed dormancy genes in the regions to thus improve the dormancy of japonica rice.
Abbreviations: CIM, composite interval mapping • cM, centimorgans • LOD, log likelihood ratio • NILs, near-isogenic lines • PCR, polymerase chain reaction • PHS, preharvest sprouting • PVE, percentages of variance explained by QTLs • QTLs, quantitative trait loci • SSR, simple sequence repeats
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INTRODUCTION
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SEED DORMANCY, defined as the inability of viable seed to germinate under environmental conditions favorable to germination, is one of the most important traits in breeding programs of cereal crops because it is associated with preharvest sprouting (PHS), affecting the production of rice under humid climates. In Southeast Asia, due to the long spell of rainy weather in early summer and autumn, PHS frequently happens and leads to great loss of yield and low quality in rice. Rice seed dormancy is known to be induced by some environmental factors like temperature during later stage of ripening (Ikehashi, 1972). In the domestication of rice a series of mutations would have occurred to form an acceptable level of weakened dormancy, thereby a group of cultivars may have lost a part of functioning genes while the other group inactivated other part of them. Often, rice workers see that the present level of seed dormancy is inadequate to prevent pre-harvest sprouting, but face the difficulty in strengthening genetic bases for the dormancy.
A few reports have been published on the genetics of rice seed dormancy. Wan et al. (1997) reported that isozyme loci Pgi1 (chromosome 3), Amp3 and C (chromosome 6), Est9 (chromosome 7), and Acp2 (chromosome 12) are linked with dormancy genes. Recently, molecular marker–assisted studies have been performed to identify QTLs for dormancy. Lin et al. (1998) found five regions on the RFLP map of chromosomes 3, 5, 7, and 8, which were supposed to harbor seed dormancy genes. Cai and Morishima (2000) demonstrated that all chromosomes except for chromosomes 4 and 10 harbor seed-dormancy genes and chromosomes 3, 5, 6, 9, and 11 carry more than one dormancy locus. The seed dormancy of rice is a transient or elusive trait during seed storage, being variable by testing times. Its induction is affected by the temperature during maturation (Ikehashi, 1972), in which a high temperature of around 30°C during later stage of maturity induces strong dormancy while a low temperature of around 20°C does not induce an appreciable dormancy in cultivars of otherwise producing dormant seed. Therefore, an estimated level of seed dormancy is determined not only by inheritance but also by conditions for seed maturation.
In this report, we identify genetic markers for seed dormancy using two successive generations of an indica x japonica cross. The QTLs controlling seed dormancy were identified by linkage analysis to SSR. Simple sequence repeats marker–linked genes with stable effects on seed dormancy could be useful for further study of cloning and functional analysis.
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MATERIALS AND METHODS
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Plant Materials
Indica cultivars have dominant genes for seed dormancy (Wan et al., 1997). A three-way cross, IR50/Tatsumimochi//Miyukimochi, was made to find the positive alleles in indica cultivar IR50 which enhance the dormancy of different japonica cultivars Tatsumimochi and Miyukimochi. The F1 population of 166 plants and their parents were planted in the experimental farm at Weigang Campus of Nanjing Agricultural University in 2000. Twelve F2 plants derived from each F1 plant were planted in the same field in 2001. Leaves of individual plants in the F1 population were collected for the extraction of total DNA. The climates during the seed maturation were normal in 2000 and 2001; the temperature during the late stage of maturity ranged from 20°C to 30°C.
Evaluation of Seed Dormancy
The dormancy was assessed following Ikehashi (1972) and Wan et al. (1997). Two panicles from each F1 and parental plants were collected on the 35th day after heading when the grains were fully filled and were immediately stored at 4°C to maintain dormancy. For evaluation of dormancy, 100 seeds from each F1 plant were placed on doubled sheets of filter paper moistened with distilled water in a petri dish of 6-cm diameter, and maintained at 30°C and 100% relative humidity for 7 d. Each plant was tested with three repeats. Germination was determined by the emergence of radicle and/or plumule. The degree of seed dormancy was scored as the mean percentage of germinated seeds.
Each F2 progeny from the 166 F1 plants derived from the three-way cross was treated as a population and the level of dormancy in each F2 population was given by the average percentage germination of bulked seed from their 12 progeny plants. Parental plants were also tested in the F2 environments. Since maximum segregation occurs in the F2 generation and thus does not permit replication per se, we measured germination three times per F2 population by bulking F3 seeds among the 12 F2 plants comprising each population. The number of F1 plants sampled for DNA was 166, and the 166 F1 plants and F2 populations were phenotyped respectively for QTL analyses. Theoretically there is a sampling error with the three-way cross F2 population, but the sample size of 12 plants should be sufficient to detect QTLs with major effects (Hayashi and Ukai, 1999). A larger sample size could detect minor QTLs, however; it would greatly increase the workload.
DNA Preparation and Simple Sequence Repeats Assay
For DNA extraction, one 
3-cm long leaf was harvested from each plant and ground in 0.4 M NaOH, then 160 µL 100 mM Tris-HCl (pH7.5) was applied. DNA solution with a concentration of 5 ng µL–1 was used as the template DNA after the storage at –20°C. The original sources and motifs for all the SSR markers used in this study were based on the gramene database (http://www.gramene.org) and McCouch et al. (2002) (or http://www.dna_res.kazusa.or.jp/9/6/05/spl_figure1/fig1.pdf). Polymerase chain reaction (PCR) amplifications were performed as described in Chen et al. (1997) with minor modifications. Briefly, 10 µL reactions contained 0.2 µM of each primer, 50 µM each of dNTPs, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 10 ng of DNA, and 0.5 unit of Taq polymerase. The PCR consisted of the following cycle: 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 2 min, and finally by 5 min at 72°C for the final extension. Polymerase chain reaction products were run on 8% polyacrylamide nondenaturing gels, and marker bands were revealed using the silver staining method based on Sanguinetti et al. (1994) and scored on a light box with fluorescent lamps.
Construction of Linkage Map
Informative markers distributed throughout the 12 chromosomes were selected and scored for 166 F1 plants. We simplified the three-way F1 genotypes into the two classes, IJ (heterozygous for alleles of indica and japonica) and JJ (homozygous for alleles of japonica), because the parents chosen in this experiment were typical indica and japonica cultivars, meanwhile little polymorphism was found between the two japonica cultivars with the markers in this experiment. Linkage analyses were performed with MAPMAKER/EXP v3.0 (Lander et al., 1987). Markers were allocated to linkage groups with a minimum threshold log likelihood ratio (LOD) score of 2.0 and a maximum recombination fraction 
= 0.25 using the group command. The use of SSR markers previously mapped on the 12 rice chromosomes (Chen et al., 1997) allowed for direct identification of the linkage groups.
QTL Analysis
To normalize the variance, the percentages of germinated seeds of each F1 plant and F2 offspring were transformed to the arc sine [=arc sine (
)1/2]. A composite interval mapping (CIM) was performed with Cartographer (Zeng, 1994) to detect QTLs for the variations of dormancy in the population of three-way F1 cross and their progeny population (F2). An LOD value of 2.0 was used as criteria to indicate the putative QTL position. The additive effects and the percentage of variation explained by individual QTLs were estimated. Epistasis in the parameters was analyzed using the program QTL mapper (Wang et al., 1999). For the designation of QTLs, we followed the recommendation by McCouch et al. (1997).
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RESULTS
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Linkage Map Construction
A molecular map with 108 SSR markers, distributed across all chromosome arms of the rice genome, was constructed using the three-way cross population. The total map length was 1708 centimorgans (cM) with an average distance between markers of 15.8 cM. Two gaps on chromosomes 2 and 3 of 47.9 cM and 54.5 cM, respectively, were due to limited marker polymorphisms in those regions. The marker order of this map agreed with a previously published map (Temnykh et al., 2000). The percentage of heterozygous regions ranged from 37 to 63%, not significantly different from the expected 50%. Segregation distortion was observed at 11 loci on chromosomes 3, 5, 6, 7, 8, 9, and 11 at P = 0.05. The frequency of indica alleles increased in regions on chromosome 3 (long arm), 5, 6, 7, and 9, while that of japonica alleles increased on chromosomes 3 (short arm) and 8 (Fig. 1)
. Such distorted segregations in mapping populations have been frequently reported, which affect the detection of QTLs (Xu et al., 1997; Harushima et al., 2002), but in the present population only 10% of markers showed a significant deviation from the expected 1:1 ratio.
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Fig. 1. Simple sequence repeats (SSR) linkage map and putative quantitative trait loci (QTLs) for seed dormancy. White and black circles represent putative region of QTLs for seed dormancy detected in the three-way cross and F2 populations, respectively. Black bars represent two epitatic loci. Loci with significant segregation distortion from the expected segregation ratio (P < 0.05) are marked with I or J, where the frequency of indica allele and that of japonica allele increased, respectively.
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Seed Dormancy Segregation
Germination rates of twelve F2 plants from each F1 plant were evaluated. The mean values for IR50, Tatsumichi, and Miyukimochi were 16.3 ± 2.1%, 80.1 ± 3.8%, and 61.0 ± 3.5%, respectively, confirming that IR50 has a high level of seed dormancy while Tatsumimochi and Miyukimochi produce virtually nondormant seed. Continuous variations were observed both in the F1 population and in their progeny population (Fig. 2
and Fig. 3)
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Fig. 2. Frequency distribution of seed dormancy in F1 generation of a rice population constructed from crosses among IR50/Tachimimochi//Miyukimochi. (The arrows denote the dormancy levels of the parents.)
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Fig. 3. Frequency distribution of seed dormancy in F2 generation of a rice population constructed from crosses among IR50/Tachimimochi//Miyukimochi. (The arrows denote the dormancy levels of the parents.)
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QTLs for Seed Dormancy
Three putative QTLs associated with the seed dormancy were detected in the F1 population based on composite interval mapping. The QTLs were located in the vicinity of RM315 and RM104 on chromosome 1, RM251 and RM7 on chromosome 3, and RM11 and RM10 on chromosome 7 (Fig. 1, Table 1), where IR50 alleles reduced germination. The additive effect of the QTL on chromosome 3, defined as the dormancy difference between the value of IR50 genotype (II) and the mean of the two japonica genotypes (JJ), was the most significant and percentages of the variance explained by the QTL (PVE) was 21%.
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Table 1. Putative QTLs controlling seed dormancy found in two generations of a rice population constructed from crosses among IR50/Tachimimochi//Miyukimochi and evaluated under field conditions at Nanjing, China.
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In the F2 population, two QTLs were detected in the vicinity of RM315 and RM104 on chromosome 1 and RM7-RM282 on chromosome 3 (Fig. 1, Table 1). The additive effects of IR50 alleles and PVE in the QTL on chromosome 3 were larger than those on chromosome 1.
The map locations of the putative QTLs on chromosome 1 and 3 detected in the F1 coincided with those detected in the F2 population. The putative QTL on chromosomes 7 was not identified in the F2 generation.
Based on the marker genotypes lying closest to the peak in each of the three QTL-containing genomic regions, t tests were conducted to characterize the mode of gene action at the three loci (Table 2). Supporting the linkage of these markers to QTLs for seed dormancy, the germination score was significantly differentiated by genotypes at the three markers RM104, RM7, and RM10 in the F1 population and at the two markers RM104 and RM7 in the F2 population.
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Table 2. Mean percentage germination of seed classified by genotypes in the two generations of a rice population constructed from crosses among IR50/Tachimimochi//Miyukimochi.
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Epistatic Effects on Seed Dormancy
In the three-way cross F1 generation, epistasis between loci on chromosomes 1 and 11 explained about 2.8% of the total variation (Table 3). The epistasis value e = 0.33 was relatively large compared with the additive effects in Table 1. In the F2 generation, no epistasis was detected. The significant epistasis for seed dormancy was caused by a japonica-type homozygote at one locus and a indica–japonica heterozygote at the other locus, which showed relatively high levels of dormancy, while japonica homozygote at the two loci and indica–japonica heterozygote at the two loci resulted in low dormancy (Table 3).
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Table 3. Epistasis associated with seed dormancy in F1 generations of a rice population constructed from crosses among IR50/Tachimimochi//Miyukimochi.
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DISCUSSION
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Seed dormancy in rice is a transient, genetically complex trait, detectable only briefly after seed maturation. In the past, one orthologous Vp1 gene (McCarty et al., 1991) for the dormancy-related maize transcription factor viviparous-1 was detected on rice chromosome 1 (Bailey et al., 1999). Four QTLs (Wan et al., 1997) on chromosomes 3, 6, 7, and 12, respectively, and five QTLs (Lin et al., 1998) on chromosomes 3, 5, 7 (two QTLs), and 8, respectively, all associated with dormancy in rice, were detected. A recent study (Cai et al., 2000) reported that many chromosomes in rice contain one or two QTLs for seed dormancy. It seems very difficult right now to review these results to find a set of stably functioning genes for seed dormancy. Meanwhile, all the previous studies were conducted on morphological or RFLP marker analyses rather than PCR-based marker analysis, increasing the utility of these markers. However genetic x environmental interactions on the QTL analysis may limit their effectiveness similar to the limitations of previous markers in earlier studies.
The QTL qSD-1 is located in the genomic region of the orthologous Vp1 gene since the Vp1 gene was located between the C86 and C12 markers on rice chromosome 1 (Quarrie et al., 1997; Bailey et al., 1999), also corresponding to the region of RM104 (McCouch et al., 2002). It is difficult to compare the other QTLs in this paper with others due to the different parents and markers and lack of sequence information to assess homology. The QTL qSD-7 was detected only in F1 generation, similar to the results of Wan et al. (1997), who reported that dormancy controlled by one QTL on chromosome 7 was easily broken during storage. The qSD-7 QTL was not identified in the F2 generation reflecting either genotype x environment interactions or Type I error (P < 0.05). Seed dormancy is one of the postharvest seed-germination traits. The SSR markers linked to dormancy QTLs reported in this study may facilitate selection of cultivars with adequate level of seed dormancy through marker-assisted selection because of its technical simplicity, small amount of starting DNA required, relatively low cost for the user, and rapid turn-around time. Development of NILs different from those with specific QTLs associated with rice seed dormancy in this study is underway to perform the fine mapping of genes controlling dormancy to elucidate the stable biological functions of the dormancy genes.
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ACKNOWLEDGMENTS
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We are indebted to the editor and anonymous reviewers for their important improvement of this article. This study was sponsored by National high-tech project (No. 2003AA207020, 2001AA241024, 2002AA221001-06), China; High-tech project (No. BG2001301, Jiangsu Province), China.
Received for publication February 22, 2004.
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INTRODUCTION
