Genetic Analysis of Two Weak Dormancy Mutants Derived from Strong Seed Dormancy Wild Type Rice N22 (Oryza sativa) F

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1 Journal of Integrative Plant Biology 211, 3 (): Research Article Genetic Analysis of Two Weak Dormancy Mutants Derived from Strong Seed Dormancy Wild Type Rice (Oryza sativa) F Bingyue Lu 1, Kun Xie 1, Chunyan Yang 1, Long Zhang 1, Tao Wu 1,XiLiu 1, Ling Jiang 1 and Jianmin Wan 1,2 1 National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 219, China 2 National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 11, China Corresponding authors Tel (Fax): ; rice@njau.edu.cn, wanjm@njau.edu.cn F Articles can be viewed online without a subscription. Available online on 21 March 211 at and doi: /j x Abstract Two weak dormancy mutants, designated and, were obtained from the rice cultivar after treatment with 4 Gy 6 Co gamma-radiation. Compared to the cultivar, the dormancy of the mutant seeds was more readily broken when exposed to a period of room temperature storage. The mutants also showed a reduced level of sensitivity to abscisic acid compared to the cultivar, although was more insensitive than. A genetic analysis indicated that in both mutants, the reduced dormancy trait was caused by a single recessive allele of a nuclear gene, but that the mutated locus was different in each case. The results of quantitative trait locus (QTL) mapping, based on the F 2 population from x Nanjing3, suggested that lacks the QTL qsdn-1 and carries a novel allele at QTL qsdn-9, while a similar analysis of the x Nanjing3 F 2 population suggested that lacks QTL qsdn-, both qsdn-1 and qsdn- are major effect seed dormancy QTL in. Therefore, these two mutants were helpful to understand the mechanism of seed dormancy in. Lu B, Xie K, Yang C, Zhang L, Wu T, Liu X, Jiang L, Wan J (211) Genetic analysis of two weak dormancy mutants derived from strong seed dormancy wild type rice (Oryza sativa). J. Integr. Plant Biol. 3(), Introduction Seed dormancy has evolved in plants as a way to ensure survival of the seed in the soil. Seed dormancy also represents an agronomically important trait in many crop species. Unraveling the genetic basis of seed dormancy by conventional methods is complicated because of its polygenic nature, and the large influence exerted on the trait by the pre- and postharvest environment (Koornneef et al. 22). In Arabidopsis thaliana, a large number of mutants affecting seed dormancy have been generated artificially, and the genetic, physiological, and molecular characterization of these mutants are starting to shed light on the complexity of the regulation of this process. Many mutations affecting dormancy actually effect abscisic acid (ABA) synthesis and regulation (Kermode 2; Nambara et al. 21). Thus, at least in A. thaliana, ABA appears to play an important role in the expression of seed dormancy. The current knowledge about the underlying genes causing dormancy in crop species is limited. Particular effort has been directed towards understanding the dormancy trait in cereals, since in most cases, their adequate level of dormancy prevents pre-harvest sprouting when rainfall is high around the period of crop maturity. In rice, Agrawal et al. (21) reported a C 211 Institute of Botany, Chinese Academy of Sciences

2 Genetic Analysis of Two Weak Dormancy Mutants Derived from Rice 339 viviparous (non-dormant) mutant, and showed that its phenotype was due to a defective gene encoding for zeaxanthin epoxidase (part of the ABA synthesis pathway). Recently, a delayed-germination mutant, OsDSG1, was identified in rice as an E3 ligase targeting ABI3 (Park et al. 21). Similarly in wheat, Rikiishi and Maekawa (21) managed to isolate a reduced seed dormancy mutant, RSD32, which showed not only reduced seed dormancy, but also sensitivity to ABA upon germination. Similar mutants associated with seed dormancy have been reported in barley (Molina-Cano et al. 1999). All of these examples indicate that ABA is an important factor in controlling cereal seed dormancy. Many studies have been performed to detect quantitative trait locus (QTL) for rice seed dormancy (Wan et al. 1997; Lin et al. 199; Gu et al. 24; Guo et al. 24; Wan et al. 26). However, progress has been slow due to complicated genetic mechanisms. To date, only one rice dormancy gene, Sdr4, has been successfully cloned (Sugimoto et al. 21). The expression of this gene is positively regulated by OsVP1, a global regulator of seed maturation, which in turn interacts with supposed regulators of seed dormancy and acts to repress the expression of the genes, which are triggered after germination. Analyses of the activity of the weedy rice (also known as red rice; Oryza sativa L.) QTL qsd12 suggests that it promotes ABA accumulation during early seed development (Gu et al. 21). Thus, it appears that in rice, as well as in A. thaliana, seed dormancy is associated with ABA. Creating rice seed dormancy mutants is a practical approach to research the dormancy mechanisms., the long established indica rice cultivar (O. sativa ssp. Indica), expresses a strong level of seed dormancy. Intact indica seeds harvested 3 d after heading displayed <2% germination following a 7 d period of imbibition (Wan et al. 26). The action of one or two major genes has been proposed to be responsible for this dormancy (Seshu and Sorrells 196; Gu et al. 23). In previous study, Wan et al. (26) constructed three simple sequence repeat (SSR)-based linkage maps using Nanjing3///Nanjing3 BC 1 (PI), USSR///USSR BC 1 (PII) and USSR/ F 2 (PIII) population to detect the locus controlling seed dormancy in. Five putative QTLs associated with seed dormancy, namely qsdn-1, qsdnj-3, qsdn-, qsdn-7, and qsdn-11 were detected in these three populations through composite interval mapping (Wan et al. 26). In support of these results, Lu et al. (21) showed that qsdn-1 and qsdn- displayed major effects on seed dormancy in. In another study by Xie et al. (21), three seed dormancy QTLs, qsd-1, qsd-2 and qsd-3, were detected through the Nanjing3///Nanjing3 BIL population. Furthermore, qsd-1 was detected to be a major dormancy QTL in the advanced backcross population of Nanjing3/// (BC 6 F 2 ), and resolved into qsd-1 1 and qsd-1 2 (Xie et al. 21). Two mutants, and, were obtained from seeds through treatment with 4Gy 6 Co gamma-radiation. Seeds obtained from and express weaker dormancy compared to the wild-type. The phenotype of has previously been partially described by Qin et al. (21). In this report, the specific phenotype and ABA sensitivity of both mutants were described, and the genetic basis of seed dormancy was studied. Results Screening of and mutants seeds treated with 4Gy 6 Co gamma-radiation produced mutant populations, generation M1 to M, which were screened on the basis of its reduced seed dormancy phenotype. In the first screening, whole seeds of the M2 population ( plants) and that acquired physiological maturity were imbibed for germination tests. Plants with high germination rates were saved. In generation M3-M4, two genetically stable mutant lines were identified, the seeds of these two mutant lines had significantly higher germination rates than that of at 3 d after heading, and displayed reduced seed dormancy. These lines were designated as and. Germination, morphological and phonological characterization of the two mutants The wild-type cultivar has very strong seed dormancy. Intact seeds harvested 3 d after heading displayed <2% germination 7 d after imbibition (Wan et al. 26). On the other hand, the mutants, and, displayed very weak dormancy compared with the germination rate was 43% and 4%, respectively, 7 d after imbibition. The levels of the mutants seed dormancy differed significantly from (Figure 1A). The seed dormancy will normally reduce during storage. After harvest the seeds of, and were stored in room temperature (2 3 C) and the germination rates of the seeds were monitored every 1 d. The germination rates of,, and are shown in Figure 1B. The degree of dormancy of the mutants were always lower than that of the wild type, and concurrently the rates of release from dormancy of the mutants were always higher than that of the wild type. There were no significant differences between the two mutants and in morphological and phonological characterization (Table 1). Germination response of and to exogenous ABA In order to determine ABA sensitivity, the seeds of,, and were treated in high temperature ( Cfor

3 34 Journal of Integrative Plant Biology Vol. 3 No. 211 A B Days of imbibition (d) Days of room temperature storage (d) Figure 1. The germination rates of,, and. (A) The germination rates of, and after 3 d heading. (B) The germination rates of,, and during storage in room temperature. Error bars represent SE. Table 1. Morphological and phonological characterization of, and Material name PH (cm) TP KW (g) FLA (cm 2 ) HD (d) GL (mm) GW (mm) 177 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.66 FLA, flag leaf area; GL, grain length; GW, grain width; HD, heading date; KW, kernel weight; PH, plant height; TP, tillers per plant. The data are presented as means ± SD. 7 d), and non-dormancy seeds were incubated in water or different concentration of ABA. The germination percentages of,, and were 96.67%, 97.33% and 1% for incubation with distilled water, respectively. The germination rates of,, and were 94.67%, 93.33% and 6.67% for incubation with 1 µm of ABA, respectively. There were no significant differences between mutants and the wildtype. The germination of was severely inhibited when incubated with µm of ABA, only 21.33%, which is significantly lower than for the germination rates of and at 7.33% and 64.67%, respectively. When incubated with 1 µm of ABA, the germination rates of and were 6% and 3%, respectively, which was significantly higher than the rate of 3.33% of. Germination of was fully inhibited at 2 µm of ABA, whilst the germination rates of and were 4.67% and 16.67%. When inhibited at 1 µm of ABA, was fully inhibited, while the germination percentage of was 2.67% (Figure 2). These results indicate that the sensitivity of the mutants to ABA is weaker than the wild-type, and that is more insensitive to ABA than ABA concentration (μm) Figure 2. Abscisic acid (ABA) sensitivity of,, and. Germination rate for seeds incubated at different concentration of ABA after broken dormancy. Error bars represent SE. Different genes controlling seed dormancy in and The phenotypes of and were very similar. To unravel whether the mutant genes of and were

4 Genetic Analysis of Two Weak Dormancy Mutants Derived from Rice 341 A B Figure 3. The germination rates of / F 2 and / F 2 after 3 d heading. (A) / F 2. (B) / F 2. the same, reciprocal crosses were done between and. In two crosses, the plants had a strong seed dormancy, similar to, which suggests that different recessive genes controlled the reduced dormancy phenotype. / F 2 segregated into 1:1 ratio (4 plants with germination rates ranging from % to %: 6 plants with germination rates ranging from 6% to %, χ 2 =.4 < χ 2. = 3.4 P >.). / F 2 also segregated into 1 : 1 ratio ( plants with germination rates ranging from % to %: 6 plants with germination rates ranging from 6% to %, χ 2 =.4 <χ 2. = 3.4 P >.). The double recessive plants revealed higher germination rates (Figure 3). All of these results indicate that the underlying genes in and are different. The similar reciprocal crosses result also reflects the reduced germination phenotype, which were embryo-determined but not cytoplasmic in terms of inheritance. Genetic dissection of and Using and as the female parents, the mutants were crossed with the wild-type. All individuals from these crosses had wild-type germination rates; segregation of the germination rate was bimodal in the distribution curves in the two F 2 populations. In the / F 2 population, 73 wild-type and 21 mutant plants were observed, this segregation ratio fitted to 3:1 (χ 2 =.3 <χ 2. = 3.4). 72 wild-type and 269 mutant plants were observed in the F 2 population derived from / F 2, that also fitted to 3:1 ratio (χ 2 = 2.4 < χ 2. = 3.4) (Figure 4). All the results indicated the reduced A B Figure 4. Frequency distributions of the germination rate of whole seeds at 3 d after heading in the F 2 populations derived from a cross between and mutants. (A) Indicated / F 2. (B) Indicated / F 2.

5 342 Journal of Integrative Plant Biology Vol. 3 No. 211 A 3 B Nanjing3 1 1 Nanjing Figure. The germination rates of /Nanjing3 F 2 and /Nanjing3 F 2 after 3 d heading. (A) /Nanjing3 F 2. (B) /Nanjing3 F 2. seed dormancy phenotype of and were inherited as monogenic and recessive. Analysis of QTL in the two mutants In present study two segregated populations, /Nanjing3 F 2 and /Nanjing3 F 2, were developed to detect dormancy QTL among the mutants. The two populations both showed continuous variation in germination percentage and frequency distribution skewed towards low germination (Figure ), the germination rates of /Nanjing3 and /Nanjing3 were 24% and 3.67%, respectively. In /Nanjing3 F 2, 3 QTLs, qsdnj-3, qsdn-, and qsdn-9 were detected on chromosomes 3,, and 9, respectively. The QTLqSdn-9 was determined to be a novel dormancy locus, and it was mapped between SSR markers RM73 and RM1 with a likelihood-odds ratio (LOD) score of.4, explaining 11.% of the overall trait variation. The major dormancy QTL qsdn-1 was not detected in /Nanjing3 F 2. Two QTLs, qsdn-1 and qsdnj-3, were detected in /Nanjing3 F 2, the position of qsdnj-3 was accorded with the QTL in /Nanjing3 F 2, and qsdn- was not detected in /Nanjing3 F 2. The following QTLs: qsdn-2, qsdn-7 and qsdn-11 were not detected in the two populations (Figure 6, Table 2). Discussion Mutagenesis can provide an informative means of unraveling the genetic basis of complex traits, such as seed dormancy. Despite the importance of this trait for rice end-use quality, few mutants have been described to date. A number of viviparous mutants, induced by treatment with either EMS, gamma rays or thermal neutrons, were reported by Miyoshi et al. (2). These all exhibited pre-harvest sprouting, but no obvious defects for other agronomic traits. In this study, two novel mutants, and isolated from indica cultivar were examined for physiological and genetic characteristics based on seed dormancy. Comparison with showed that and have weaker seed dormancy and ABA sensitivity. The two mutants have similar germination rate, but has lower ABA sensitivity than. Genetic analyses indicated the reduced-dormancy trait of the mutants was caused by a single, recessive mutation at two distinct loci, and was embryo determined. The plant hormone ABA plays a fundamental role in seed dormancy. Many mutants related to ABA biosynthesis and responses to ABA have been studied in Arabidopsis (Kermode 2; Finkelstein et al. 2; Penfield and King 29). Taken together, these reports indicate that seed dormancy is regulated by intricate networks, including ABA biosynthesis, response to ABA. Recently, ABA- or gibberellic acid (GA)- mediated epigenetic processes regulate seed dormancy and germination also are reported (Xing et al. 27; Chinnusamy et al. 2). In rice, the qsd12 derived from weedy rice promotes ABA accumulation in early developing seeds to induce primary dormancy in rice (Gu et al. 21). Sdr4 was the first cloned dormancy QTL in rice. The ABA insensitivity of sdr4 seeds with high germination was controlled by OsVP1, VP1/ABI3, which encodes a transcription factor with the B3 domain (Sugimoto et al. 21). The germination of seeds was inhibited by the increased concentration of ABA in, while and showed lower inhibitory effects of ABA on germination. Germination of was fully inhibited at 2 µm of ABA, while the germination rates of and were at 4.67% and 16.67%, respectively. When immersed

6 Genetic Analysis of Two Weak Dormancy Mutants Derived from Rice 343 Chr.1 RM49 Chr.2 RM14 3 Chr.3 RM132 Chr. RM13 Chr.7 RM214 Chr.9 RM444 Chr.11 RM RM374 RM4 RM23 RM121 RM29 RM496 RM292 RM23 RM493 RM11162 RM RM4 RM237 RM1297 RM12 RM11669 RM1216 RM26 RM31 RM431 RM14 RM414 7 RM19 RM11 OSR14 RM6247 RM7636 RM72 RM13 RM116 RM1234 RM341 RM262 RM643 RM26 RM2 RM31 RM24 RM112 RM42 RM2 RM213 RM RM231 RM4 RM4 OSR16 RM71 RM2326 OSR13 RM21 RM22 RM411 RM16 RM33 3 RM4 RM16 RM36 RM2 RM71 RM227 RM6 RM 1 RM413 RM93 RM169 RM9 RM164 3 RM3 RM421 RM362 RM17 RM3321 RM742 RM4 RM26 RM3664 RM317 RM6313 RM334 RM263 1 RM1 RM44 RM11 RM12 RM4 RM RM234 RM11 RM24 RM172 RM73 RM1 RM434 RM27 RM27 RM3 RM32 OSR2 RM21 RM cm RM77 RM167 RM3133 RM36 RM27 RM29 RM229 RM21 RM961 RM26 RM224 Figure 6. Comparison of quantitative trait locus (QTL) for seed dormancy in different populations. 1, in /Nanjing3 F 2 ; 2, in /Nanjing3 F 2 ; 3, in Nanjing3///Nanjing3 BC 1 (Wan et al. 26); 4, in USSR///USSR BC 1 (Wan et al. 26);, in USSR/ F 2 (Wan et al. 26); 6, in Nanjing3/// BC 6 F 2 (Xie et al. 21); 7, in Nanjing3///Nanjing3 BC 1 F 7 (Xie et al. 21);, in Nanjing3///Nanjing3 BC 4 F 2 (Lu et al. 21). with 1 µm of ABA, was fully inhibited, but remained viable to germinate. Unlike the ABA-deficient mutants of Arabidopsis, the and mutants showed no wilty phenotype. Moreover, ABA-deficient mutants show a normal sensitivity to ABA. These findings suggest that and are ABA-insensitive mutants. The dormant cultivars show higher sensitivity to ABA than non-dormant cultivars (Walker-Simmons 197). This ABA-insensitivity would be predicted to result in a less dormant phenotype, as was the case for and. The greater ABA insensitivity shown by presumably reflects differences between the two mutants in the nature of the mutational events. Table 2. Quantitative trait locus (QTL) for seed dormancy detected in /Nanjing3 F 2 and /Nanjing3 F 2 /Nanjing3 F 2 /Nanjing3 F 2 QTL name MI NML LOD PVE (%) a d MI NML LOD PVE (%) a d qsdn-1 RM4-RM11669 RM qsdnj-3 OSR13-RM411 RM RM21-RM411 RM qsdn- RM3321-RM6313 RM qsdn-9 RM73-RM1 RM a, additive effect; d, dominant effect; MI, marker interval; NML, nearest mark locus of its corresponding QTL; PVE, percentage of phenotypic variance explained.

7 344 Journal of Integrative Plant Biology Vol. 3 No. 211 The seed dormancy of is controlled by one or two major genes (Seshu and Sorrells 196; Gu et al. 23). In previous study, four putative QTLs associated with the seed dormancy, qsdn-1, qsdn-, qsdn-7, and qsdn-11, were detected in using three different populations by Wan et al. (26). qsdn-1 and qsdn- as major dormancy QTL had been proved using Nanjing3///Nanjing3 (BC 4 F 2 ) populations (Lu et al. 21), qsdn-1 was mapped between RM11669 and RM1216, a more precise region, this location was slightly different compared with previous study using Nanjing3 ///Nanjing3 BC 1 population by Wan et al. (26), but according to the mapping results using USSR///USSR BC 1 population and USSR/ F 2 population, the location of qsdn- was associated with previous studies. Later, Xie et al. (21) detected the dormancy QTL using Nanjing3///Nanjing3 BIL population, and three QTLs, qsd-1, qsd-2, and qsd-3, were mapped in chromosomes 1, 2, and 3, respectively. Based on previous studies, comparing the dormancy QTL locations of mutants with the wild-type could be helpful to reveal the underlying gene of and. Two SSR-based linkage maps derived from two F 2 -type populations of /Nanjing3 F 2 and /Nanjing3 F 2. In /Nanjing3 F 2, 3 QTLs were detected, qsdnj-3, qsdn-, and qsdn-9. Compared with the previous studies of, qsdn-9 was detected as a novel dormancy locus. There are mainly three possibilities: (i) qsdn-9 itself exists in, but this QTL is unstable, easily affected by the environment; (ii) the gene mutated in qsdn-9 locus; and (iii) it exists epistatically and affects the mutant gene and qsdn-9. In /Nanjing3 F 2, only qsdn-1 and qsdnj-3 were detected when comparing with the previous study of. The marker interval of qsdn-1 was associated with the results in Nanjing3///Nanjing3 BC 1 population in the previous study (Wan et al. 26), this location was also similar to qsd-1 (Xie et al. 21). In summary (Figure 6), qsdn-1 could be simultaneously detected in different populations (Wan et al. 26; Lu et al. 21; Xie et al. 21), despite the location having varied somewhat. qsdn-2 and qsdn-7 were only detected in one population and the effect was minor. qsdn-, a major dormancy QTL, was also detected in different populations, which had already been proven by Lu et al. (21). qsdn-11 was only detected using USSR///USSR BC 1 and USSR/ F 2 populations, but not in Nanjing3///Nanjing3 BC 1 populations (Wan et al. 26), neither in the present study nor recent studies (Lu et al. 21; Xie et al. 21), indicating that there are no differences in terms of this locus between Nanjing3 and. Furthermore, the allele of qsdnj-3 increased the degree of dormancy from the non-dormant Nanjing3. Therefore, qsdn-1 and qsdn- could be inherited as the major dormancy locus, but qsdn-1 in and qsdn- in were not detected. Whether the reduced dormancy phenotype caused by qsdn-1 and qsdn- mutated or not will require further investigation. As the next step, the genes associated with dormancy in qsdn-1 and qsdn- loci will be analyzed. Materials and Methods Materials multiplication and evaluation of seed dormancy The and F 2 of /; the and F 2 of /; the and F 2 of /; the and F 2 of /; the and F 2 of /Nanjing3; the and F 2 of /Nanjing3 and the parents,, and Nanjing3 were planted in the experimental farm at Tuqiao of Nanjing Agricultural University in 2. Leaves of individual plants of the /Nanjing3 F 2 and the /Nanjing3 F 2 were collected for the DNA extraction. The temperature during the late stage of seed maturity ranged from 2 to 3 C, which is normal for rice growth and seed maturation. Parental plants were all cultivated under the same conditions. Flowering date of each plant was marked by the emergence of the first panicle from the leaf sheath. The dormancy levels of selected genotypes were assessed following the method by Wan et al. (Wan et al. 1997, 2). Filled intact grains from two panicles of each plant were collected on the 3th day after heading, for evaluation of dormancy, seeds from each plant were placed on doubled sheets of filter papers moistened with distilled water in a Petri dish of 9 cm diameter, and maintained at 3 C and 1% relative humidity for 7 d. Each plant was tested with three replications. Germination rate was determined by the emergence of radicle or/and plumule and the degree of seed dormancy was scored routinely as the mean percentage of germinated seeds from three replications. The harvested seeds were exposed to C for 7 d to break dormancy (Lu et al. 21), then they were used to detect sensitivity of the mutants to ABA. Fifty seeds were also incubated with ABA (, 1,, 1, 2,,1 µm), all germination rate tests consisted of three replications and were counted daily for 7 d, the same method used as before. After harvest the seeds of and the mutants were stored in room temperature, the germination tests were performed every 1 d, and the method of germination rate tests was conducted as before. The data were analyzed by Microsoft Excel. DNA preparation and SSR assay For DNA extraction, 1 3 cm long leaves were harvested separately from each plant of /Nanjing3 F 2, the /Nanjing3 F 2 and the parents. The methods are referred to by Dellaporta et al. (193), with some modifications. After the DNA samples were completely dissolved in TE buffer, we used a spectrophotometer to test the concentration of

8 Genetic Analysis of Two Weak Dormancy Mutants Derived from Rice 34 DNA solution. The DNA solution was diluted with water into concentrations of 2 ng/µl, then used for PCR amplification. The original sources and motifs for all of the SSR markers used in this study were based on the gramene database ( and McCouch et al. (22). Polymerase chain reaction (PCR) amplifications were performed as described in Chen et al. (1997) with minor modifications. Briefly, 1 µl reactions contained 1 ng of template DNA,.2 µm of each primer, 2. mm of each dntp, 1 buffer (free Mg 2+ ) of 1 µl, 2 mm of MgCl 2 and.u of rtaq DNA polymerase (Takara, Dalian). The PCR consisted of the following cycle, 9 C for min, followed by 3 cycles of 94 C for 3 s, C for 3 s, 72 C for 1 min, and finally by 7 min at 72 Cforthe final extension. PCR products were run on % polyacrylamide non-denaturing gels, and marker bands were revealed by the silver staining method based on Sanguinetti et al. (1994) and scored on a light box with fluorescent lamps. SSR markers were used for developing a genetic linkage map. Background genotypes of mutants and wide-type were analyzed using 661 SSR markers distributed on 12 chromosomes. QTL mapping and statistic analysis The molecular maps with 177 and 163 SSR markers, distributed across all chromosome arms of the rice genome, were constructed using /Nanjing3 F 2 and /Nanjing3 F 2, respectively. The total map lengths were and cm with average distances between markers of 11.4 and 1.2 cm, respectively. Linkage analysis was performed with mapmaker/exp3. (Lander et al. 197). QTL analysis was performed by composite interval mapping (CIM) using QTL Cartographer V2. (Wang et al. 27). A LOD score of 2. was used for suggesting the presence of putative QTL. The threshold LOD scores for detection of definitive QTL were also calculated based on 1 permutations (Churchill and Doerge 1994). Confidence intervals (CI) were obtained using positions ± 1 LOD away from the peak. More than one QTL with overlapping CI were treated as one QTL. Acknowledgements This research was supported by grants from the National Natural Science Foundation of China (347112, ), the High-tech Research and Development (63) Program (projects 29AA1111) of China, National Key Transform Program (2ZX1-6), the Jiangsu Cultivar Development Program (projects BE and BE234), and the 111 Project (B2). Received 22 Dec. 21 Accepted Mar. 211 References Agrawal GK, Yamazaki M, Kobayashi M, Hirochika R, Miyao A, Hirochika H (21) Screening of the rice viviparous mutants generated by endogenous retrotransposon Tos17 insertion. Tagging of a zeaxanthin epoxidase gene and a novel OsTATC gene. Plant Physiol. 12, Chen X, Temnykh S, Xu Y, Cho YG, McCouch SR (1997) Development of a microsatellite framework map providing genomewide coverage in rice (Oryza sativa L.). Theor. Appl. Genet. 9, Chinnusamy V, Gong Z, Zhu JK (2) Abscisic acid-mediated epigenetic processes in plant development and stress responses. J. Integr. Plant Biol., Churchill GA, Doerge RW (1994) Empirical threshold values for quantitative trait mapping. Genetics 13, Dellaporta SL, Wood J, Hicks JB (193) A plant DNA minipreparation: Version II. Plant Mol. Biol. Rep. 1, Finkelstein R, Reeves W, Ariizumi T, Steber C (2) Molecular aspects of seed dormancy. Annu. Rev. Plant Biol. 9, Guo LB, Zhu LH, Xu YB, Zeng DL, Wu P, Qian Q (24) QTL analysis of seed dormancy in rice (Oryza sativa L.). Euphytica 14, Gu XY, Liu T, Feng J, Suttle JC, Gibbons J (21) The qsd12 underlying gene promotes abscisic acid accumulation in early developing seeds to induce primary dormancy in rice. Plant Mol. Biol. 73, Gu XY, Kianian SF, Foley ME (24) Multiple loci and epistases control genetic variation for seed dormancy in weedy rice (Oryza sativa L.). Genetics 166, Gu XY, Chen ZX, Foley ME (23) Inheritance of seed dormancy in weedy rice. Crop Sci. 43, Kermode AR (2) Role of abscisic acid in seed dormancy. J. Plant Growth Regul. 24, Koornneef M, Bentsink L, Hilhorst H (22) Seed dormancy and germination. Curr. Opin. Plant Biol., Lander E, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newburg L (197) MAPMAKER: An interactive computer package for constructing primary genetic linkage maps of experimental and nature populations. Genomics 1, Lin SY, Sasaki T, Yano M (199) Mapping quantitative trait loci controlling seed dormancy and heading date in rice, Oryza sativa L., using backcross inbred lines. Theor. Appl. Genet. 96, Lu BY, Xie K, Yang CY, Wang SF, Liu X, Zhang L, Jiang L, Wan JM (21) Mapping two major effect grain dormancy QTL in rice. Mol. Breeding, online, DOI: 1.17/s McCouch SR, Teytelman L, Xu Y, Lobos KB, Clare K, Walton M, Fu B, Maghirang R, Li Z, Xing Y, Zhang Q, Kono I, Yano M, Fjellstrom R, DeClerck G, Schneider D, Cartinhour S, Ware D, Stein L (22) Development and mapping of 224 new ssr markers for rice (Oryza sativa L.). DNA Res. 9, Miyoshi K, Nakata E, Nagato Y (2) Characterization of viviparous mutants in rice. Breed. Sci.,

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