National Agricultural Research Center for Tohoku Region, 3 Shimofurumichi, Yotsuya, Omagari, Akita , Japan 2)

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1 Breeding Science 52 : (2002) Quantitative Trait Loci for Nonstructural Carbohydrate Accumulation in Leaf Sheaths and Culms of Rice (Oryza sativa L.) and their Effects on Grain Filling Kenji Nagata* 1), Hiroyuki Shimizu 2) and Tomio Terao 3) 1) National Agricultural Research Center for Tohoku Region, 3 Shimofurumichi, Yotsuya, Omagari, Akita , Japan 2) National Agricultural Research Center for Hokkaido Region, 1 Hitsujigaoka, Toyohira, Sapporo, Hokkaido , Japan 3) National Agricultural Research Center (Hokuriku Research Center), Inada, Joetsu, Niigata , Japan Quantitative trait loci (QTLs) that affect the accumulation of nonstructural carbohydrates (NSCs) in the leaf sheaths and culms of rice (Oryza sativa L.), enhancing subsequent grain filling, were analyzed in a population of back-crossed inbred lines (BCILs) from the cross Sasanishiki (japonica)/habataki (indica)//sasanishiki/// Sasanishiki. QTLs affecting NSC accumulation were detected on chromosomes 1, 4, 5, 7, 11 and 12. Of these, the indica alleles of QTLs on chromosomes 7 and 12 increased NSC accumulation, but were detected for only one year in the two-year study. These loci also showed a strong effect on the number of days to heading, suggesting that their effect on NSC accumulation was linked to environmental conditions before heading. The QTLs on chromosomes 1, 4, 5 and 11 did not affect appreciably the number of days to heading. The QTL on chromosome 1 might be identical with the QTL that increases the number of spikelets per panicle in the indica allele, implying that it has pleiotropic effects. The indica allele of this QTL reduced NSC accumulation, which led to an increased percentage of incompletely filled spikelets. In contrast, the indica allele of the QTLs found on chromosomes 5 and 11 increased NSC accumulation and did not affect appreciably the number of spikelets. These indica allele QTLs also exerted some effect on the reduction of the percentage of incompletely filled spikelets, suggesting the possibility that the expression of these QTLs could improve grain filling in rice. Key Words: QTL, rice, nonstructural carbohydrates, grain filling, number of days to heading. Introduction Communicated by Y. Ukai Received January 28, Accepted August 8, *Corresponding author ( knagata@affrc.go.jp) Grain filling is an important process in determining the ultimate yield of rice (Matsushima 1957). Extensive investigations have shown that grain filling and yield are affected by the amount of carbohydrates supplied by photosynthesis after heading, and by the reserves of nonstructural carbohydrates (NSCs) that accumulate in leaf sheaths and culms before heading (Matsushima 1957, Wada 1969, Ohyama 1977, Weng et al. 1982, Song et al. 1990, Kusutani et al. 1993, Sumi et al. 1996, Tsukaguchi et al. 1996, Kusutani et al. 1999). NSCs that accumulate in leaf sheaths and culms before heading are usually translocated to panicles (Song et al. 1990), and are reported to play an important role in compensating for the lack of a source supply after heading (Kobata and Takami 1983, Sumi et al. 1996). We also showed that the NSC accumulation before heading prevents incomplete grain filling when dry matter production after heading is limited (Nagata et al. 2001). NSC accumulation may be particularly important for grain filling in high-yielding cultivars that bear a large number of spikelets, which requires a large supply of carbohydrates. We previously investigated NSC accumulation in cultivars with a large sink, and suggested that a larger NSC accumulation per spikelet would improve grain filling (Nagata et al. 2001). To achieve maximum grain filling in highyielding rice, the genetic basis of NSC accumulation must be elucidated. Previous studies showed that indica cultivars accumulate a larger amount of NSCs before heading in leaf sheaths and culms than do japonica cultivars (Ishikawa et al. 1993). Therefore, for breeding programs designed to produce cultivars with a higher level of grain filling, it would be useful to identify the indica loci linked to the increase of NSC accumulation. A technique for analyzing quantitative trait loci (QTLs) has been developed using restriction fragment length polymorphism (RFLP) markers (Soller et al. 1976, Lander and Botstein 1986, Nienhuis et al. 1987, Lander and Botstein 1989, Haley and Knott 1992). The application of this technique in a crossed population of indica and japonica may contribute to the identification of the loci affecting NSC accumulation. QTL analysis has been used to study many agronomic traits in rice (Yano et al. 1995, Lin et al. 1996, Lu et al. 1996, Wu et al. 1996, Xiao et al. 1996, Zhuang et al. 1997, Redoña and Mackill 1998, Xiao et al. 1998, Moncada et al. 2001). We previously applied this technique to examine sink size and grain filling in rice, and detected QTLs that control grain filling with or without affecting the sink size (Nagata et al. submitted for publication). The effects of these QTLs on NSC accumulation have not yet been analyzed in rice or in other graminaceous crops, except for perennial ryegrass (Turner et al. 1999).

2 276 Nagata, Shimizu and Terao In this study, the QTLs of back-crossed inbred lines (BCILs) of rice, derived from inter-subspecific crosses between indica and japonica cultivars were analyzed in order to identify the QTLs responsible for NSC accumulation in the leaf sheaths and culms. The QTLs detected in this study were compared with those previously identified and found to control sink size and grain filling. Materials and Methods Plant materials and field experiments The plant population consisted of 85 BCILs derived from the cross Sasanishiki/Habataki//Sasanishiki///Sasanishiki (Sasanishiki Habataki) (Hirayama et al. 1999). The semidwarf indica cultivar (Habataki) has a large number of spikelets per panicle (Kobayashi et al. 1990), while the japonica cultivar (Sasanishiki) is characterized by a large number of panicles per plant and properties that make it more suitable for consumption in Japan. Seeds of BC 2 F 4, obtained using the single seed descent method after the production of BC 2 F 1 from Sasanishiki Habataki, were supplied by the National Institute of Agrobiological Sciences, and BC 2 F 4 and BC 2 F 6 were grown in a paddy field at the Hokuriku National Agricultural Experiment Station (Joetsu, Japan) in 1998 and 2000, respectively. These lines were planted in double rows, and double rows of each of the parent cultivars were grown in every ten lines as controls. Fertilizer containing 5 g m 2 each of N, P 2 O 5 and K 2 O as a basal dose was applied just before transplanting in both years. At heading, the date when 50 % of the panicles had emerged was recorded, and five plants from each of the 85 lines, as well as five plants of each parent from five positions, were sampled. For each plant, the dry weight, the NSC content of the leaf sheaths and culms and the number of spikelets were determined. The NSC content in milled dry samples of leaf sheaths and culms was measured spectrophotometrically using p-hydroxybenzoic acid hydrazide to develop color in monosaccharides, as described by Nagata et al. (2001). The total amount of NSCs in the leaf sheaths and culms per plant was calculated by multiplying the NSC content by the dry weight of the leaf sheaths and culms per plant. The amount of NSCs per spikelet was calculated by dividing the total amount of NSCs in the leaf sheaths and culms by the number of spikelets. The ripening percentage and the percentages of sterile and incompletely filled spikelets were determined using the samples of BCILs from Sasanishiki Habataki in 1998 and 1999, as described in a previous study (Nagata et al. submitted for publication). Data for the air temperature and solar radiation conditions in the fields at the Hokuriku National Agricultural Experiment Station, where this experiment was carried out, were obtained from the Laboratory of Agricultural Meteorology at the Hokuriku National Agricultural Experiment Station. Fig. 1 shows the average daily mean air temperature and daily total solar radiation in early, mid- and late July and August, for 1998 and The periods during which head- Fig. 1. Average daily mean air temperature (lines) and daily total incident solar radiation (bars) in early, mid-, and late July and August, in 1998 and Closed bars and circles with solid lines, 1998; open bars and squares with broken lines, The heading period in the back-crossed inbred lines (BCILs) and parents is also shown: H, Habataki; S, Sasanishiki. Air temperature and solar radiation were measured at a meteorological observation site in the experiment fields at the Hokuriku National Agricultural Experiment Station.

3 Quantitative trait loci for nonstructural carbohydrate accumulation of rice 277 ing of the BCILs and parents occurred in 1998 and in 2000 are also shown. Solar radiation was particularly low in early July in 1998, compared to 2000, and the air temperature in mid-july was markedly lower in 1998 than in 2000, although the solar radiation during this time recovered to almost the same level as that recorded in In August, both the daily solar radiation and daily air temperature, on the average, were significantly lower in 1998 than in RFLP linkage analysis and QTL mapping The segregation data for 238 RFLP markers and their linkage map, constructed by Hirayama et al. (1999), were used to analyze the BCILs. QTL analysis was conducted with the simple interval mapping procedure using QGene (ver v; Nelson 1997), and a LOD score of 3.0 was used to detect putative QTLs. The additive effect and percentage of phenotypic variance associated with a QTL were also estimated using QGene. Details were reported in a previous paper (Nagata et al. submitted for publication). Results Phenotypic variation The frequency distribution of the NSC content in the leaf sheaths and culms, the total amount of NSCs in the leaf sheaths and culms per plant, the amount of NSCs per spikelet, and the number of days to heading from transplanting for the BCILs from Sasanishiki Habataki are shown in Fig. 2. Habataki showed a larger NSC accumulation and a higher number of days to heading than Sasanishiki in both years. In the BCILs, the mean NSC accumulation and number of days to heading were close to the mean of both parents in 1998, but were as low as, or lower than those of the lower parent (Sasanishiki) in The reason for this phenomenon is not clear, but differences in the meteorological conditions that led to earlier heading in 2000 (Fig. 1), particularly in the BCILs compared to the parents (Fig. 2 D1 and D2), may be incriminated. Nevertheless, transgressive segregation in the BCILs in both directions was observed for the measured traits in both years. Mapping of QTLs for NSC accumulation and number of days to heading Table 1 and Fig. 3 show the putative QTLs, or the peaks of LOD, for NSC accumulation and the number of days to heading in the BCILs from Sasanishiki Habataki, detected by simple interval mapping. Nine putative QTLs were found on chromosomes 1, 5, 7, 8 and 12 in 1998 (Table 1, Fig. 3). The analysis in 2000 revealed the presence of 14 putative QTLs on chromosomes 1, 4, 5, 7, 11 and 12 (Table 1, Fig. 3). Five of the QTLs were detected in both years. The QTLs detected in both years included a QTL for the total amount of NSCs on chromosome 5 between the markers C1018 and C466 and a QTL for the amount of NSCs per spikelet in the same region, as well as three QTLs for the number of days to heading, one on chromosome 7 between R2401E and G1068S, and two on chromosome 12 between C1336EH and R2672 and between R1436RH and R1709H (Table 1, Fig. 3). Near the QTLs for the total amount of NSCs and the amount of NSCs per spikelet on chromosome 5, a QTL for the NSC content was also detected, although this was significant only in In addition, QTLs for the total amount of NSCs were detected near each of the three QTLs for the number of days to heading on chromosomes 7 and 12, but only in 2000 (Fig. 3). Two QTLs for the amount of NSCs per spikelet were also detected near the QTLs for the number of days to heading on chromosome 12. The indica allele increased the NSC accumulation or delayed heading. This finding strongly suggests that the effects of QTLs, leading to a longer vegetative growth period before heading, resulted in the accumulation of a larger amount of NSCs in leaf sheaths and culms, when the environmental conditions consistently favored carbohydrate assimilation. Indeed, a significant positive relationship was observed between the number of days to heading and the total amount of NSCs in 2000 (r = 0.394**), whereas no such relationship was observed in 1998 (r = 0.02 ns ). Other QTLs were detected in one year for the total amount of NSCs per plant and the amount of NSCs per spikelet on chromosome 1 between the markers S14064EH and C470, for the amount of NSCs per spikelet on chromosome 4 between C1100SH and R514, for the total amount of NSCs on chromosome 5 between G1458 and C1268E, for the number of days to heading on chromosome 8 between C277 and C1121, and for the NSC content and the total amount of NSCs on chromosome 11 between G1465S and C82 (Fig. 3). In the QTL on chromosome 1, the japonica allele increased the NSC accumulation both per plant and per spikelet (Table 1, Fig. 3), while in the QTLs on chromosomes 4, 5 and 11 the indica allele increased this accumulation (Fig. 3). Effects of QTLs for NSC accumulation on grain filling The QTLs for NSC accumulation were compared with the QTLs found to affect sink size and grain filling in a previous study (Nagata et al. submitted for publication). Interesting regions were found on chromosomes 1, 5 and 11. Fig. 4 shows the interval mapping of QTL analysis on chromosomes 1, 5 and 11 for the number of spikelets per plant (Fig. 4 A1, A5 and A11), the percentage of incompletely filled spikelets (Fig. 4 B1, B5 and B11), and the total amount of NSCs (Fig. 4 C1, C5 and C11). On chromosome 1, QTLs with a large effect on the number of spikelets per plant (A1) and on the percentage of incompletely filled spikelets (B1), as shown in a previous paper (Nagata et al. submitted for publication), were detected at the same location as the QTL for the total amount of NSCs (C1). This finding indicated that the indica allele at this locus increased the sink size, but decreased NSC accumulation, and hence, decreased grain filling. The two loci on chromosomes 5 and 11, at which the indica allele increased the total amount of NSCs (Fig. 4 C5

4 278 Nagata, Shimizu and Terao Fig. 2. Frequency distribution of the traits related to nonstructural carbohydrate (NSC) accumulation and the number of days to heading in the BCILs of rice derived from the cross Sasanishiki/Habataki//Sasanishiki///Sasanishiki, and their parental cultivars. A1 and A2, NSC content in leaf sheaths and culms; B1 and B2, total amount of NSCs in leaf sheaths and culms per plant; C1 and C2, amount of NSCs per spikelet; D1 and D2, number of days to heading. A1, B1, C1 and D1 are the results of the 1998 experiment, while A2, B2, C2 and D2 are the results of the 2000 experiment. The bars and closed arrowheads indicate the ranges of distribution and the means, respectively, of the parents Habataki (H) and Sasanishiki (S). The open arrows indicate the means of the BCILs. and C11), did not affect the number of spikelets at all (Fig. 4 A5 and A11), but exerted a reducing effect on the percentage of incompletely filled spikelets (Fig. 4 B5 and B11). Although the LOD score of the peak of both loci for the percentage of incompletely filled spikelets was around 2.0, and was not significant, the peaks were stable for the experiments in both years (Fig. 4 B5 and B11). A QTL for the amount of NSCs per spikelet on chromosome 4 (Fig. 3) also did not affect the number of spikelets (data not shown); it exerted some effect on the percentage of incompletely filled spikelets, but the effect was small and unstable (data not shown). Discussion The objective of this study was to identify QTLs controlling NSC accumulation in leaf sheaths and culms before heading in rice, and to analyze their subsequent effects on grain filling. Several significant regions affecting the NSC accumulation before heading were identified using BCILs

5 Quantitative trait loci for nonstructural carbohydrate accumulation of rice 279 Table 1. Putative quantitative trait loci (QTLs) detected for traits related to NSC accumulation and number of days to heading for BCILs of rice developed from the cross Sasanishiki/Habataki//Sasanishiki///Sasanishiki Traits 1) NSC content in leaf sheaths and culms (nscc) Chromosome number Location between Distance 2) LOD Additive effect 3) Variance explained (%) Distance LOD Additive effect Variance explained (%) 5 C1018 R G1465S C Total amount of NSCs in leaf sheaths and culms per plant (nscp) Amount of NSCs per spikelet (nscs) 1 R1613 C G1458 C1268E C1018 C R646 R G1465S C C1336EH R S1436EH R1709H S14064EH R C1100SH R C1018 R C1336EH R R1436EH R1709H Number of days to heading from transplanting (dh) 7 R2401E G1068S C277 C C1336EH R S1436EH R1709H ) Abbreviations shown here are also used in Fig. 3. 2) Distance from left markers. 3) + and signs indicate the positive and negative additive effect of the Habataki allele, respectively. derived from the cross between Sasanishiki (japonica) Habataki (indica). These were located on chromosomes 1, 4, 5, 7, 11 and 12 (Table 1, Fig. 3). The QTLs affecting NSC accumulation on chromosomes 7 and 12 coincided with the QTLs for the number of days to heading (Table 1, Fig. 3), which strongly suggests that these QTLs have pleiotropic effects. The indica allele of these loci increased NSC accumulation with delayed heading. A longer period of vegetative growth before heading might result in a larger NSC accumulation. Consequently, environmental conditions before heading might affect the expression of the effects of these QTLs. These QTLs for NSC accumulation on chromosomes 7 and 12 were detected only in 2000, while the QTLs for the number of days to heading were detected in both years (Fig. 3). This phenomenon might be explained by the differences in the meteorological conditions between the two years, as the experiments were conducted in the same field with the same fertilizer application in both years. In 2000, both solar radiation and air temperature were constantly high from July through August (Fig. 1). Accordingly, carbohydrate production was constantly high, and the longer the period for vegetative growth, the larger the NSC accumulation. Therefore, these QTLs for NSC accumulation were actively expressed in In 1998, however, low air temperatures with high solar radiation occurred in July, which might have led to increase NSC accumulation in the early heading lines because such conditions promote the transformation of photoassimilates to NSCs rather than to structural components (Kumura 1956, Sato 1966). Although the vegetative growth period was prolonged, an insufficient amount of NSCs might have accumulated during the period of low solar radiation observed in August Consequently, delayed heading due to the effect of the indica allele for the QTLs on chromosomes 7 and 12 may not have influenced NSC accumulation in 1998, so that their effect may not have been expressed. The QTLs on chromosomes 1, 4, 5 and 11 did not affect the number of days to heading. Therefore, the duration of vegetative growth was not the reason for their effect on NSC accumulation. Of these, the japonica allele of the QTL on chromosome 1 increased NSC accumulation (Fig. 3 and Fig. 4 C1), this QTL might be identical with the QTL for which the indica allele drastically increased the number of spikelets per plant (Fig. 4 A1) (Yagi et al. 2001, Nagata et al. submitted for publication). In addition, the indica allele for this locus reduced the ripening percentage by increasing the percentage of incompletely filled spikelets (Fig. 4 B1) (Nagata et al. submitted for publication). This suggests that the increase in the number of spikelets per panicle in the indica allele for this locus decreased the NSC accumulation, proba-

6 280 Nagata, Shimizu and Terao Fig. 3. RFLP linkage map showing the locations of QTLs for the traits related to NSC accumulation and the number of days to heading for the BCILs of rice derived from the cross Sasanishiki/Habataki//Sasanishiki///Sasanishiki. Markers are shown on the right side of the chromosomes. Arrowheads indicate the peak positions of the LOD, and dark and black bars indicate the regions with one LOD support interval in the 1998 and 2000 experiments, respectively. Trait designations are abbreviated as follows: nscc, NSC content; nscp, total amount of NSCs per plant; nscs, amount of NSCs per spikelet; dh, number of days to heading. Abbreviations enclosed in boxes indicate the traits that increased in the Habataki alleles, and the others in the Sasanishiki alleles. The chromosomes are arranged so that the short arm is at the bottom. bly because some of the NSCs were used to produce larger panicles during the panicle-developing period before heading. This low NSC accumulation might be one of the reasons for poor grain filling with the indica allele for this locus, because a low NSC accumulation before heading is reported to reduce grain filling (Sumi et al. 1996, Tsukaguchi et al. 1996). Hence, the japonica allele for this locus reduces the number of spikelets per panicle, although NSC accumula-

7 Quantitative trait loci for nonstructural carbohydrate accumulation of rice 281 Fig. 4. Interval mapping of QTL analysis on chromosomes 1, 5 and 11 for the traits related to sink size, incompletely filled spikelets and total amount of NSCs for the BCILs from the cross Sasanishiki/Habataki//Sasanishiki///Sasanishiki. A, number of spikelets per plant; B, percentage of incompletely filled spikelets; C, total amount of NSCs per plant. The suffixes 1, 5 and 11 denote chromosomes 1, 5 and 11, respectively. The number of spikelets per plant and the percentage of incompletely filled spikelets were plotted using data obtained in a previous study (Nagata et al. submitted for publication). Solid and broken lines in A and B indicate the LOD scores for the 1998 and 1999 experiments, whereas those in C indicate the LOD scores for the 1998 and 2000 experiments, respectively. LOD scores of loci showing positive and negative additive effects with the Habataki allele are indicated above and below, respectively. Chromosomes are arranged so that the short arm is on the left side. The locations of the markers on each chromosome are shown at the bottom. Thin lines indicate significant levels for LOD score = 3.0. Ticks on the x-axis represent 20 cm. tion and grain filling might be improved. Thus, the effect of this locus on NSC accumulation seems to be pleiotropic, and may not occur alone. In contrast, the QTLs on chromosomes 5 and 11 for NSC accumulation did not affect appreciably the sink size (Fig. 4 A5 and A11), but they exerted some effect on incompletely filled spikelets (Fig. 4 B5 and B11). The larger amount of NSCs accumulated before heading, resulting from the indica allele for these loci, reduced the percentage of incompletely filled spikelets. The QTL for NSC accumulation on chromosome 5 was detected in both years (Table 1, Fig. 3 and Fig. 4 C5). Although the QTL for NSC accumulation on chromosome 11 was detected only in 1998 (Table 1, Fig. 3), a peak of LOD was also observed at almost the same location in 2000 (Fig. 4 C11). Accordingly, environmental conditions are considered to have exerted a reduced effect on these QTLs on chromosomes 5 and 11. Therefore, these QTLs for NSC accumulation might be useful for improving grain filling in rice. The QTL for the amount of NSCs per spikelet on chromosome 4 might also be useful, but this needs to be confirmed. Although none of the QTLs for NSC accumulation detected in this study were linked, the population of 85 BCILs used in this experiment contained distorted segregation markers on chromosomes 3, 4, 6 and 9 (Hirayama et al. 1999). This segregative distortion may reduce the efficiency in detecting significant QTLs, and the percentage of variance explained in the detected QTLs. In addition, the number of measured plants per line in this experiment (five plants per line) might be insufficient. Increasing the number of measured plants might help to determine the presence of other minor QTLs for NSC accumulation. In conclusion, two types of QTLs affect NSC accumulation in the leaf sheaths and culms in rice: the first acts in association with the heading date, while the second behaves independently. Among the latter, the QTLs found on chromosomes 5 and 11 may be valuable for improving grain filling because they do not exert deleterious effects on other traits, such as sink size, and are environmentally stable. Acknowledgments This experiment was supported in part by a Grant-in- Aid from the Ministry of Agriculture, Forestry and Fisheries

8 282 Nagata, Shimizu and Terao of Japan (Bio-Design Program; no. BDP-02-I-1-5). The authors would like to thank Dr. M. Yano of the Rice Genome Research Program (RGP), a joint project of the National Institute of Agrobiological Sciences of Japan and the Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries of Japan, for supplying the Sasanishiki Habataki BCILs seeds, and to Drs. M. Yano, S.Y. Lin and T. Hirayama of the RGP for their permission to use their marker genotype data. The authors would also like to thank the Laboratory of Agro-Meteorology at the National Agricultural Research Center (Hokuriku Research Center) for supplying the meteorological data. In addition, the authors are grateful to Mr. K. Fukunaga, Ms. K. Nozaki, Ms. U. Watanabe, Ms. S. Hayashi and Ms. K. Takatsuto for their excellent technical assistance, and to Drs. T. Hirose, H. Tabuchi and M. Chiba for their helpful discussions. Literature Cited Haley, C.S. and S.A. 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