Rice Novel Semidwarfing Gene d60 Can Be as Effective as Green Revolution Gene sd1

Gene effects on the yield performance were compared among promising semidwarf genes, namely, novel gene d60, representative gene sd1 with different two source IR8 and Jukkoku, and double dwarf combinations of d60 with each sd1 allele, in a Koshihikari background. Compared with the culm length of variety Koshihikari (mean, 88.8 cm), that of the semidwarf or double dwarf lines carrying Jukkoku_sd1, IR8_sd1, d60, Jukkoku_sd1 plus d60, or IR8_sd1 plus d60 was shortened to 71.8 cm, 68.5 cm, 65.7 cm, 48.6 cm, and 50.3 cm, respectively. Compared with the yield of Koshihikari (mean, 665.3 g/m2), that of the line carrying Jukkoku_sd1 allele showed the highest value (772.6 g/m2, 16.1% higher than Koshihikari), while that of IR8_sd1, d60 and IR8_sd1 plus d60, was slightly decreased by 7.1%, 5.5%, and 9.7% respectively. The line carrying Jukkoku_sd1 also showed the highest value in number of panicles and florets/panicle, 16.2% and 11.1% higher than in Koshihikari, respectively, and these effects were responsible for the increases in yield. The 1000-grain weight was equivalent among all genetic lines. Except for the semidwarf line carrying Jukkoku_sd1, semidwarf line carrying d60 was equivalent to line carrying IR8_sd1in the yield of unpolished rice, and yield components such as panicle length, panicle number, floret number /panicle. Therefore, the semidwarfing gene d60 is one of the best possible choices in practical breeding.


Introduction
Semidwarfing prevents plants from lodging at their full-ripe stage, making them lodging-resistant to wind and rain, enhances their adaptability for heavy manuring and markedly improved the global productivity of rice and wheat between 1960-1990 (up to double yields of rice and quadruple yields of wheat) [1,2]. Semidwarf rice contributes stable production in the monsoonal regions of Asia, where typhoons frequently occur during the yielding season and also brings benefits such as erect leaf angle, reduced photoinhibition, and possibility to plant at higher densities to japonica varieties grown in California and also in South America [3]. However, gene source of semidwarfness is limited. The International Rice Research Institute (IRRI) developed a semidwarf rice variety IR8 in 1966 by using Taiwanese native semidwarf variety Dee-geo-woo-gen (DGWG). IR8 called as Miracle Rice, has been improved with lodging resistance and light-reception attitude, and it brought the Green Revolution in tropical Asia [2]. In Japan, semidwarf cultivars in the Kyushu region were developed in the 1960s using the native semidwarf variety Jukkoku [4]. In the Tohoku region, semidwarf cultivars were developed in the 1970s using the semidwarf mutant Reimei induced by Fujiminori-gamma-ray irradiated [5]. In the United States, Calrose 76 was developed in 1976 by Calrose-gamma-ray irradiated [6,7].

Effects of Semidwarf and Double Dwarf Genes on Growth
The trends in full-length growth, depicted by growth curves, were comparable among all lines. (Figure 1). The full length in lines carrying one or two semidwarf genes was already shorter than that of Koshirikari lines at the time of transplanting (June 7, 28 days after sowing), and the differences became prominent around 64-70 days after sowing (July 13 and 19) ( Figure 1, Table 1). The full length of d60-carrying line was longer than that of sd1-carrying lines at the time of transplanting. However, the full length of line carrying Jukkoku_sd1 and that of line carrying IR8_sd1 exceeded that of line carrying d60 on June 23 (43 days after sowing) and on July 13 (64 days after sowing), respectively: full length in lines carrying either Jukkoku_sd1 or IR8_sd1 was longer than that in line carrying d60 at the time of final measurement (August 23, 103 days after sowing). Days to heading ranged from 86.5 days of line carrying IR8_sd1 to 90.5 days of those carrying d60. Such a four-day difference was thought to be little. Therefore, the differences appeared in morphological traits, such as culm length and panicle length, were attributed to genetic reason.
3 Figure 1. Effect of growth of semidwarf and double dwarf gene lines. Ten plants were randomly selected, and the distance between the ground and the highest standing point (i.e., the full length) was measured every week for approximately three months until the panicle emerged. The full length of d60-carrying line was longer than that of sd1-carrying lines at the time of transplanting. However, the full length of line carrying Jukkoku_sd1 and that of line carrying IR8_sd1 exceeded that of line carrying d60 on June 23 (43 days after sowing) and on July 13 (64 days after sowing), respectively: full length in lines carrying either Jukkoku_sd1 or IR8_sd1 was longer than that in line carrying d60 at the time of final measurement (August 23, 103 days after sowing). The full length in lines carrying one or two semidwarf genes was already shorter than that of Koshirikari lines at the time of transplanting 28 days after sowing, and the differences became prominent around 64-70 days after sowing. Finally, the full length of semidwarf and double dwarf lines were significantly shorter than Koshihaikri. Color in the boxes of genetic lines coincide the color of growth curve in Figure 1. *: statistically significant at the 5% level.
Integration of a semidwarf gene (or genes) resulted in a reduction in culm length: the mean culm length of Koshihikari was 88.8 cm, while that of lines carrying Jukkoku_sd1, IR8_sd1, d60, Jukkoku_sd1 plus d60, or IR8_sd1 plus d60 was 71.8 cm, 68.5 cm, 65.7 cm, 48.6 cm, or 50.2 cm, respectively. Leaf length was shorter in line carrying Kinuhikari_sd1 (9-16% reduction compared with Koshihikari) or d60 (9-18% reduction compared with Koshihikari) than in those carrying Jukkoku_sd1 (1-9% reduction compared with Koshihikari ( Figure 2). Furthermore, leaves of the semidwarf and double dwarf lines were slightly shorter and straighter (pointing upwards) than in Koshihikari (Figure 3), indicating improved light-reception attitude by the integration of semidwarf gene (or genes). Panicle length was slightly longer (by 2.5%) in line carrying Jukkoku_sd1 and slightly shorter in lines carrying Kinuhikari_sd1 (by 2.4%) or d60 (by 3.0%), compared with Koshihikari (Table  2). However, the reduction in panicle length was quite less than that in culm length (22.8% decrease in lines carrying Kinuhikari_sd1 vs a 26.1% decrease in lines carrying d60). Therefore, the negative effects of semidwarf genes sd1 and d60 on panicle length were negligible.  The full length in lines carrying one or two semidwarf genes was already shorter than that of Koshirikari lines at the time of transplanting 28 days after sowing, and the differences became prominent around 64-70 days after sowing. Finally, the full length of semidwarf and double dwarf lines were significantly shorter than Koshihaikri. Color in the boxes of genetic lines coincide the color of growth curve in Figure 1. *: statistically significant at the 5% level.
Integration of a semidwarf gene (or genes) resulted in a reduction in culm length: the mean culm length of Koshihikari was 88.8 cm, while that of lines carrying Jukkoku_sd1, IR8_sd1, d60, Jukkoku_sd1 plus d60, or IR8_sd1 plus d60 was 71.8 cm, 68.5 cm, 65.7 cm, 48.6 cm, or 50.2 cm, respectively. Leaf length was shorter in line carrying Kinuhikari_sd1 (9-16% reduction compared with Koshihikari) or d60 (9-18% reduction compared with Koshihikari) than in those carrying Jukkoku_sd1 (1-9% reduction compared with Koshihikari ( Figure 2). Furthermore, leaves of the semidwarf and double dwarf lines were slightly shorter and straighter (pointing upwards) than in Koshihikari (Figure 3), indicating improved light-reception attitude by the integration of semidwarf gene (or genes). Panicle length was slightly longer (by 2.5%) in line carrying Jukkoku_sd1 and slightly shorter in lines carrying Kinuhikari_sd1 (by 2.4%) or d60 (by 3.0%), compared with Koshihikari (Table 2). However, the reduction in panicle length was quite less than that in culm length (22.8% decrease in lines carrying Kinuhikari_sd1 vs a 26.1% decrease in lines carrying d60). Therefore, the negative effects of semidwarf genes sd1 and d60 on panicle length were negligible.

Effects of Semidwarf and Double Dwarf Genes on Yield
The yield components of each genotype are summarized in Table 2. The weight of unpolished rice/1000 grains and the proportions of fertile florets differed only slightly between lines. The effect of these genes on the proportion of fertile florets and the weight of unpolished rice/1000 grains were negligible. The number of panicles/plants was 17.9 in Koshihikari: 20.8 in line carrying Jukkoku_sd1 (+16.2% vs Koshihikari), and 15.4 in Jukkoku DW line (−14.0% vs Koshihikari) ( Figure 4, Table 3). In addition, the floret number/panicle was 87.3 in line carrying Jukkoku_sd1 (+11.1% vs Koshihikari) and 72.1 in Jukkoku DW line (−8.3% vs Koshihikari) ( Figure 5, Tables 2 and 4). The number of panicles was larger in line carrying Jukkoku_sd1, while floret density was larger in all semidwarf varieties than in Koshihikari ( Figure 6, Table 5). Thus, an increase in both the number of panicles/plant and the floret number/panicle resulted in an increase in the number of panicles/m 2 and a consequent increase in yield ( Figure 7, Table 6).        8 Figure 6. Effect of semidwarf and double dwarf genes to floret density. The floret density was larger in all lines carrying one semidwarf gene than that of Koshihikari. Floret number/panicle Figure 6. Effect of semidwarf and double dwarf genes to floret density. The floret density was larger in all lines carrying one semidwarf gene than that of Koshihikari.   The yield of line carrying d60 was comparable to that in line carrying IR8_sd1. *: statistically significant at the 5% level.
The yield of unpolished rice was 665.3 g/m 2 in Koshihikari, 772.6 g/m 2 in line carrying Jukkoku_sd1 (+15.9% vs Koshihikari), 617.9 g/m 2 in line carrying Kinuhikari_sd1 (−7.1% vs Koshihikari), and 628.5 g/m 2 in line carrying d60 (−5.5% vs Koshihikari) (Figure 7, Table 6). The introduction of Kinuhikari_sd1 or d60 into Koshihikari appears to cause a slight reduction in yield. On the other hand, the yield of DW lines was markedly lower than that of Koshihikari: for Jukkoku DW line (−24.5% vs Koshihikari) and 600.5 g/m 2 for IR8 DW line (−9.7% vs Koshihikari) (Figure 7, Table 6). When using the alternative equation, the yield index was higher in all semidwarf-genecarrying lines than in Koshihikari (Figure 8). The high yield index and lodging resistance of semidwarf varieties suggest that introduction of sd1 and d60 into non-dwarf genomes will be beneficial for increasing crop yield. Moreover, only minor differences in the grain appearance were observed among lines including Koshihikari, indicating that the grain quality of semidwarf lines is equivalent to that of Koshihikari. Taken together, semidwarf genes sd1 and d60 are useful in the agricultural industry.  The yield of line carrying d60 was comparable to that in line carrying IR8_sd1. *: statistically significant at the 5% level.
The yield of unpolished rice was 665.3 g/m 2 in Koshihikari, 772.6 g/m 2 in line carrying Jukkoku_sd1 (+15.9% vs Koshihikari), 617.9 g/m 2 in line carrying Kinuhikari_sd1 (−7.1% vs Koshihikari), and 628.5 g/m 2 in line carrying d60 (−5.5% vs Koshihikari) (Figure 7, Table 6). The introduction of Kinuhikari_sd1 or d60 into Koshihikari appears to cause a slight reduction in yield. On the other hand, the yield of DW lines was markedly lower than that of Koshihikari: for Jukkoku DW line (−24.5% vs Koshihikari) and 600.5 g/m 2 for IR8 DW line (−9.7% vs Koshihikari) (Figure 7, Table 6). When using the alternative equation, the yield index was higher in all semidwarf-gene-carrying lines than in Koshihikari (Figure 8). The high yield index and lodging resistance of semidwarf varieties suggest that introduction of sd1 and d60 into non-dwarf genomes will be beneficial for increasing crop yield. Moreover, only minor differences in the grain appearance were observed among lines including Koshihikari, indicating that the grain quality of semidwarf lines is equivalent to that of Koshihikari. Taken together, semidwarf genes sd1 and d60 are useful in the agricultural industry.
The yield index was higher in line carrying IR8_sd1 than in those carrying Jukkoku_sd1 (Figure 8, Table 7). Although line carrying Jukkoku_sd1 gave a higher yield than those carrying IR8_sd1, the higher yield index associated with IR8_sd1 than Jukkoku_sd1 suggests that the efficiency of the distribution to sink organs (e.g., seeds) is higher. Thus, the yield favors a gain in dry matter, which may be also higher in plants carrying IR8_sd1 than in those carrying Jukkoku_sd1. Furthermore, the yield index for Jukkoku DW and IR8 DW are high--45.6% and 46.5% higher, respectively than that of Koshihikari ( Figure 8, Table 7). In order to increase a markedly low yield in DW lines, the use of conditions that favor a gain in dry matter, such as intensive cultivation with heavy fertilization to increase the number of tillers, may be effective. The yield index was higher in line carrying IR8_sd1 than in those carrying Jukkoku_sd1 ( Figure  8, Table 7). Although line carrying Jukkoku_sd1 gave a higher yield than those carrying IR8_sd1, the higher yield index associated with IR8_sd1 than Jukkoku_sd1 suggests that the efficiency of the distribution to sink organs (e.g., seeds) is higher. Thus, the yield favors a gain in dry matter, which may be also higher in plants carrying IR8_sd1 than in those carrying Jukkoku_sd1. Furthermore, the yield index for Jukkoku DW and IR8 DW are high--45.6% and 46.5% higher, respectively than that of Koshihikari (Figure 8, Table 7). In order to increase a markedly low yield in DW lines, the use of conditions that favor a gain in dry matter, such as intensive cultivation with heavy fertilization to increase the number of tillers, may be effective. Figure 8. Yield index of semidwarf and double dwarf gene lines. The yield index was higher in line carrying IR8_sd1 than in those carrying Jukkoku_sd1. The yield index for Jukkoku DW and IR8 DW are 45.6% and 46.5%, respectively higher than that of Koshihikari. The high yield index and lodging resistance of semidwarf varieties suggest that introduction of sd1 and d60 into non-dwarf genomes will be beneficial for increasing crop yield. *: statistically significant at the 5% level.

Discussion
As exemplified by IR8, which was the variety behind the Green Revolution, many of the rice varieties cultivated worldwide commonly carry the semidwarf gene sd1. Another semidwarf gene d60 is non-allelic to sd1 and is of particular interest as a different source of semidwarfism to give Yield index (%) Figure 8. Yield index of semidwarf and double dwarf gene lines. The yield index was higher in line carrying IR8_sd1 than in those carrying Jukkoku_sd1. The yield index for Jukkoku DW and IR8 DW are 45.6% and 46.5%, respectively higher than that of Koshihikari. The high yield index and lodging resistance of semidwarf varieties suggest that introduction of sd1 and d60 into non-dwarf genomes will be beneficial for increasing crop yield. *: statistically significant at the 5% level.

Discussion
As exemplified by IR8, which was the variety behind the Green Revolution, many of the rice varieties cultivated worldwide commonly carry the semidwarf gene sd1. Another semidwarf gene d60 is non-allelic to sd1 and is of particular interest as a different source of semidwarfism to give genetic diversity among the semidwarf varieties. In this study, semidwarf lines, namely Jukkoku (Jukkoku _sd1), sd1 of Kinuhikari (IR8_sd1: Kinuhikari maintains sd1 of IR8 origin), d60 or sd1 plus d60 into the Koshihikari background, were used to investigate influence of these semidwarf genes on phenotypic traits, in relation to yield.
This study showed that all tested semidwarf lines had shorter culm lengths than Koshihikari, indicating improved lodging resistance. The effect on culm length carrying d60(65.7 cm) is slight shorter than in those carrying sd1 (Jukkoku_sd1, 71.8 cm; IR8_sd1, 68.5 cm), Among the genetic lines, line carrying Jukkoku_sd1showed the highest yield of unpolished rice 772.6 g/m 2 , which is 16.1% higher than in Koshikikari. The Jukkoku_sd1 line also showed highest value in the number of panicles, the number of florets per panicle than in Koshihikari. Therefore, it was highly possible that the increasing yield of Jukkoku_sd1 line was ascribed to the increasing numbers of panicles and florets. Although the yield of unpolished rice of d60 line, 628.5 g/m 2 was 5.5% lower than that of Koshihikari (665.3 g/m 2 ), but this is almost equivalent yield performance of IR8_sd1 (617.9 g/m 2 ). Ogi et al. (1993) [21] and Murai et al. (2004) [22] reported characteristics of isogenic line carrying sd1 derived from DGWG, the source of IR8 sd1. These isogenic lines showed almost same number of panicles as that of original varieties, 'Norin 29 and 'Shiokari'. Therefore, it was concluded that Jukkoku_sd1 especially has potential increasing panicle numbers compared to IR8_sd1. Hence, Jukkoku_sd1 appears to confer a pleiotropic effect of increasing panicle number very well in the Koshihikari genetic background. The difference of such as effect between sd1 alleles may be ascribed to that IR8_sd1 suffered 383 bp deficit in the region exon 1-2 of GA20-ox [18], whereas, Jukkoku_sd1 has only a SNP against the wild type GA20-ox [18] and the transcripts existed [23].
This study demonstrated that d60 confers slightly shorter culm length than IR8_sd1, but almost equivalent yield performance with IR8_sd1 together with effects on yield-related phenotypic traits comparable to IR8_sd1, which actually contributed to green revolution [2]. Although many dwarf genes are accompanied with a reduction in panicle length, yield of unpolished rice, and grain thresh ability (which is likely attributed to excessive dwarfing effects), d60 does not exert such negative effects on yield-related phenotypic traits of rice plant. In conclusion, d60 is applicable to practical breeding and one of choice for expanding genetic diversity of rice varieties.

Cultivation
Rice seeds were taken from stocks kept in a refrigerator. Seeds of each line were immersed in enough water just to cover the seeds. Water was exchanged every day for seven days (May 2 to May 8) during seed soaking and stimulation of germination. Seedlings were grown in nursery boxes (30 × 15 × 3 cm) for approximately 20 days. Seedlings were then individually transplanted into a paddy field (120 m 2 : 6.0 × 20.0 m) of the University Farm on June 8. Two 4-m 2 plots (2 × 2 m) with transplanting densities 22.2 seedlings/m 2 (one seedling per 30 × 15 cm, 78 seedlings per field) were prepared for each genetic line (two instances). The paddy field was fertilized by 4.0 kg of basal fertilizer containing nitrogen, phosphorus, and potassium (weight ratio, nitrogen:phosphorus:potassium = 2.6:3.2:2.6) at the rate with 4.3 g/m 2 nitrogen, 5.3 g/m 2 phosphorus, and 4.3 g/m 2 potassium evenly across the field. A herbicide (Joystar L Floable, Kumiai Chemical Industry, Tokyo, Japan) was applied on June 20 to kill weeds grown uncontrollably, and the water level was then kept at a high enough level to cover the weeds for a week.

Growth Analysis
Ten seedlings were randomly selected for each line at the time of transplantation, and the full length was measured individually. After transplantation, ten plants were randomly selected, and the distance between the ground and the highest standing point (i.e., the full length) was measured every week for approximately three months until the panicle emerged. The time when the tip of the panicle first emerged from the flag leaf sheath was recorded as the heading time for all plants.

Plant Phenotyping
After ripening, ten plants typical of each genotype were sampled twice. Sampled plants were air-dried, and were assessed or measured the following traits. Culm length: the length between the ground surface and the panicle base of the main culm was measured at the time of sampling. Leaf length: the lengths of the upper five leaves, arising from the main culm, were individually measured. Length and weight of panicle: the length between the panicle base and the tip of the panicle, and the weight of the panicle, were measured. Total panicle number: the number of panicles were counted by sampled individuals and panicle numbers per 1 m 2 area (panicles/m 2 ) were counted twice in each plot of the paddy field. Total floret number: florets were counted to obtain total floret number. Floret number/panicle: the total floret number (including both sterile and fertile florets) was divided by the total panicle number. Proportion of fertile florets: each floret was assessed to determine its fertility. Floret density (floret number/cm): the number of florets per panicle was divided by the length of the panicle. Presence of awns: florets with an awn were counted when counting the florets. Grain threshability: was manually tested during examination of phenotypic traits. Appearance of grains: the size, color, and presence of an awn were observed for assessment of grain quality. Total weight of winnowed paddy: the total weight of winnowed paddy was weighed after grain selection using the salt solution (salt content of 1.06 g/m 3 ). Weight of sieved unpolished rice/1000 grains: obtained by multiplying the total weight of winnowed paddy by 0.84. Weight of plant parts above the ground: the weight of the plant parts above the ground was measured. Yield index: the winnowed paddy weight was divided by the weight of the plant part above the ground to obtain the yield index. The means of traits were statistically compared using the t-test.

Yield
Yield of unpolished rice was calculated using the following equation.