1. Introduction
The development of high-yielding semidwarf varieties of wheat and rice led to a rapid increase in the global production of grains, which more than doubled from 1960 to 1990 (Khush, Reference Khush1999). The semidwarf rice variety, IR8, was developed and released by the International Rice Research Institute (IRRI) using the Chinese cultivar ‘Dee-geo-woo-gen’ (DGWG). IR8 is known as ‘miracle rice’ because it responds well to fertilizer and produces an increased yield without culm elongation. Widespread adoption of IR8 brought about a ‘Green Revolution’, particularly in the monsoonal regions of Asia, where typhoons frequently occur during the harvest season (Athwal, Reference Athwal1971; Khush, Reference Khush1999).
The semidwarf characteristic is a very important agronomic trait for crop breeding. In other countries, many other short-culm cultivars, such as the Japanese indigenous landrace ‘Jikkoku’ (Okada et al., Reference Okada, Yamakawa, Fujii, Nishiyama, Motomura, Kai and Imai1967), or γ-ray-induced semidwarf cultivars such as ‘Reimei’ in Japan and ‘Calrose 76’ in the USA (Foster & Rutger, Reference Foster and Rutger1978) were developed using independent sources of semidwarfism. Dwarf varieties of rice have contributed to the dramatic improvement and stabilization of yields worldwide, and although derived from several native or mutant maternal lines, dwarf stature is controlled by a single dwarf gene, sd1 (Kikuchi & Futsuhara, Reference Kikuchi, Futsuhara, Matsuo, Shimizu, Tsunoda, Murata, Kumazawa, Futsuhara, Hoshikawa, Yamaguchi and Kikuchi1997; Ashikari et al., Reference Ashikari, Sasaki, Ueguchi-Tanaka, Itoh, Nishimura, Datta, Ishiyama, Saito, Kobayashi, Khush, Kitano and Matsuoka2002; Monna et al., Reference Monna, Kitazawa, Yoshino, Suzuki, Masuda, Maehara, Tanji, Sato, Nasu and Minobe2002; Sasaki et al., Reference Sasaki, Ashikari, Ueguchi-Tanaka, Itoh, Nishimura, Swapan, Ishiyama, Saito, Kobayashi, Khush, Kitano and Matsuoka2002; Spielmeyer et al., Reference Spielmeyer, Ellis and Chandler2002). The sd1 alleles on the long arm of chromosome 1 (Cho et al., Reference Cho, Eun, Kim, Chung and Chae1994a, Reference Cho, Eun, McCouch and Chaeb; Maeda et al., Reference Maeda, Ishii, Mori, Kuroda, Horimoto, Takamure, Kinoshita and Kamijima1997) encode a defective C20-oxidase in the gibberellin (GA) biosynthesis pathway (GA 20-oxidase, OsGA20ox2) (Ashikari et al., Reference Ashikari, Sasaki, Ueguchi-Tanaka, Itoh, Nishimura, Datta, Ishiyama, Saito, Kobayashi, Khush, Kitano and Matsuoka2002; Monna et al., Reference Monna, Kitazawa, Yoshino, Suzuki, Masuda, Maehara, Tanji, Sato, Nasu and Minobe2002; Sasaki et al., Reference Sasaki, Ashikari, Ueguchi-Tanaka, Itoh, Nishimura, Swapan, Ishiyama, Saito, Kobayashi, Khush, Kitano and Matsuoka2002; Spielmeyer et al., Reference Spielmeyer, Ellis and Chandler2002). The sd1 gene confers the semidwarf phenotype with no detrimental effects on grain yield (Hedden, Reference Hedden2003a, Reference Heddenb), but other options for dwarf breeding are limited.
A narrow genetic base, or monoculture, frequently leads to outbreaks of diseases or pests. The most dramatic recent pest epidemic to hit food crops because of genetic uniformity was the 1970 attack of southern corn leaf blight (SCLB) in the US maize (Zea mays L.) crop. Pathogen outbreaks also struck crops of potato in Ireland in the late 1840s; coffee in Sri Lanka in the 1870s; banana throughout the Caribbean; wheat in the USA in 1916; rice in Bengal and India in 1942; and oats in the USA in 1946 (Kaneko et al., Reference Kaneko, Jinno, Furusawa, Morotomi and Yamaguchi2000). Therefore, a wider range of genetic diversity to cope with future environmental changes is essential. In Japan, ‘Koshihikari’, released in 1953, has been a leading cultivar since 1979. It was grown on 601 100 ha in 2009, accounting for over 37% of the total Japanese rice-growing area. ‘Koshihikari’ is also grown in various other countries (e.g. in the United States and Australia; available at: http://www.tdb.maff.go.jp/toukei/a02stopframeset). The demand for ‘Koshihikari’ is high because of its good nutritional quality, high resistance to pre-harvest sprouting, tolerance of cool temperatures at booting stage and wide adaptability. In contrast to its advantages, ‘Koshihikari’, with a culm length from 90 to 100 cm, has a serious disadvantage of poor lodging resistance (Kashiwagi et al., Reference Kashiwagi, Togawa, Hirotsu and Ishimaru2008). Lodging caused by autumnal rains and typhoons hinders harvest and reduces yield and grain quality. Therefore, genetic improvement of ‘Koshihikari’ to resist lodging is critical.
To find a new dwarf gene to replace sd1, analyses focused on ‘Hokuriku 100’, a mutant line with culms ∼15 cm shorter in length, close to 20% shorter than those of ‘Koshihikari’. ‘Hokuriku 100’ is the most promising semidwarf mutant selected from 60Co γ-irradiated ‘Koshihikari’ in a large-scale mutation breeding operation to enhance lodging resistance (Samoto & Kanai, Reference Samoto and Kanai1975). The author discovered a novel dwarf gene, d60, together with a gametic lethal gene, gal, in a cross between semidwarf mutant ‘Hokuriku 100’ and its original tall variety ‘Koshihikari’ (Tomita et al., Reference Tomita, Tanisaka, Okumoto, Yamagata, Iyama and Takeda1989; Tanisaka et al., Reference Tanisaka, Tomita and Yamagata1990; Tomita, Reference Tomita and Khush1996). The gene d60 was thought to have a unique mode of inheritance that complements the gametic lethal gene, gal. According to this hypothesis, the F1 between ‘Hokuriku 100’ (genotype d60d60GalGal, Gal: non-lethal mutant allele) and ‘Koshihikari’ (D60D60galgal, D60: tall allele) should show 25% sterility because of the deterioration of gametes bearing both gal and d60. The F2 progenies also segregated in a ratio of one semidwarf (1 d60d60GalGal) : two tall and 25% sterile (2 D60d60Galgal) : six tall (2 D60d60GalGal : 1 D60D60GalGal : 2 D60D60Galgal : 1 D60D60galgal) (Tomita, Reference Tomita and Khush1996), which was skewed from the expected Mendelian ratio for a single recessive gene of one semidwarf : three tall. The induced semidwarfing gene d60 confers a fine plant phenotype with erect leaves, about 15 cm shorter than those of the original variety, and may be a promising alternative to the widespread gene, sd1. In this study, based on the genetic hypothesis mentioned earlier, BCnF1 plants (D60d60Galgal) selected for pollen sterility caused by d60 and gal and used as pollen parents to allow introgression of d60 by successive backcrosses. This study also aimed to pyramid both semidwarfing genes, d60 and sd1, into ‘Koshihikari’. Finally, a useful parental line ‘Minihikari’ was developed as a new genetic source for both d60 and sd1. ‘Minihikari’ displays super-short stature through successful combination of sd1 with d60, which are genetically and functionally independent.
2. Materials and methods
(i) Development of a isogenic lines in ‘Koshihikari’ genetic background
(a) Development of an sd1 isogenic line
The author previously developed ‘Koshihikari’ sd1, which was released as a cultivar ‘Hikarishinseiki’ (Tomita, Reference Tomita2009; rice cultivar no. 12273, Ministry of Agriculture, Forestry and Fisheries of Japan). Short-culmed ‘Koshihikari’-like lines, homozygous for sd1 and with same heading dates as those of ‘Koshihikari’, were selected from 100 self-pollinated F3 lines raised from 100 randomly selected F2 plants of ‘Kanto 79’ (an early heading mutant line derived from ‘Koshihikari’) × ‘Jikkoku’ (a cultivar with a semi-dwarf gene, sd1) and fixed at the F4 generation in 1989 (Fig. 2). The short-culmed ‘Koshihikari’-like line and its descendants were backcrossed with ‘Koshihikari’ (Sd1Sd1) as the recurrent parent eight times between 1990 and 1997. Sd1sd1-heterozygous plants could be selected by their slightly erect leaves in the first generation of each backcross (BCnF1) and then backcrossed by ‘Koshihikari’ as the female parent.
(b) Development of a d60 isogenic line
Semidwarfism in the rice cultivar ‘Hokuriku 100’ is controlled by the single semidwarfing gene, d60. However, d60 was thought to pleiotropically activate the gametic lethal gene, gal. According to this hypothesis, the F2 progenies of a cross between ‘Hokuriku 100’ (genotype d60d60GalGal, Gal: gametic non-lethal allele) and the original tall variety ‘Koshihikari’ (D60D60galgal, D60: is the tall allele) exhibit segregation distortion in a ratio of one semidwarf (1 d60d60GalGal) : two tall and 25% sterile (2 D60d60Galgal) : six tall (2 D60d60GalGal : 1 D60D60GalGal : 2 D60D60Galgal : 1 D60D60galgal), because of the deterioration of the F1 male and female gametes that carry both gal and d60 (Fig. 1).
Fig. 1. Introgression of the d60 dwarfing gene in relation to the gametic lethal-gal gene by backcross breeding.
In this study, based on the genetic hypothesis mentioned earlier, BCnF1 plants (D60d60Galgal) were selected for pollen sterility caused by d60 and gal and were used as pollen parents to introgress d60 by successive backcrosses. Between 1987 and 1998, ‘Koshihikari’ was crossed with ‘Hokuriku 100’ (d60d60GalGal), and then backcrossed seven times to ‘Koshihikari’ (D60D60galgal) as the recurrent parent to minimize transmission of the flanking region of d60 (Fig. 2). D60d60Galgal-heterozygous plants, recognized by pollen sterility prior to anthesis, were selected in the first generation of each backcross (BCnF1). The F1(D60d60Galgal) progenies of ‘Koshihikari’ × ‘Hokuriku 100’ were backcrossed to ‘Koshihikari’ (D60D60galgal) to produce 30 BC1F1 individuals that segregated in a ratio of one tall and 25% sterile (D60d60Galgal) : two tall (1 D60D60Galgal : 1 D60D60galgal). Tall and 25% sterile BC1F1 plants (D60d60Galgal) were then selected for pollen sterility prior to anthesis and backcrossed with ‘Koshihikari’ as female parent to produce 30 BC2F1 seeds. ‘Koshihikari’ was used as the female parent in each backcross generation. For each BCnF2 generation, 200–300 individuals were always grown out during the following year to confirm that short-culmed plants (d60d60) appeared in the BCnF2 generation, while backcrossing of 5–10 pollen sterile individuals from the BCn +1 F1 was underway.
Fig. 2. Development of variety ‘Minihikari’, an isogenic line with sd1 and d60 dwarfing genes in the ‘Koshihikari’ genetic background.
(c) Development of a line isogenic for sd1 and d60
In 2000, ‘Hikarishinseiki’ (‘Koshihikari sd1’) was crossed with ‘Koshihikari’ d60 and the double dwarf ‘Koshihikari’, containing both sd1 and d60, was selected from F2 to F4 generation and fixed in 2004 (Fig. 1). The super-short-culmed ‘Koshihikari’, designated as ‘Minihikari’, underwent varietal registration by the Ministry of Agriculture, Forestry and Fisheries (MAFF).
(d) Evaluation of sd1 introgression
The two alleles at the sd1/OsGA20ox2 locus on chromosome 1 of each line were distinguished by PCR amplification of the first exon of sd1 followed by digestion of the PCR product with PmaCI. Primer design was based on the sequence reported for sd1 from the rice variety ‘Nipponbare’ (http://rgp.dna.affrc.go.jp). Reaction mixtures contained 10 ng of template genomic DNA, 1 μm each primer (F: 5′-GCTCGTCTTCTCCCCTGTTACAAATACCCC-3′; R: 5′-ACCATGAAGGTGTCGCCGATGTTGATG ACC-3′), 0·4 mm dNTPs, 1 × GC buffer I, 2·5 mm MgCl2, and 0·5 U LA Taq polymerase (Takara, Kyoto, Japan) in a total volume of 20 μl. The PCR reaction program consisted of 35 cycles of 30 s at 94 °C, 30 s at 58 °C and 1 min at 72 °C. The ‘Jikkoku’ allele for sd1 was detected by digestion of the PCR products with PmaCI (CACGTG) according to Tomita (Reference Tomita2009).
(ii) Field evaluation of isogenic lines
Performance tests were conducted in a paddy at Tottori University, Koyama, during 2009 and 2010. ‘Minihikari’, ‘Koshihkari d60’, ‘Hikarishinseiki’ and ‘Koshihikari’ were sown on 18 April 2009 and 22 April 2010, and 128 plants per plot were transplanted with three replications on 14 and 18 May, respectively. In both years, planting density was 22·2 individual plants/m2 (30 × 15 cm), planted one by one. Fertilizers including N, P2O5 and K2O were applied as dressings at 5, 13·6 and 7·2 kg/10 acre, respectively. Each replicate consisted of an area of 16 m2 in each year, and plants were transplanted into three replications in a random order. The date at which 50% of all panicles had emerged from the flag leaf sheath was recorded as the heading date. Days-to-heading was the number of days from sowing to heading date. Culm length, panicle length, number of panicles, leaf length and leaf width were measured on ten randomly selected plants in each plot. Based on the inclination angle between the base of the plant and the neck of panicles, plants were categorized into five groups by degree of lodging: standing (0), almost 70 (1), almost 50 (2), almost 30 (3), almost 10 (4), lodged (5). Thousand-grain weight, grain yield of brown rice, grain quality and eating quality were measured on bulks of 50 plants.
(iii) Data analyses
Data were analysed using analysis of variance (ANOVA) followed by post hoc tests to determine the statistical significance of the data, followed by multiple comparison analysis using the Holm method (Nagata & Yoshida, Reference Nagata and Yoshida2009), which is an improved version of the Bonferroni method.
3. Results
(i) Development of isogenic lines in ‘Koshihikari’ genetic background
(a) Development of an sd1 isogenic line
The dwarf sd1 homozygous strain (‘Jikkoku’-type ‘Koshihikari’), derived from the cross between Kanto 79’ (Sd1Sd1) × ‘Jikkoku’ (sd1sd1) and with a heading date the same as ‘Koshihikari’, was selected by the pedigree breeding method and fixed in the F4 generation (Tomita, Reference Tomita2009). After obtaining the ‘Koshihikari’ × (‘Koshihikari’ × ‘Jikkoku’-type ‘Koshihikari’) BC1F1 (1 Sd1Sd1 : 1 Sd1sd1), continuous backcrosses were made between an Sd1sd1 individual as the pollen parent in BCnF1 and the recurrent parent ‘Koshihikari’ until BC8 (Fig. 2). The subsequent generation BCnF2 of pollen parent was developed simultaneously and a segregation of 3(1 Sd1Sd1 : 2 Sd1sd1) : 1 sd1sd1 was verified (BC8F2 = 202 : 77, χ2 = 1·01, 0·30<P < 0·50). The semidwarf phenotype ‘Koshihikari sd1’ (sd1sd1) was developed by the BC8F3 generation, at which point the plant genomes should be ≥99·8% ‘Koshihikari’, based on theoretical expectations after eight recurrent backcrosses.
(b) Development of a d60 isogenic line
The semidwarf gene d60 from ‘Hokuriku 100’ has the potential to replace sd1, which is now widely distributed. In this study, semidwarf isogenic strains of ‘Koshihikari’ into which either sd1 or d60, or both, had been introgressed, were developed to compare the effects of sd1 and d60 on aspects of the plant phenotype and on lodging resistance. The recessive d60 mutation and the gametic lethal gene gal, which is present in ‘Koshihikari’ and other rice varieties, was thought to cause complementary lethality to both male and female gametes (Tomita, Reference Tomita and Khush1996). Accordingly, the pollen and ovule fertility of ‘Koshihikari’ (D60D60galgal) × ‘Hokuriku 100’ (d60d60GalGal) F1 progeny is ∼70%, and the F2 segregates six fertile, long culm (4 D60D60 : 2 D60d60GalGal): two partial sterile, long culm (F1 type = (D60d60Galgal): one semidwarf (d60d60GalGal) (Fig. 1).
‘Koshihikari’ × (‘Hokuriku 100’ × ‘Koshihikari’) BC1F1 segregated 11 : 6, which approximately fit a ratio of two fertile, long culm (D60D60Galgal : D60D60galgal): one partial sterile long culm (D60d60Galgal) (Fig. 3). Partially sterile individuals were thought to be heterozygous d60, and the BC1F2 it segregated clearly into six fertile, long culm (4D60D60 : 2D60d60GalGal): two partial sterile, long culm (D60d60Galgal): one semidwarf (d60d60GalGal) (154 : 51 : 26, χ2 = 0·20, 0·90<P < 0·95, Fig. 4). Subsequently, continued backcrosses were performed until the BC7 with individuals selected in the BCnF1 for ∼70% pollen fertility with ‘Koshihikari’ (Fig. 1). It is not necessary to grow out and select a semidwarf d60 individual from BCnF2 if a pollen parent with ∼70% pollen fertility (Fig. 3) is chosen from the BCnF1 to cross with the recurrent parent. The semidwarf phenotype (d60d60GalGal) was fixed by the BC7F3 generation, by which time plants should carry ≥99·6% ‘Koshihikari’ genome.
Fig. 3. Pollen observed in double heterozygous D60 and Gal in BCnF1 plants.
Fig. 4. Genotypic distribution for culm length in the F3 (red) derived from the partial sterile, semidwarf (sd1sd1D60d60Galgal) following the cross between ‘Koshihikari d60’ (Sd1Sd1d60d60GalGal) and ‘Koshihikari sd1’ (sd1sd1D60D60galgal), and in the BC1F2 (green) derived from the partial sterile (Sd1Sd1D60d60Galgal) following the cross between ‘Koshihikari’ (Sd1Sd1D60D60galgal) and ‘Hokuriku 100’ (Sd1Sd1d60d60GalGal). Red shows sd1 homozygous and pale red shows partial sterility among them. Green shows Sd1 homozygous and pale green shows partial sterility among them.
(c) Development of the line isogenic for sd1 and d60
In the F2 of ‘Koshihikari d60’ (Sd1Sd1d60d60GalGal) × ‘Koshihikari sd1’ (sd1sd1D60D60galgal), the phenotypes (1 Sd1Sd1 : 2 Sd1sd1 : 1 sd1sd1) × 6 (4 D60D60 : 2 D60d60GalGal) : 2 (D60d60Galgal) : 1 (d60d60GalGal) = 18 fertile long culm: six partial sterile, long culm: nine fertile, semidwarf: two partial sterile, semidwarf: one double dwarf segregated. Among these, the F3 from the partial sterile, semidwarf (sd1sd1D60d60Galgal) segregated into six fertile, semidwarf (4 sd1sd1D60D60 : 2 sd1sd1D60d60): two partial sterile, semidwarf (sd1sd1D60d60Galgal): one double dwarf (sd1sd1d60d60GalGal) (137 : 56 : 22, χ2 = 1·84, 0·30<P < 0·50, Fig. 4), and in the next generation (F4) the double dwarf genotype (sd1sd1d60d60GalGal) was fixed (Fig. 5). This super-short-culmed ‘Koshihikari’, designated as variety ‘Minihikari’, was registered by the Ministry of Agriculture, Forestry and Fisheries (MAFF 2010).
Fig. 5. Phenotype at maturity of ‘Koshihikari’ (a) and three isogenic lines in the ‘Koshihikari’ genetic background: sd1 (b), d60 (c), combination of sd1 and d60 (d).
The sd1 allele from ‘Jikkoku’ has a G → T substitution in the first exon, which results in a single amino acid change from Gly to Val. PmaCI recognizes the substituted sequence (CACGTG) in the sd1 allele from ‘Jikkoku’. This revealed the sd1 allele in the double dwarf genotype (Minihikari) as two unique fragments, while the wild-type allele of ‘Koshihikari’ produced an undigested single fragment (Fig. 6). These results show that d60 and gal are inherited independently in the homozygous sd1 background.
Fig. 6. DNA sequence analysis of sd1 in the ‘Minihikari’ (‘Koshihikari sd1d60’) genome. The sd1 allele from ‘Jikkoku’ has a G → T substitution in the first exon. PmaCI recognizes the substituted sequence (CACGTG) and digested the sd1 allele from ‘Minihikari’ into two fragments, while the wild-type allele of ‘Koshihikari’ was not digested and remained as a single fragment.
(ii) Field evaluation of isogenic lines
The agronomic characteristics of ‘Minihikari’ and ‘Koshihikari’ are shown in Table 1. Isogenic lines created by backcrossing d60, sd1 or both, into ‘Koshihikari’, resulted in the d60sd1 line with an additive, extreme dwarf phenotype (Fig. 5), which demonstrates that d60 and sd1 are functionally independent and not related to the GA1 biosynthesis pathway. Relative to ‘Koshihikari’, the culm length of ‘Koshihikari sd1’ was 26·8% shorter, that of ‘Koshihikari d60’ was 26·0% shorter, and that of the double dwarf ‘Koshihikari sd1d60’, ‘Minihikari’, was 39·9% shorter (Fig. 5).
Table 1. Comparison of agronomic characters of ‘Koshihikari’ and isogenic ‘Koshihikari’ strains carrying semidwarfing genes d60 or sd1
a Grain quality was classified into nine grade: 1, excellent good to 9, especially bad low quality.
b Lodging degree was determined based on the inclination angle of plant: 0, standing; 1, almost 70; 2, almost 50; 3, almost 30; 4, almost 10; and 5, lodged.
c Leaf blast score was determined based on the percentage of infected leaf area: 0 : 0%, 1 : 1%, 2 : 2%, 3 : 5%, 4 : 10%, 5 : 20%, 6 : 40%, 7 : 60%, 8 : 80%, 9 : 90% and 100%.
d Panicle blast score was determined based on the percentage of infected kernels: 0 : 0%, 1 : 1%, 2 : 2%, 3 : 5%, 4 : 10%, 5 : 20%, 6 : 40%, 7 : 60%, 8 : 80%, 9 : 90% and 100%.
e Value of taste was determined using a Taste-meter MA-90B (Tokyo Rice-producing Machine Factory, Japan).
f Eating quality shows the aggregate evaluation in 11 categories: 5, excellent through – 5, especially poor.
g Amylose and protein content were measured by Near Infrared Spectrometer AN800 (Kett Electric Laboratory, Japan).
Both the early heading date and maturity dates of ‘Minihikari’ were the same as those of ‘Koshihikari’. The culm lengths of ‘Koshihikari d60’ (69·4 cm) and ‘Minihikari’ (56·4 cm) averaged 74 and 60% of the length of ‘Koshihikari’ (i.e. 26 and 40% shorter), respectively. ‘Minihikari’ and ‘Koshihikari d60’, had the highest degree of lodging, 0·0, which was clearly higher than that of ‘Koshihikari’, 4·4. The panicle lengths of ‘Koshihikari d60’ (17·1 cm) and ‘Minihikari’ (17·0 cm) averaged 91 and 90% of that of ‘Koshihikari’, respectively. The number of panicles of ‘Koshihikari d60’ (472/m2) and ‘Minihikari’ (515/m2) averaged 102 and 111% of that of ‘Koshihikari’, respectively. The thousand-grain weight of ‘Koshihikari d60’ (23·0 g) and ‘Minihikari’ (23·0 g) averaged 100% of that of ‘Koshihikari’, and the brown rice yield of ‘Koshihikari d60’ (62·9 kg/a) and ‘Minihikari’ (60·1 kg/a) averaged 96 and 92% of that of ‘Koshihikari’, respectively. The relatively greater number of panicles and sustainable yield may have resulted from the conversion of nutrients that were not directed to leaves and stems.
The leaves of ‘Minihikari’ averaged 18% wider than those of ‘Koshihikari’, were more erect, and retained a deep green colour longer (Fig. 5). The improved light-intercepting attitude of ‘Minihikari’ may thus improve its photosynthetic efficiency. Ripening period and grain quality (Table 1) were not significantly different among genotypes, according to ANOVA tests. Grain size and quality were also not significantly different. The brown rice quality of both varieties was ranked ‘medium’ (with a score of 4·3), and both scored ‘minimum’ for contents of ‘white belly’ and cracked grain. The flavour of ‘Koshihikari d60’ and ‘Minihikari’ scored an average of −0·17 and ±0·00 points against ‘Koshihikari’, respectively (Table 1), and ‘Koshihikari d60’ and ‘Minihikari’ ranked in the same ‘upper medium’ quality grade as ‘Koshihikari’. Resistance to pre-harvest sprouting and grain shattering of both cultivars are both rated ‘difficult’ (Table 1). Flavour and quality of ‘Koshihikari d60’ and ‘Minihikari’ were rated as identical to those of ‘Koshihikari’. Thus, the semidwarfing gene d60 reduces lodging in ‘Koshihikari d60’ and ‘Minihikari’, while retaining the flavour and quality of ‘Koshihikari’.
4. Discussion
‘Koshihikari’ remains a favourite rice cultivar in Japan. Production of the two major cultivars, ‘Koshihikari’ and ‘Hitomebore’, accounts for about 50% of the total rice production in Japan; so the genetic base of the rice crop in Japan is narrow. This raises the risk of crop loss due to disease and pests, and to lodging in ‘Koshihikari’ caused by frequent typhoons. Such major damage is a nationwide problem, and so the genetic improvement of lodging resistance in ‘Koshihikari’ has been a major rice breeding goal in Japan. This paper describes the development of semidwarf ‘Minihikari’, which was released in 2010.
To compare the effect of d60 and sd1 gene expression in the ‘Koshihikari’ genetic background, d60 and sd1 were introgressed into ‘Koshihikari’ by backcrossing each 7–8 times between 1985 and 2000 to ‘Koshinishiki’ as the recurrent parent. The short-culmed isogenic strains were generated, including ‘Koshihikari sd1’ (later named and registered as ‘Hikarishinseiki’) and ‘Koshihikari d60’. ‘Koshihikari sd1’ and ‘Koshihikari d60’ were then crossed in 2001 to show that the two short-culm genes were not genetically identical, and to produce a new line carrying both d60 and sd1 at the F4 generation. The culm length of this new line was very short, ∼40 cm shorter than that of ‘Koshihikari’, showing the additive effect of both genes and demonstrating that d60 was functionally independent from sd1. This extremely short-culmed ‘Koshihikari’, carrying the rice semidwarf genes sd1 and d60, was named ‘Minihikari’. Compared with ‘Koshihikari’, ‘Minihikari’ exhibited markedly high lodging resistance. We filed for registration of the cultivar with the Ministry of Agriculture, which was granted in October 2010 (varietal registration number 19985). ‘Minihikari’ proves that d60 is independent from sd1 and carries both genes in the genetic background of ‘Koshihikari’.
‘Minihikari’ is the first short-culmed isogenic form of ‘Koshihikari’ carrying both an sd1 and d60 allele while retaining over 99·8% of the ‘Koshihikari’ genome. ‘Minihikari’ was registered to promote its use as a maternal parent containing both the useful semi-dwarfing genes d60 and sd1, which will expand the gene pool for short-culm phenotypes. ‘Minihikari’ has remarkably enhanced lodging resistance over that of ‘Koshihikari’. Moreover, its increased panicle number gave it an average of 3% increase in grain yield, combined with the highly prized flavour of ‘Koshihikari’. The number of panicles of ‘Koshihikari d60’ (472/m2) and ‘Minihikari’ (515/m2) averaged 102 and 111% of that of ‘Koshihikari’, respectively. Murai et al. (Reference Murai, Komazaki and Sato2004) and Ogi et al. (Reference Ogi, Kato, Maruyama and Kikuchi1993) reported that an isogenic line carrying sd1 derived from DGWG had more panicles than its parent cultivar ‘Norin 29’, while another sd1 isogenic line had the same number of panicles in the ‘Shiokari’ genetic background. The semidwarfing gene sd1 encodes a defective C20 oxidative enzyme, OsGA20ox2, which functions in the biosynthesis of gibberellin GA1 (Sasaki et al., Reference Sasaki, Ashikari, Ueguchi-Tanaka, Itoh, Nishimura, Swapan, Ishiyama, Saito, Kobayashi, Khush, Kitano and Matsuoka2002; Spielmeyer et al., Reference Spielmeyer, Ellis and Chandler2002), and the defective enzyme results in a short culm. Hence, sd1 appears to confer a pleiotropic effect of significantly increased panicle number in the ‘Koshihikari d60’ genetic background.
In this study, ‘Koshihikari’ × (‘Hokuriku 100’ × ‘Koshihikari’) BC1F1 segregated in a ratio of two fertile, long culm (D60D60Galgal : D60D60galgal): one partial sterile, long culm (D60d60Galgal). Moreover, the BC1F2 progeny segregation fit a six fertile, long culm (4D60D60 : 2D60d60GalGal): two partial sterile, long culm (D60d60Galgal): one semidwarf (d60d60GalGal) ratio. Subsequently, continuous backcrosses were successfully performed until the BC7 generation using individuals selected in the BCnF1 for ∼70% pollen fertility with ‘Koshihikari’. This shows that d60 and the gametic-lethal gene gal cause complementary lethality to both male and female gametes, a form of hybrid sterility. However, hybrid sterility caused by two loci, namely d60 and gal, is quite different from the genetic systems of hybrid sterility previously discovered in rice, which consist of a single gene locus, such as S5, S-a or S10 (Kitamura, 1962; Ikehashi & Araki, Reference Ikehashi, Araki and Khush1986; Sano, Reference Sano1990; Zhang et al., Reference Zhang, Lu, Liu, Feng and Zhang2006; Chen et al., Reference Chen, Ding, Ouyang, Du, Yang, Cheng, Zhao, Qiu, Zhang, Yao, Liu, Wang, Xu, Li, Xue, Xia, Ji, Lu, Xu and Zhang2008; Long et al., Reference Long, Zhao, Niu, Su, Wu, Chen, Zhang, Guo, Zhuang, Mei, Xia, Wang, Wu and Liu2008). Hybrid sterility between O. sativa and O. glumaepatula is only a rare case caused by duplicate genes, S27 and S28 (Yamagata et al., Reference Yamagata, Yamamoto, Aya, Win, Doi, Sobrizal Ito, Kanamori, Wu, Matsumoto, Matsuoka, Ashikari and Yoshimura2010), but they have no dwarfing effects. In this study, BCnF1 plants (D60d60Galgal) were selected for pollen sterility caused by d60 and gal, and were used as pollen parents to backcross d60 in successive backcrosses. This method was shown to be effective in this study and can be used for other cultivars in addition to ‘Koshihikari’.
If a gametic non-lethal allele Gal had not mutated simultaneously with the mutation from D60 to d60, d60 would have been eliminated due to lethality in M1 gametes. Earlier studies in mutation breeding of semidwarf ‘Koshihikari’, conducted by several organizations prior to work done at Hokuriku National Agricultural Experiment Station, resulted in no introduction of new varieties. Subsequently, Samoto & Kanai (Reference Samoto and Kanai1975) increased the scale of mutation breeding by screening 200 000 M1 plants. The semidwarf line ‘Hokuriku 100’ was then selected in M5 plants derived from 298 short mutants selected from among 80 000 M2 plants. Samoto & Kanai (Reference Samoto and Kanai1975) found that the rate of short-statured mutants they observed, 0·3%, was much lower than the 11·0% observed in wheat (Kaizuma et al., Reference Kaizuma, Hirano and Gotoh1967) and the 5·2% observed in barley (Toda et al., Reference Toda, Nakada, Miki and Tsukada1972). The low rate of recovery of short-statured mutants could be due to gametic lethality caused by interactions between gal and induced dwarf genes.
That is, the non-lethal allele Gal is essential to the transmission of d60. Thus, d60 and Gal are rare and valuable mutant genes for the advancement of semidwarf breeding to replace sd1. The semidwarf gene d60 from ‘Hokuriku 100’ has potential to replace the widespread sd1, and provides more options for dwarf breeding acquired through simultaneous mutation of two genes, one of which is essential for the heritability of the other, such as Gal is for d60. In this study, both semidwarfing genes d60 and sd1 were pyramided successfully in the ‘Koshihikari’ genome to develop a new and useful genetic resource for breeding dwarf rice.
This work was supported by Adaptable and Seamless Technology Transfer Program through Target-driven R & D (grant numbers 08150094 and 08001167) from Japan Science and Technology Agency.
5. Declaration of Interest
None.
