Review

The role of QTLs in the breeding of high-yielding rice

Kotaro Miura1, Motoyuki Ashikari2 and Makoto Matsuoka2
1

Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka Kenjyojima, Eiheiji-cho, Yoshida-gun, Fukui, 910-1195, Japan 2 Bioscience and Biotechnology Center, Nagoya University, Furo-cho, Chikusa, Nagoya, Aichi, 464-8601, Japan

Food shortages have once again become a serious problem, not only because of world population growth but also as a result of escalating demand for crops as a substrate for biofuels. The production of improved plant varieties, especially major crops such as rice, is urgently required to increase yield. The completion of the sequencing of the rice genome has made it possible to clone and analyze quantitative trait loci (QTLs). Furthermore, the development of high-throughput sequencing and genotyping technologies has improved spectacularly the accuracy of QTL analysis. In this review article, we focus on the potential roles of major QTLs in the selection for agronomic traits in rice and discuss the application of high-throughput technologies for high-resolution genetic analysis. Rice quantitative trait loci (QTLs) for high-yielding crop breeding Rice (Oryza sativa L.) is one of the most important food crops in the world, providing over 21% of the caloric needs of the population of the world and up to 76% of the caloric intake of the population of southeast Asia [1]. Food shortages have once again become a serious problem because demand from population growth and the production of biofuels exceeds global food supply [2,3]. To meet global food demand, grain production will need to increase by a further 50% worldwide by 2025 [4]. To achieve this ambitious goal, plant varieties with greatly improved agronomic traits will be required. Many important agronomic traits, such as crop yield and stress tolerance, are controlled simultaneously by multiple genes (namely, QTLs) and are strongly inuenced by the environment [5]. To understand the mechanism of agronomic traits, therefore, the dissection of complex traits into single chromosome loci and the isolation and characterization of each QTL is important. In 2002, draft genomic sequences of two rice subspecies, O. sativa ssp. japonica (cv. Nipponbare) and O. sativa ssp. indica 93-11, were reported [6,7], and subsequently the International Rice Genome Sequencing Project completed the nal sequence of the entire rice genome of Nipponbare [8]. This achievement has provided us with a vast amount of information on the rice genome and allowed us to perform detailed genetic analysis. Using this information, researchers have now succeeded in isolating many important QTL
Corresponding author: Matsuoka, M. (makoto@agr.nagoya-u.ac.jp).

genes, which have the potential to greatly improve rice production. In addition to sequencing the rice genome, sorghum (Sorghum bicolor) [9] and maize (Zea mays) [10] sequences have been released, and work of the International Barley Sequencing Consortium [11] and International Wheat Genome Sequencing Consortium [12] is well underway. QTL analysis of these important cereal crops will be carried out soon, and thus the study of major rice QTLs is an important model for the future study of these cereal crops. In this review article, we will summarize the studies of major rice QTLs that show potential for use in future breeding programs. QTLs for grain number Grain number is one of the most important traits in the determination of crop productivity. In combination with grain weight and panicle number per plant in rice, grain number per panicle is an important factor determining yield production. Previously, we identied Grain number 1a (Gn1a) as a major QTL of grain number using the backcross inbred lines (BILs) of Koshihikari (lower grain number) and Habataki (higher number). This work was a pioneering study of the molecular analysis of grain number determination in rice [13]. Gn1a encodes an enzyme, namely cytokinin oxidase/dehydrogenase (OsCKX2), which degrades the phytohormone cytokinin. The reduced expression of Gn1a causes cytokinin accumulation in the inorescence meristem and increases the number of reproductive organs, resulting in enhanced grain number. The nearly isogenic line (NIL)-Gn1a in the Koshihikari genetic background has a higher grain number per panicle (45% higher than Koshihikari) and higher grain number per plant (34% higher than Koshihikari) [13]. The natural variation of DEP1 (DENSE AND ERECT PANICLE1) was identied from two F2 populations derived from japonica indica crosses (Q169 93-11 and W101 NJ6) [14]. The mutation allele of DEP1, coding for a truncated phosphatidylethanolamine-binding proteinlike domain protein, shows three dominant pleiotropic phenotypes: dense panicle, high grain number per panicle and erect panicle. Consequently, plants carrying the mutant allele show increased grain yield (+40.9%), although they also have a decreased 1000 grain weight [14]. WFP (WEALTHY FARMERS PANICLE) and IPA1 (IDEAL PLANT ARCHITECTURE) were independently identied, and it was later found that these are the same
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Review
QTL that encodes the transcription factor SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (OsSPL14) [15,16]. WFP from indica variety ST-12 and IPA1 from Shaoniejing increase the number of grains per panicle because of a higher number of primary branches per panicle. The OsSPL14 mRNA contains a target sequence of microRNA OsmiR156, which is highly expressed in the vegetative stage and which disappears in the early reproductive stage. By this mechanism, OsSPL14 expression is restricted in the early reproductive stage in the wild type plant (WT). We reported that the OsSPL14WFP allele shows an approximately nine times higher expression in the early reproductive stage than does the WT, probably because of an epigenetic effect in its promoter region, and this leads to the promotion of enhanced branch formation in the panicle [15]. Because the high expression of OsmiR156 suppresses OsSPL14 expression, there is no signicant effect on OsSPL14WFP expression in the vegetative stage. IPA1 had a single nucleotide polymorphism (SNP) in the microRNA target sequence [16]. This mutation in OsSPL14 confers resistance to the microRNA and leads to ectopic expression in the vegetative stage and higher expression in the reproductive stage [16]. OsSPL14ipa1 showed increased primary branches in the panicle, as with OsSPL14WFP; however, it also showed reduced tiller number and stronger culm, probably caused by its ectopic expression in the vegetative stage [16]. In a BC2F2 population derived from backcrossing Nipponbare to ST12, the grain number per plant of plants homozygous for the OsSPL14WFP allele was 3142 grains, which is 41% higher than the 2232 grains for plants homozygous for the Nipponbare allele. Furthermore, the BC2F2 plants carrying both Gn1a and OsSPL14WFP alleles produced 3396 grains per plant (52% higher than the plants carrying the Nipponbare alleles) [15]. The NIL of OsSPL14ipa1 in the XS11 background also showed increased grain number per plot (11% higher than that in XS11). In addition to grain number, OsSPL14ipa1 showed an increased number of vascular bundles and sclerenchyma cells [16]. Consistently, the culm mechanical strength of the NIL OsSPL14ipa1 was higher than that of its recurrent parent [16]. Taken together, these observations demonstrate that the modied plant architecture conferred by the higher expression of OsSPL14 has the potential to improve rice yields. QTLs for grain size and weight Grain weight is also an important factor affecting grain yield in rice. The grain weight of rice mainly depends on its size and lling rate. Recent studies have made signicant progress in the identication of genes regulating grain size and lling rate. GS3 was the rst major QTL in this category to be isolated, and it has been found that it contributes to both grain length and weight [17]. GS3 was identied in progeny derived from a cross between Minghui 63 (large grain) and Chuan7 (small grain). In the BC3F2 population with the Chuan7 background, the grain length of homozygous plants carrying the Minghui 63 allele was 10.2 mm, which was 39% longer than homozygous plants with the Chuan 7 allele (7.32 mm) [17]. Consequently, the 1000 grain weight of homozygous plants carrying the Minghui 63 allele increased to 25.6 g, which was 46%
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heavier than homozygous plants with the Chuan 7 allele (17.5 g) [17]. Mao et al. identied two more alleles, in addition to Minghui 63 and Chuan7, and classied four types of GS3 alleles [18]. GS3-1 (Zhenshan 97) shows medium grain length and has 231 amino acids in the coding region. The predicted protein of the GS3-1 allele is composed of four putative domains: a plant-specic organ size regulation (OSR) domain in the N terminus, a transmembrane domain, a tumor necrosis factor receptor/nerve growth factor receptor (TNFR/NGFR) family cysteine-rich domain and a von Willebrand factor type C (VWFC) in the C terminus. GS3-2 (Nipponbare) also shows medium grain length and has a 3 bp insertion in the coding region. GS3-3 (Minghui 63) shows long grain and has a C!A substitution 165 bp downstream of the translation start site, causing the premature termination of the predicted protein and resulting in the loss of all four domains. GS3-4 (Chuan 7) shows short grain and has a 1 bp deletion 357 bp downstream of the translation start site, causing the loss of the TNFR/NGFR and VWFC domains. From this observation and transgenic analysis, it was revealed that these domains have different functions in grain size regulation. The OSR domain is both necessary and sufcient for the protein function as a negative regulator. The TNFR/NGFR and VWFC domains show an inhibitory effect on the OSR function, whereas the loss-of-function mutations of these domains produced very short grains [18]. Another major QTL for grain size is GW2, which inuences grain width and weight [19]. This QTL was identied in progeny from a cross between WY3 (large grain) and Fengaizhan-1 (small grain). GW2 encodes the RING-type protein with E3 ubiquitin ligase activity, which is known to be involved in the degradation processes of the ubiquitin proteasome pathway [19]. The loss-of-function allele of GW2 increased cell number, resulting in a larger (wider) spikelet hull, and accelerated grain lling. NIL(GW2) in the Fengaizhan-1 background showed increased grain width (26.2% wider than Fengaizhan-1) and 1000 grain weight (49.8% heavier than Fengaizhan-1). Although the number of grains per panicle was lower in NIL(GW2) (29.9% lower than Fengaizhan-1), the total grain yield per plant increased (19.9% higher than Fengaizhan-1) [19]. Two homologs of GW2, ZmGW2-CHR4 and ZmGW2-CHR5, have been identied in the maize genome [20]. Linkage analysis has demonstrated that ZmGW2CHR4 is located within a consistent QTL of 100 kernel weight (HKW). In addition, association analysis using the diverse panel of 121 maize inbred lines revealed that an SNP in its promoter region is signicantly associated with kernel width and HKW [20], whereas its expression level is signicantly negatively correlated with these traits. Thus, it is possible that the GW2 genes in rice and maize play an important role in kernel size and weight variation. qSW5 [21] (also reported as GW5 [22]) is another QTL for grain width and weight and has also been reported as a rice domestication-related gene. This QTL was identied in progeny from a cross between Nipponbare (wide grain) and Kasalath (slender grain). The Nipponbare allele of qSW5 has a 1.2 kb deletion, which overlaps with a predicted gene of unknown function in the Kasalath genomic sequence. The transformation of the RNAi construct into

Review
Kasalath leads to an increase in grain weight, suggesting that the functional qSW5 gene has a negative effect on grain weight. The NIL (qSW5-Kas) showed decreased 100 grain weight (11% lower than Nipponbare) and total grain weight (13% lower than NIL (qSW5-Nip)). The decrease in grain weight with the functional qSW5 allele is because of the decreased cell number of the outer glumes, suggesting that this gene could also be involved in the proliferation of glume cells. A gene regulating grain lling GRAIN INCOMPLETE FILLING 1(GIF1) was mapped to chromosome 4 and encodes a cell wall invertase required for carbon partitioning during early grain lling [23]. Grains from a loss-offunction gif1 mutant accumulated lower levels of glucose, fructose and sucrose compared with wild type grains. This lower accumulation of sugar in the gif1 mutant leads to a large reduction in grain weight. The cultivated GIF1 allele shows a restricted expression pattern during grain lling compared with the wild rice allele because of accumulated mutations in the regulatory sequence of the gene by domestication. The higher expression of the wild rice GIF1 allele leads to smaller grains compared with the cultivated allele [23]. It has been interpreted that the restricted expression of the GIF1 in the ovular vascular trace is the key factor to increased grain weight. This also suggests that the tissue-specic higher expression levels of GIF1 could provide a means for increasing grain lling. QTLs for heading date Heading date is a crucial trait for the adaptation of rice to different cultivation areas and cropping seasons. To date, many QTLs contributing to heading date in rice have been detected [2429] and cloned [3033] from progeny from a cross between japonica and indica subspecies. Heading date 1 (Hd1) encodes a protein with a zinc nger and the CCT (CONSTANS, CONSTANS-LIKE, TOC1) motif and is an ortholog of Arabidopsis (Arabidopsis thaliana) CONSTANS [30]. Hd3a is a rice ortholog of FT, and its expression is regulated by Hd1 [32,34]. Hd6 encodes a casein kinase 2 alpha [31]. Tamaki et al. [35] demonstrated that Hd3a protein functions as a origen-type mobile owering signal. Another major QTL Early heading date1 (Ehd1) encodes a B-type response regulator [33]. Expression analysis has revealed that Ehd1 functions upstream of Hd3a [33]; however, no ortholog of the rice Ehd1 has been identied in Arabidopsis. Two QTLs, Ghd7 (grain number, plant height and heading date) and DTH8 (days to heading) encode a CCT domain protein and a putative HAP3 subunit of the CCAAT box-binding transcription factor, respectively [36,37]. The functional alleles of these genes delay heading date under long-day conditions and increase plant height and panicle size. The functional Ghd7 is highly expressed under long-day conditions and it almost completely suppresses the expressions of Ehd1 and Hd3a [36]. Similarly, the functional allele of DTH8 suppresses the expressions of Ehd1 and Hd3a under the light condition [37]. Taken together, these results indicate that Ehd1 could be an integrator of different pathways in the regulation of owering, which is downregulated by Hd1, Ghd7 and DTH8. Then, under long-day conditions, the expression of Hd3a or

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its ortholog(s) is activated by Ehd1, and the transition from vegetative to oral development occurs. QTL for lodging resistance Lodging resistance is an important agronomic trait that must be included in breeding programs to obtain high yields. Although reducing plant height has been the major target to improve lodging resistance thus far, as in the famous case of semi-dwarf-1 (sd-1) used in varieties produced in the green revolution of the 1960s [38], semi-dwarf mutations also reduce biomass production, resulting in a negative effect on yield components. An alternative strategy to achieve lodging resistance is the isolation of genes directly controlling culm strength. Recently, we cloned the major QTL STRONG CULM2 (SCM2) from the high-lodging-resistant indica cultivar Habataki [39]. The positional cloning of SCM2 revealed that it is identical to APO1, which has been reported to encode an F-box-containing protein orthologous to the Arabidopsis UFO gene and to control the panicle structure [40]. The NIL-SCM2 in the Koshihikari background resulted in enhanced culm strength (59% higher than Koshihikari) [39]. The NILSCM2 plants also showed pleiotropic phenotypes, such as increased brunch number (26% higher than Koshihikari) and grain number (21% higher than Koshihikari) per panicle [39]. Although the SCM2 allele of APO1 showed some desirable effects on crop yield, overexpression mutants, apo1-D and Ur1, showed a dramatic decrease in tiller number [41,42], probably caused by the ectopic and higher expression of APO1. The identication and application of SCM2, a weak allele of APO1 overexpression mutation, taught us that it is important to choose a suitable allele carrying a desirable trait and losing inadequate traits from variations of alleles. QTL for disease resistance QTLs controlling quantitative disease resistance are a valuable source of broad spectrum and durable disease resistance. To date, approximately 30 R genes for bacterial blight resistance have been identied, and six of these (Xa1, Xa3/Xa26, xa5, xa13, Xa21 and Xa27) have been isolated and characterized [43]. In addition, more than 50 R genes for blast resistance have been identied, and eight of these (Pib, Pi-d2, Pi-ta, Pizt, Pi2, Pi9, Pi36 and Pi37) have been characterized [43]. Recently, two QTLs for broad-spectrum blast resistance, pi21 and Pb1, were cloned. pi21 was identied in the progeny from a cross between Aichiasahi (susceptible) and Owarihatamochi (resistant), which is a QTL gene contributing to blast disease resistance, and is different from other previously isolated R genes. Pi21 does not contain the typical structure of R genes such as NBS-LRR, but encodes a proline-rich protein containing a putative heavy metal-1 binding domain and a putative proteinprotein interaction motif [44]. The rate of penetration of the hyphae of Magnaporthe oryzae into the cells of NIL-pi21 plants did not differ from that of its host plant. However, the rate of the invasion of hyphae from penetrated cells into adjacent cells, as an indicator of hyphal growth, was signicantly arrested in NIL-pi21 [44], indicating that Pi21 could be involved in a mechanism
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to resist hyphal invasion, although the molecular function of the Pi21 protein has not yet been determined. Pb1 is a broad-spectrum panicle blast resistance gene, identied from a 60 kb duplicated chromosomal region of the indica cultivar Modan [45]. Pb1 encodes coiledcoil (CC)-NBS-LRR and contains two putative CC domains in the N terminus. The Pb1 protein sequence differed from previously reported R proteins, particularly in the NBS domain, in which the P-loop was apparently absent and some other motifs were degenerated. Rice cultivars into which Pb1 has been introduced are rather weakly resistant to leaf blast but they do show remarkable resistance to panicle blast [45]. QTL for salt tolerance Rice is cultivated under diverse biotic and abiotic stresses, and soil salinity is one of the important stresses that limits rice growth and yield. To date, several QTLs for salt tolerance have been reported. The QTL for salt tolerance SKC1 (shoot K+ content) was cloned from progeny of a cross between a salt-tolerant indica variety Nona Bokra and a susceptible japonica variety Koshihikari [46]. SKC1 encodes an HKT-type transporter. Detailed biochemical analyses revealed that SKC1 is a selective transporter for Na+, which is localized at the plasma membrane and preferentially expressed in the parenchyma cells surrounding the xylem vessels. NIL-SKC1 in the Koshihikari background showed signicantly lower Na+ content in shoots compared with Koshihikari under salt stress conditions at the seedling stage [46]. Furthermore, Lee et al. [47] detected QTLs for salt tolerance at the seedling stage by using recombinant inbred lines (RILs) derived from a cross between Milyang 23 (indica/japonica) and Gihobyeo (japonica). Although both these parents were susceptible to salt, the RILs showed a wide range of salt sensitivity. Of the 164 RILs, 22 were tolerant, 47 were intermediate and 93 were sensitive. Using these BILs, two QTLs were detected on chromosomes 1 and 3, namely qST1 and qST3, respectively, but the isolation of these QTL genes has not yet been achieved. Cold tolerance Cold stress is a crucial factor at several stages of the growth of rice plants. QTLs for three aspects of cold tolerance, lowtemperature germinability, cold tolerance at the seedling stage (CTSS) and cold tolerance at the booting stage, have been reported thus far. Three QTLs for low-temperature germinability were detected on chromosomes 3 and 4, qLTG-3-1, qLTG-3-2 and qLTG-4, from a cross between temperate japonica varieties, Italica Livorno (vigorous) and Hayamasari (weak) [48]. The germination rate at 15 8C for 7 days of Italica Livorno and Hayamasari was 98.7% and 26.8%, respectively [48]. The most effective QTL qLTG-3-1 accounted for 35.0% of the total phenotypic variation for low-temperature germinability, whereas two additional QTLs, qLTG-3-2 and qLTG-4, accounted for 17.4% and 5.5% of the total phenotypic variation, respectively [48]. The map-based cloning of qLTG-3-1 revealed that it encodes an unknown function protein containing two known conserved domains, GRP (glycinerich cell wall protein) and Tryp alpha amyl of the protease
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inhibitor/seed storage/LTP family, and a conserved amino acid motif [49]. A high level of qLTG-3-1 expression occurred in the embryo during seed germination, and it was tightly associated with the vacuolation of the tissues covering the embryo. This could cause tissue weakening, resulting in a reduction in the mechanical resistance to growth of the coleoptile [49]. Koseki et al. reported three QTLs for CTSS [50]. The major locus qCtts11 was located on the long arm of chromosome 11 and explained approximately 40% of phenotypic variation. The introduction of the cold tolerance W1943 (Oryza rupogon) allele of qCtss11 to Guang-lu-ai 4 (cold sensitive indica cultivar) signicantly increased CTSS. Map-based cloning narrowed down the qCtss11 locus to a 60 kb region containing six predicted genes, but the causal gene has not yet been identied [50]. Saito et al. reported two closely linked QTLs for cold tolerance at the booting stage, Ctb1 and Ctb2 on chromosome 4, from progeny derived from crosses between a coldtolerant japonica variety Norin-PL8 and a cold-sensitive japonica variety Kirara397 [51]. Ctb1 was narrowed down to a 56 kb region by map-based cloning using NILs and this locus contains seven candidate genes [52]. Zhou et al. also reported a QTL for cold tolerance at the booting stage on chromosome 7, namely qCTB7 [53]. This QTL was identied in progeny derived from a cross between the cold tolerant variety Kunmingxiaobaigu and the sensitive japonica variety Towada. qCTB7 explained 9% and 21% of the phenotypic variances in the F2 and F3 generations, respectively. Map-based cloning narrowed down the qCTB7 locus to a 92 kb region containing 12 predicted genes [53]. Submergence tolerance Submergence stress because of ash ooding is one of the major constraints to rice production in south and southeast Asia. It causes a reduced oxygen supply and thereby inhibits respiration. A major QTL against such stress Submergence1 (Sub1) was detected from progeny derived from a cross between a submergence-tolerant indica variety FR13A and an intolerant japonica variety M-202 [54]. The FR13A locus contains a cluster of three genes, Sub1A, Sub1B and Sub1C. All three genes encode putative ethylene response factors (ERFs). The overexpression of the Sub1A-1 allele in a submergence-intolerant japonica variety conferred enhanced tolerance to the plants, the downregulation of Sub1C and the upregulation of Alcohol dehydrogenase 1, indicating that Sub1A-1 is a primary determinant of submergence tolerance [54]. In contrast to Sub1, which restricts plant elongation during ash ooding, deepwater rice has the unique ability to elongate its internodes with increasing water depth. Recently, we identied the major QTL for the deepwater response from the progeny derived from a cross between deepwater rice C9285 and non-deepwater rice Taichung 65 [55]. The most effective QTL, which is on chromosome 12, contains two genes, SNOKEL1 (SK1) and SNOKEL2 (SK2). These genes possess a putative nuclear localization signal and a single APETALA2/ERF domain. The ERF domains in the SK genes showed a high similarity to those of Arabidopsis thaliana ERF1, O. sativa ERF1 and

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SUB1A-1. Although the overexpression of both SK1 and SK2 results in internode elongation in air, SK2 was more effective at internode elongation in water than was SK1 [55]. Additionally, a wild rice W0106 (Oryza nivara), which carries the active SK1 and inactive SK2 caused by transposon insertion, showed little response to deepwater. These observations indicate that SK2 is important for the deepwater response [55]. QTL for cadmium accumulation in grain The accumulation of cadmium (Cd) in rice grains is a serious concern for food safety. Cd can be accumulated in the human body over time from the ingestion of food containing Cd [56]. The disease Itai-Itai, which has occurred in Japan in the past, is the result of the intake of Cdcontaining rice. Therefore, reducing Cd accumulation in rice is an important issue for human health [56]. To date, several QTLs for Cd accumulation in grain have been identied in japonica indica crosses. Ishikawa et al. [57] detected three QTLs on chromosomes 3, 6 and 8 for Cd concentration in brown rice using chromosome segment substitution lines derived from Koshihikari and Kasalath. Kashiwagi et al. [58] identied three QTLs for Cd concentration in brown rice from BILs derived from crosses between Nipponbare and Kasalath; two on chromosome 4 and one on chromosome 11. Ishikawa et al. [59] identied two QTLs for Cd concentration in grains on chromosomes 2 and 7 from a cross between the low-Cd-accumulating cultivar Sasanishiki (japonica) and high-Cd-accumulating cultivar Habataki (indica). Ueno et al. [56] detected a QTL with a large effect for Cd accumulation on chromosome 7 from a cross between the indica rice cultivar Anjana Dhan (high-Cd concentration) and japonica cultivar Nipponbare (low-Cd concentration) and revealed the encoded protein to be a putative heavy metal transporter OsHMA3 [60]. Heterologous expression in yeast showed that the transporter from the low-Cd cultivar is functional, but the transporter from the high-Cd cultivar had lost its function, probably because of a single amino acid mutation [60]. Yield-related domestication genes To date, several domestication-related genes in rice have been reported. Among these, QTLs for yield-related traits have also been reported. During the domestication of rice, many of the negative yield traits in wild rice were lost. However, when benecial genes are introduced into cultivated rice from wild rice it is necessary to eliminate any associated negative traits by marker-assisted selection. Thus, information on yield-related domestication genes is necessary for effective breeding programs. The loss of the seed shattering trait was a key yieldrelated event during rice domestication. Two major QTLs for seed shattering (sh4 and qSH1) have been cloned to date [61,62]. sh4 was detected in the F2 population derived from a cross between cultivated rice (O. sativa) and wild rice (O. rupogon) [61]. This QTL explains 69% of the total phenotypic variance in the F2 population. The wild rice allele of sh4 encodes a putative transcription factor Myb3 DNA-binding domain, whereas the cultivated rice allele has a SNP in the domain. This mutation causes the misformation of the abscission layer between the pedicel and

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spikelet [61]. qSH1 was detected in the F2 population derived from a cross between Nipponbare (japonica) and Kasalath (indica) and explains 68.6% of the total phenotypic variance [62]. qSH1 encodes a BEL1-type homeobox gene, and the causal mutation was found in the 50 upstream region of the gene in Nipponbare. This mutation causes the absence of the abscission layer in Nipponbare [62]. The transition from the prostate growth of ancestral wild rice (O. rupogon) to erect growth of O. sativa cultivars was one of the critical domestication events in rice. This evolutionary step improved plant architecture and increased grain yield. PROG1 (PROSTRATE GROWTH 1) is a semi-dominant gene isolated from an F2 population derived from a cross between Teqing (elite indica variety) and introgression lines that carried a wild rice (O. rupogon) genomic segment of the short arm of chromosome 7 in Teqing genetic background [63,64]. PROG1 encodes a zincnger nuclear transcription factor and it is expressed predominantly in the axillary meristems [64]. prog1 variants identied in O. sativa are disrupted in gene expression and protein function and lead to erect growth, greater grain number and higher grain yield in cultivated rice [63]. Future perspectives Most of the QTLs described in Table 1 have been cloned from progeny derived from a cross between japonica and indica subspecies, because the intersubspecic diversity is signicantly larger than is the intrasubspecic diversity, which makes it easy to produce molecular markers in the regions of interest in the rice genome. However, a recently developed advanced PCR marker system [8,6569] and genotyping technology [68,70] have made it possible to produce markers and to perform QTL analysis using intrasubspecic crosses. For example, QTLs have been identied [71] for days to heading in BILs from crosses between Nipponbare (japonica cultivar) and Koshihikari (japonica cultivar). In addition to the development of genotyping technology, high-throughput sequencing systems have enabled researchers to carry out whole-genome resequencing [7275]. The authors of [76] performed the whole-genome sequencing of an elite Japanese rice variety Koshihikari using high-throughput sequencing, and they then designed high-throughput typing arrays [77,78] based on the SNP information from a comparison of the sequences of Koshihikari and Nipponbare. In addition, the authors of [79] performed high-throughput genotyping by whole-genome resequencing. They carried out the whole-genome genotyping of 150 rice RILs using 287 PCR-based markers and whole-genome resequencing using an Illumina Genome Analyzer. The whole-genome resequencing method provided a resolution of recombination breakpoints within an average of 40 kb. In comparison to the genetic map constructed with 287 PCR-based markers, the sequencingbased method was approximately 20 times faster in data collection and 35 times more precise in recombination breakpoint determination. Furthermore, a whole-genome sequencing study of 517 rice landraces [80] identied approximately 3.6 million SNPs. From this sequence information, the authors constructed a high-density haplotype map, performed genome-wide association studies
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Table 1. Rice genes responsible for major QTLs involved in agronomic traits
Trait Grain number Grain number and strong culm Grain number Grain number, low tiller number and strong culm Grain size Grain size and lling Grain size Grain lling Heading date Name of QTL Gn1a dep1 WFP Ipa gs3 gw2 qSW5/GW5 GIF1 Hd1 Encoded protein Cytokinin oxidase PEBP-like domain protein OsSPL14 OsSPL14 Transmembrane protein RING-type ubiquitin E3 ligase Unknown Cell wall invertase CONSTANS-like protein

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Nature of allele suitable for use in breeding programs Low expression Loss of function High expression High and ectopic expression Loss of function Loss of function Loss of function Restricted expression in the ovular vascular trace Loss-of-function allele leads to late heading Lossof-function allele leads to early heading Low expression leads to late heading Loss-of-function allele leads to late heading Functional allele Functional allele Loss of function High expression Loss of function Functional allele Gain of function Functional allele Gain of function Gain of function Functional allele Loss of function Low expression in abscission layer between pedicel and spikelet Loss of function

Refs [13] [14] [15] [16] [17] [19] [21,22] [23] [30]

Heading date Heading date Heading date Grain number, plant height and heading date Days to heading Plant height Lodging resistance Disease resistance Disease resistance Salt tolerance Cold tolerance Submerge tolerance Internode elongation under submerge condition Cadmium accumulation Seed shuttering Seed shuttering Prostrate growth

Hd6 Hd3a Ehd1 Ghd7 DTH8 sd1 SCM2 pi21 Pb1 SKC1 qLTG3-1 Sub1A SK2 OsHMA3 sh4 qSH1 PROG1

asubunit of protein kinase FT-like B-type response regulator CCT domain protein CCT domain protein Gibberellin 20 oxidase F-box protein Proline-rich protein CC-NBS-LRR protein HKT-type transporter GRP and LTP domain ERF-related factor ERF-related factor Putative heavy metal transporter Myb3 transcription factor BEL1-like homeobox protein Zinc nger transcription factor

[31] [32,34,35] [33] [36] [37] [38] [39] [44] [45] [46] [49] [54] [55] [60] [61] [62] [63,64]

(GWAS) for 14 agronomic traits and detected six previously identied loci. These studies indicate that the next generation of genome sequencing and GWAS strategy are powerful tools for dissecting complex traits in rice. Such new technologies have the potential to accelerate the detection and cloning of QTLs in rice and allow us to accumulate QTLs, which will be required to make precise changes in agronomic traits. Although several QTLs have been identied from cultivated indica varieties (e.g. Gn1a, sd1, DEP1), this means these genes are effective for most japonica varieties but not for indica varieties. To address such issues, the further exploration of benecial genetic resources from a wider range of genetic backgrounds is necessary. This should include an expansion of the function of benecial genes by transgenic strategies in combination with QTL-based breeding technology [2]. Furthermore, to make a real contribution to world food security, the vigorous exchange of genetic resources among countries and collaboration of plant breeders are required. For the future, it will be necessary to test the actual productivity of various combinations
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of QTLs in various genetic backgrounds for each country. The optimum combinations of QTLs can then be used to develop commercial varieties suited to the diverse challenges of agriculture in the 21st century.
Acknowledgments
This work was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Genomics for Agricultural Innovation, IPG0003).

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