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Sake-brewing cultivars among varieties of Japanese rice (Oryza sativa L.) have traits adapted to the sake-brewing process, such as a high white-core expression rate (WCE). Our previous study detected putative quantitative trait loci (QTLs) associated with a high WCE derived from Yamadanishiki, a popular brewing rice cultivar. Because the occurrence of white-core grains depends on air temperature and the position of the grain on the panicle, phenotyping of WCE must consider these variable conditions. In this study, qWCE6, a QTL for the WCE on chromosome 6, was validated for the first time, and the phenotyping method examined for its suitability in fine-mapping. A clear tendency towards high WCE was observed in late-heading substituted lines which headed under low daily mean temperature at the experimental location. White-core grains were often expressed by the primary spikelets on the upper panicle, producing a high percentage of superior grains. The segregating population for qWCE6 in late heading revealed a distinct difference in WCE between the Koshihikari and Yamadanishiki homozygous alleles at qWCE6 as determined from that locality. Further, two insertion/deletion markers were developed for the marker-assisted selection of qWCE6. Our results will be useful for informing the breeding of sake-brewing rice cultivars.

Bacterial grain rot (BGR), caused by the bacterial pathogen Burkholderia glumae, is one of the most destructive rice (Oryza sativa) diseases in Japan; however, there are no BGR-resistant cultivars for use in Japan. We previously developed a cut-panicle inoculation method to assess the levels of BGR resistance in the World Rice Collection (WRC). Here, we evaluated major Japanese cultivars for BGR resistance and found that none showed "strong" or "medium to strong" resistance; most were categorized as "medium to weak". On the basis of the screening results, standard cultivars for BGR resistance were selected according to resistance level and relative maturity. Our results indicate that it is necessary to introduce quantitative trait loci (QTLs) from indica or tropical japonica resistant cultivars into Japanese temperate japonica to develop BGR-resistant cultivars for Japan. We previously developed a near-isogenic line (RBG2-NIL) by introducing the genomic region containing RBG2 from 'Kele' (indica) into 'Hitomebore'. In this experiment, we confirmed the resistance level of RBG2-NIL. The resistance score of RBG2-NIL was "medium to strong", indicating its effectiveness against BGR.

The fungal pathogen Pyricularia oryzae causes blast, a severe disease of rice (Oryza sativa L.). Improving blast resistance is important in rice breeding programs. Inoculation tests have been used to select for resistance genotypes, with DNA marker-based selection becoming an efficient alternative. No comprehensive DNA marker system for race-specific resistance alleles in the Japanese rice breeding program has been developed because some loci contain multiple resistance alleles. Here, we used the Fluidigm SNP genotyping platform to determine a set of 96 single nucleotide polymorphism (SNP) markers for 10 loci with race-specific resistance. The markers were then used to evaluate the presence or absence of 24 resistance alleles in 369 cultivars; results were 93.5% consistent with reported inoculation test-based genotypes in japonica varieties. The evaluation system was successfully applied to high-yield varieties with indica genetic backgrounds. The system includes polymorphisms that distinguish the resistant alleles at the tightly linked Pita and Pita-2 loci, thereby confirming that all the tested cultivars with Pita-2 allele carry Pita allele. We also developed and validated insertion/deletion (InDel) markers for ten resistance loci. Combining SNP and InDel markers is an accurate and efficient strategy for selection for race-specific resistance to blast in breeding programs.

The eating quality of cooked rice is important and determines its market price and consumer acceptance. To comprehensively describe the variation of eating quality in 183 rice germplasm accessions, we evaluated 33 eating-quality traits including amylose and protein contents, pasting properties of rice flour, and texture of cooked rice grains. All eating-quality traits varied widely in the germplasm accessions. Principal-components analysis (PCA) revealed that allelic differences in the Wx gene explained the largest proportion of phenotypic variation of the eating-quality traits. In 146 accessions of non-glutinous temperate japonica rice, PCA revealed that protein content and surface texture of the cooked rice grains significantly explained phenotypic variations of the eating-quality traits. An allelic difference based on simple sequence repeats, which was located near a quantitative trait locus (QTL) on the short arm of chromosome 3, was associated with differences in the eating quality of non-glutinous temperate japonica rice. These results suggest that eating quality is controlled by genetic factors, including the Wx gene and the QTL on chromosome 3, in Japanese rice accessions. These genetic factors have been consciously selected for eating quality during rice breeding programs in Japan.

A pair of complementary genes, Hwc1-1 at HWC1 locus and Hwc2-1 at HWC2 locus, cause a weakness phenomenon in rice. For this study, we performed haplotype analysis around the HWC2 locus in two core collections comprising 119 accessions. We also examined reactions to phenol and Xanthomonas oryzae pv. oryzae (Xoo) Japanese race I. To elucidate the genetic relations among all accessions, we analyzed their banding patterns of 40 Indel markers covering the rice genome. The classification by Indel markers was almost consistent with that using 4,357 SNPs. The testcross with Hwc1-1 carrier indicated that 37 accessions carried Hwc2-1 allele, whereas 82 carried hwc2-2 allele. Strong association between HWC2 and Ph genes was observed. Based on 14 DNA markers around HWC2 locus and Ph genotype, the 119 accessions were divided into 50 haplotypes. To examine the HWC2 candidate chromosomal region specifically, the ‘haplotype group’ characterized by the six DNA markers closely linked with HWC2 were analyzed. Hwc2-1 carriers had the same haplotype group. Some hwc2-2 haplotype groups were associated with resistance against the Xoo race. The relation between varietal differentiation and haplotypes around the HWC2 locus was discussed, along with its breeding significance.

Insertion-deletion (indel) polymorphisms, such as simple sequence repeats, have been widely used as DNA markers to identify QTLs and genes and to facilitate rice breeding. Recently, next-generation sequencing has produced deep sequences that allow genome-wide detection of indels. These polymorphisms can potentially be used to develop high-accuracy polymerase chain reaction (PCR)-based markers. Here, re-sequencing of 5 indica, 2 aus, and 3 tropical japonica cultivars and Japanese elite cultivar 'Koshihikari' was performed to extract regions containing large indels (10-51 bp) shared by diverse cultivars. To design indel markers for the discrimination of genomic regions between 'Koshihikari' and other diverse cultivars, we subtracted the indel regions detected in 'Koshihikari' from those shared in other cultivars. Two sets of indel markers, KNJ8-indel (shared in eight or more cultivars, including 'Khao Nam Jen' as a representative tropical japonica cultivar) and C5-indel (shared in five to eight cultivars), were established, with 915 and 9,899 indel regions, respectively. Validation of the two marker sets by using 23 diverse cultivars showed a high PCR success rate (≥95%) for 83.3% of the KNJ8-indel markers and 73.9% of the C5-indel markers. The marker sets will therefore be useful for the effective breeding of Japanese rice cultivars.

A total of 324 Japanese rice accessions, including landrace, improved, and weedy types were used to 1) investigate genetic variations in blast resistance to standard differential isolates, and 2) across the genome using polymorphism data on 64 SSR markers. From the polymorphism data, the accessions were classified into two clusters,. Accessions from irrigated lowland areas were included mainly in cluster I, and upland and Indica types were mainly in cluster II. The accessions were classified into three resistance subgroups, A2, B1 and B2, based on the reaction patterns to blast isolates. The accessions in A2 were postulated to have at least two resistance genes Pish and Pik-s, whereas those in B1 had various combinations of the resistance genes Pish, Pia, Pii, Pi3, Pi5(t), and Pik alleles. The B2 accessions were resistant to almost all isolates, and many accessions of cluster II were included, and had Pish, Pia, Pii, Pi3, Pi5(t), certain Pik, Piz and Pita alleles, and unknown genes. The frequencies of accessions of B1 originating in Hokkaido, and those of B2 originating in the Kanto and Tohoku regions were remarkably higher than in the other regions.

The appearance of brown rice grown under high temperature conditions is an important characteristic for improvement in Japanese rice breeding programs. We performed a QTL analysis of the appearance quality of brown rice using chromosome segment substitution lines of the indica cultivar 'Habataki' in the 'Koshihikari' genetic background. A line carrying a 'Habataki' segment on chromosome 7 showed a high percentage of perfect grains produced under high temperature conditions during the ripening period. To verify the role of this segment, and to narrow down the region containing the useful allele, substitution mapping was performed using multiple paired lines. As a result, the chromosomal location of a gene that we named Appearance quality of brown rice 1 (Apq1) was delimited to a 48-kb region. In addition, we developed an Apq1-near isogenic line (NIL) to evaluate the effect of Apq1 on various agronomic traits. Under high temperature conditions during the ripening period, the Apq1-NIL produced significantly higher percentages of perfect grains than 'Koshihikari'. Other agronomic traits, including yield and palatability, were similar between the Apq1-NIL and 'Koshihikari'. Therefore, the 'Habataki' allele of Apq1 will be useful in breeding programs aimed at improving the quality of grains ripened under high temperature conditions.

It is essential to elucidate genetic diversity and relationships among even related individuals and populations for plant breeding and genetic analysis. Since Japanese rice breeding has improved agronomic traits such as yield and eating quality, modern Japanese rice cultivars originated from narrow genetic resource and closely related. To resolve the population structure and genetic diversity in Japanese rice population, we used a total of 706 alleles detected by 134 simple sequence repeat markers in a total of 114 cultivars composed of 94 improved varieties and 20 landraces, which are representative and important for Japanese rice breeding. The landraces exhibit greater gene diversity than improved lines, suggesting that landraces can provide additional genetic diversity for future breeding. Model-based Bayesian clustering analysis revealed six subgroups and admixture situation in the cultivars, showing good agreement with pedigree information. This method could be superior to phylogenetic method in classifying a related population. The leading Japanese rice cultivar, Koshihikari is unique due to the specific genome constitution. We defined Japanese rice diverse sets that capture the maximum number of alleles for given sample sizes. These sets are useful for a variety of genetic application in Japanese rice cultivars.

Low temperatures at the initial stages of rice development prevent fast germination and seedling establishment and may cause significant productivity losses. In order to develop rice cultivars exhibiting cold tolerance, it is necessary to investigate genetic resources, providing basic knowledge to allow the introduction of genes involved in low temperature germination ability from accessions into elite cultivars. Japanese rice accessions were evaluated at the germination under two conditions: 13°C for 28 days (cold stress) and 28°C for seven days (optimal temperature). The traits studied were coleoptile and radicle length under optimal temperature, coleoptile and radicle length under cold and percentage of the reduction in coleptile and radicle length due to low temperature. Among the accessions studied, genetic variation for traits related to germination under low temperatures was observed and accessions exhibiting adequate performance for all investigated traits were identified. The use of multivariate analysis allowed the identification of the genotypes displaying cold tolerance by smaller reductions in coleoptile and radicle lenght in the presence of cold and high vigour, by higher coleoptile and radicle growth under cold.

Quantitative trait loci (QTLs) for low cadmium concentration in brown rice were analyzed in 2008 and 2009 using 126 RIL (F6 and F7 plants) grown in a paddy field with high Cd concentration, which were derived from a cross between a high yielding Japanese rice cultivar ‘Fukuhibiki’ and an African upland rice cultivar ‘LAC23’ with the lowest cadmium concentration in rice grains. Totally two QTLs controlling low cadmium concentration in grains were identified in both 2008 and 2009, i.e., one on chromosome 3 (qLCdG3) and one on chromosome 11 (qLCdG11), which had opposite additive effects. In qLCdG11, ‘LAC23’ alleles had the effect of reducing cadmium concentration, but qLCdG3 had the opposite effect. The explained phenotypic variances of qLCdG11 were 9.4% in 2008 and 12.9% in 2009, and those of qLCdG3 were 13.9% in 2008 and 8.3% in 2009. Cadmium concentrations in brown rice showed positive correlations with grain length and grain weight. QTLs for grain length and grain weight were detected on chromosomes 3 and 11, but their positions differed from those for cadmium concentrations. These results suggest a possibility of successful marker-assisted selection of low cadmium trait derived from ‘LAC23’.

To analyze genetic features in the Japanese rice population, which is ma inly composed of closely related ac-cessions, a core set of single-nucle otide polymorphisms (SNPs) was se lected from SNP resources based onJapanese rice cultivars. A total of 25,199 SNPs were newly detected from the comparison of genomic se-quences between two cultivars (Eiko an d Rikuu132) and Nipponbare as a re ference. A total of 81,499 non-redundant SNPs, including 67,051 SNPs of Koshihikari de tected in a previous study, were used as candidatesto select the core SNPs. Across the entire genome, 3379 SNPs were sel ected based on the chromosomal po-sition of each SNP and were investigated each alle le of 92 Japanese rice accessions and 3 from outside ofJapan. As a result, 2551 SNPs were fo und to be informative for at least 91 cultivars for reducing the potentialrisks of genotyping error. The Japanese rice accessions were classified into three groups (upland, lowlandHokkaido, and other lowland) using all 2551 SNPs. In ad dition, a core set of 768 SNPs was selected to pro-vide an even distribution among the chromosomes. Co mparison of dendrograms generated by all 2551 SNPsand the core set of 768 SNPs demonstrated that the core SNPs could be used effici ently and reliably in theclassification of the Japanese rice p opulation. The core SNPs can be used for diversity analysis and for ge-netic analysis of the biparental po pulations of Japanese rice accessions.Key Words: core SNP set, genetic diversity, Japanese rice population, Oryza sativa .IntroductionA wide range of naturally occurri ng variation exists in ricecultivars. Phenotypic variations have been utilized in ricebreeding and some have been analyzed in detail by means ofmolecular markers (reviewed by Yamamoto et al. 2009). Ingeneral, a high level of seque nce diversity has been observedbetween indica and japonica cultivars without regard to the-ories of independent (Garris et al. 2005, Londo et al. 2006)or non-independent (Gao et al. 2008) domestication. On theother hand, low diversi ty is observed among japonica popu-lations including Japanese cultivars (Glaszmann 1987). Toaccess genetic variations in Ja panese rice, several approacheshave been conducted.In the Japanese rice population, the two main agro-ecotypes are upland and lowland rice, the diversity and clas-sification of which have been analyzed using several kindsof genetic markers such as isozymes (Ishikawa et al. 1991),restriction-fragment length polymorphisms (RFLP; Ishikawaet al. 1997, Kojima et al. 2005, Kono et al. 2000), chloro-plast genotypings (Kawakami et al. 2007), and simple se-quence repeats (SSR; Kobayashi et al. 2006, Okoshi et al.2004). These studies have de monstrated that narrow geneticvariation exists among Japanese rice cultivars, which aremostly of japonica origin. Even though considerable pheno-typic variation exists among Japanese cultivars (Ebana et al.2008), it has been difficult to perform genetic dissection ofsuch variations because of th e difficulty of detecting poly-morphisms with molecular markers (Yamamoto et al. 2009).This situation has improved with the recent availability of alarge amount of SSR motif information for the rice genome,which has been revealed by the sequencing of the entireNipponbare genome (McCouch et al. 2002, InternationalRice Genome Sequencing Proj ect 2005). Single-nucleotidepolymorphisms (SNPs) are considered the most effectiveway of assessing such polymorphisms (Rafalski 2002); how-ever, until recently, little in formation has existed aboutgenome-wide SNP among Japanese cultivars.The advent of gene sequencing technology has allowed usto perform large-scale simult aneous sequencing of multiplegenomes, eventually leadin g to genome-wide discovery ofSNPs. Recently, about 160,00 0 non-redundant SNPs havebeen successfully detected am ong 20 diverse varieties andlandraces, including Japanese cultivars, using new technology(McNally e t al. 2009). More recently, 67,051 non-redundantSNPs were detected using ne xt-generation sequencing tech-nology between the elite Japa nese rice cultivars Koshihikari

To identify quantitative trait loci (QTLs) associated with culm length in Japanese japonica rice varieties, we conducted QTL analyses using a set of reciprocal backcrossed inbred lines (BILs) from crosses between Nipponbare and Koshihikari. We detected five QTLs in the two BILs that are involved in the culm-length difference between Nipponbare and Koshihikari. A QTL located on the short arm of chromosome 1, qCL1, was commonly detected near the simple sequence repeat (SSR) marker RM8068 in both BILs in three growing seasons. The Nipponbare allele of qCL1 shortened from 1.9 to 3.0 cm of culm length. Substitution lines (SLs) of Koshihikari in which the QTL was replaced with the Nipponbare allele exhibited decreased lengths of lower internodes. qCL1 was mapped within about 2.6 Mbp in the region distal from SSR marker RM10132, suggesting qCL1 differed from the dwarf or semi-dwarf genes that have been previously reported. Japanese japonica rice landraces with Nipponbare alleles of DNA markers around qCL1 had significantly shorter culm length than other landraces. The results suggest that the novel allele for short culm length was originally distributed in Japanese rice landraces and has been used for introducing the semi-dwarf phenotype and improving plant architecture during practical breeding.

A mini core collection of Japanese rice landrace was developed based on a genome-wide SSR polymorphism survey of 236 accessions. The accessions used in the initial survey were selected based on the historical record and passport data from the collection maintained at the Genebank of the National Institute of Agrobiological Sciences (NIAS). These accessions were analyzed using 32 SSR markers. A total of 247 alleles were detected and the number of alleles per locus ranged from 3 to 15 (mean 7.7). Principal coordinate analysis using SSR data enabled classification of the accessions into three major groups, two Japonica groups and one Indica group. To develop a Japanese landrace mini core collection, the SSR data on the 236 accessions were subjected to cluster analysis and 50 groups were recognized at a similarity index of 0.88. A single accession from each of the 50 groups was selected. These 50 accessions retained 87.5% of the alleles detected in the original 236 accessions, and covered the variation of the initial set of accessions in terms of several agro-morphological traits.

To identify the chromosomal regions controlling the eating quality of Koshihikari rice, we performed a quantitative trait locus (QTL) analysis using two backcross inbred lines (BILs): N-BILs (79 lines derived from a cross of Nipponbare/Koshihikari//Nipponbare) and K-BILs (89 lines derived from a cross of Nipponbare/Koshihikari//Koshihikari). We evaluated several components of the eating quality of cooked rice, namely glossiness, taste, stickiness, hardness, and overall evaluation, based on sensory tests by a trained panel, and amylose and protein contents. Ten QTLs for these components were detected in N-BILs (two regions of chromosome [chr.] 3 and one of chr. 11), and six in K-BILs (chr. 3 and chr. 6). Each QTL explained 11.6% to 32.0% of the total phenotypic variance. QTLs at the distal end of the short arm of chr. 3 were commonly identified in both BILs. The Koshihikari alleles at these QTLs increased eating quality. The genetic effect of the Koshihikari alleles was confirmed by analysis of a chromosome segment substitution line containing a Koshihikari segment of the short arm of chr. 3 in the Nipponbare background.

The weak growth occurring in hybrids derived from crosses between two normal strains is referred to as hybrid weakness. F1 hybrid weakness (F1HW) in a cross between a Peruvian rice cultivar ‘Jamaica’ and Japanese rice cultivars has been reported and was considered to be controlled by a set of complementary genes, Hwc1 and Hwc2. We observed the development of F1 plants between Nipponbare and Jamaica (NJF1s). NJF1s were characterized by short roots, rolled leaves and a short stature. Although the NJF1 embryos were normal, the growth of the primary roots was arrested and in most cases, they could not emerge from seeds of the hybrids. Histological observation of the root apical meristem (RAM) at 10 days after sowing revealed that the zone of cell division in the NJF1 plants was lost, and that the cells were not short enough to explain the reduced root length of the NJF1 plants. Thus, it was suggested that the shorter roots the NJF1 plants might be due to reduced cell division. Although no distinct morphological abnormalities in the shoot apical meristem were observed, leaf primordium production was delayed in the NJF1 plants. Inhibition of root elongation in this type of hybrid weakness was alleviated under high temperature (34°C) conditions in diverse genetic backgrounds. The threshold temperature required for the recovery of root growth from hybrid weakness seemed to range from 29 to 30°C. Among 85 NJF1 plants that germinated and grew at 34°C for 10 days, and then outdoors, 18 plants eventually flowered. These plants were dwarf and their flowering time was delayed compared with that of their parents. In spite of their abnormal development, they were fertile. Thus, it is suggested that high temperature treatment was also effective to overcome hybrid weakness and to obtain next generation in this cross combination.

To learn the genome constitution and the origin of Japanese current rice (Oryza sativa) cultivars, we studied the number of distinct genotypes and the organization of genome segments with these genotypes in these cultivars. We studied the genotype divergence in 25 Japanese elite and local cultivars by analyzing their DNA sequence divergence at 23 loci and restriction fragment length polymorphisms (RFLP) at 44 loci. These analyses showed that most of the polymorphic loci among the cultivars comprised only two sequence types or alleles, suggesting that their genomes are constituted mostly by a limited number of (possibly as few as two) genotypes. Each of the distinct genome segments appeared to have originated from distinct progenitors of the Japanese current cultivars. The graphical representation of genotypes generated based on the polymorphism data revealed that the genomes of Japanese rice cultivars were mosaics constituted mainly from the two types of progenitor chromosome segments, and that the organization of these genotypes was specific to each cultivar. In contrast to the Japanese cultivars, Chinese and Korean local japonica rice cultivars exhibited greater divergence both in the number of constitutive genotypes and in the pattern of their organization. Our results indicate that Japanese rice cultivars are composed of one subgroup of japonica cultivars that have undergone “population bottlenecks” through the course of their introduction to Japan and during subsequent selection and dissemination.

In direct seeding of rice (Oryza sativa L.), rice seedlings are exposed to low oxidation-reduction (redox) potential soil, and to low temperatures in cold areas. In order to trace the genealogy of tolerance to low redox potential among Japanese rice cultivars, we selected 12 Japanese rice cultivars that had been released over the past century and determined the ability of their seedlings to tolerate immersion in low redox culture solution of Eh =−150 to −180 mv. Seedlings with coleoptiles and radicles of about 1 to 2 cm were incubated in low redox potential culture solution for 2 days at 18 and 30°C, and then in distilled water for 3 days at the same temperatures to examine the recovery abilities. The lengths of shoots and roots were measured after pre-germination, after low redox treatment, and after recovery from damage that occurred in the de-oxidized solution. At 30°C, the tolerance of low redox potentials was positively and significantly correlated with recovery from damage that occurred in the de-oxidized solution. The high tolerance of recent cultivars to these low redox potentials at 30°C appeared to originate from Fujisaka 5. However, at 18°C, the differences among the cultivars in their tolerance of low redox potentials and in their ability to recovery became small.

To perform rapid amplified fragment length polymorphism (AFLP) analysis and efficiently clone AFLP fragments with non-radioactive primers, we modified the current AFLP protocol. When the modified procedure was applied to the rice genome, 30 to 80 AFLP fragments (∼40 to 400 bp in length) could be detected in a single PCR reaction. To assess the suitability of the technique for the analysis of the rice genome, we first applied it to quantify genetic diversity in a sample of 55 predominantly Japanese modern and domestic cultivars. In this analysis, more than 75% of the Japanese rices were differentiated by the combination of six prinrer pairs. Then we used the technique to identify and clone AFLP fragments unique to the Japanese variety Kinuhikari compared to those found in the closely related cultivar Koshihikari. In this cloning, we obtained sequences derived from chromosomal segments of an Indica genome, which had introgressed into the Kinuhikari genome. These two studies demonstrated that the modified AFLP technique is suitable for the genome analysis in rice.