Clubroot disease is caused by an obligate parasite, Plasmodiophora brassicae, and is one of the most serious diseases of Brassica crops worldwide. The pathogen is a eukaryote, and is a member of Plasmodiophorales. Recent phylogenic studies did not reveal its close relationship to fungi, but suggest a classification in Protozoa or Protoctista. The infection is thought to occur through two phases. The pathogen induces massive clubs on root, and prevents host growth. A number of clubroot resistance (CR) Chinese cabbage (Brassica rapa) cultivars have been bred using European turnips as sources of CR genes. Field populations of the pathogen have a wide variation of virulence, which causes the infection of CR Chinese cabbage cultivars. The CR gene of B. rapa is extensively studied with the aid of molecular markers. At least four independent loci are identified and mapped. They are all derived from European turnips. Crr1, and Crr2 are derived from Siloga and mapped in linkage groups R8 and R6, respectively. Crr3 from Milan White, and CRb from Gelria R are both mapped in R3. The molecular markers linked to Crr1, Crr2 and CRb showed homology to the central part of the long arm of chromosome 4 of Arabidopsis thaliana. These findings suggest that these CR genes are derived from the same region of the ancestral genome. In contrast, Crr3 is thought to have different origin, since its linkage markers showed homology to chromosome 3 of A. thaliana. Although another CR gene CRa is reported, the relationship between above mentioned four CR loci and CRa is not yet clarified. Genetic studies on CR genes in B. oleracea revealed a polygenic nature of the trait in this species and also suggest the presence of one CR gene having large effect. However, correspondence among the CR genes reported in these studies is not clarified because of the lack of information on base sequence of the linkage markers in this species. Strategies for breeding of more resistant Chinese cabbage cultivars to overcome the variation on virulence of field populations of the pathogen are presented.

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Lutein is a lipophilic antioxidant vitamin compound that may be beneficial to health in humans. For this reason, increasing the content of lutein in crops and vegetables has become a major breeding objective. The genetic variability of the lutein content in soybean is poorly documented. We analyzed the lutein content of 1,100 accessions of wild and cultivated soybeans by high performance liquid chromatography (HPLC), and identified several wild soybean strains with a high content of lutein. These wild soybean strains, characterized by a deep yellow cotyledon, also displayed peaks of β-carotene and chlorophyll pigments. On the other hand, all the analyzed cultivated soybean accessions with a yellow cotyledon exhibited a small lutein peak and substantially no β-carotene and chlorophyll peaks, whereas those with a green cotyledon displayed chlorophyll peaks. In the present study, it was shown that most of the lutein content in the seeds of the three wild soybean strains with a high content of lutein, 00033715, 00112742 and B01167, originated from the cotyledon, while almost all the β-carotene and chlorophyll pigments were localized in the seed coat, but not in the cotyledon. These results were consistent with the presence of a deep yellow cotyledon, implying that it is possible to increase the lutein content in the cotyledon of soybean by using the high lutein content character of wild soybeans. Moreover, since the high lutein content was stably maintained in the seeds obtained from the wild soybean strains grown in 2004 and 2005, it was suggested that the high lutein content of the wild soybeans was a stable trait.

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In order to obtain detailed information about the genes expressed at the initial stages of rice (Oryza sativa) seed development, we investigated gene expression profiles in the wild type and endospermless (enl) mutant, at 2–3 days after pollination (DAP) and at 4–6 DAP by microarray analysis. Using rice 22k custom oligo DNA microarray, the intensity of gene expression in the wild type seeds at 2–3 DAP (WT-3D) was compared with that in other groups. As a result, 1,620 clones (22k spots on the array glass) exhibited differential hybridization signals. Although homology searches were conducted for these clones, the function of half of them remained unknown. Based on the comparative analysis of the gene expression profile in the wild type and enl mutant, the following results were obtained. The intensity of expression of about 1,000 genes changed from the 2–3 DAP to the 4–6 DAP stages. At the earliest stage (2–3 DAP), genes for defense against diseases or response to stressagents as well as genes for transporter proteins were strongly expressed. At the succeeding stage (4–6 DAP), genes for storage proteins, genes for enzymes of starch synthesis and genes for a transcription factor (zinc finger) were strongly expressed. These results suggest that microarray analysis is useful to elucidate the molecular mechanisms of endosperm formation through the characterization of the genes expressed at each stage of seed development. Despite a change in the expression of gene intensity at both stages of development, gene functions could not be identified. Extensive investigations should be carried out to reach a definitive conclusion.

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A total of 99 recombinant inbred lines (RILs) generated from a cross between Oriental ‘Azumamugi’ and Occidental type ‘Kanto Nakate Gold’ of barley Hordeum vulgare L. cultivars were grown over a period of two years to identify quantitative trait loci (QTLs) controlling agronomic traits such as days to heading, spike characters (length, internode length, awn length and triplet number) and culm internode length. The RILs showed a wide range of variations for all the agronomic traits tested. Composite interval mapping (CIM) enabled to identify a new QTL with a major effect on rachis internode length on chromosome 2HL (qSIL.ak-2H) which was closely linked to the cleistogamy genes and Fusarium head blight resistance QTL. A new QTL for culm length was detected on chromosome 7HL (qCUL.ak-7H), which controlled the elongation, particularly that of the lower culm internodes. The dense spike 1 (dsp1) locus was precisely mapped on a molecular linkage map of chromosome 7HS. The dsp1 locus controlled only the lower culm internode length, while the uzu1 locus controlled the whole culm length. The positions of the QTLs detected in the present study were compared with those of previously reported genes.

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In order to establish a system for marker assisted selection (MAS) to develop new rice cultivars with good eating quality, we conducted QTL analyses for physicochemical properties of rice by using a recombinant inbred (RI) population consisting of 92 lines derived from a cross between closely related japonica cultivars, Moritawase (a cutivar with low eating quality) and Koshihikari (a cultivar with good eating quality). We developed a linkage map with 119 simple sequence repeat (SSR) markers. Physicochemical properties of rice were evaluated two years by measuring protein content, amylose content and textural characteristics. QTL analysis for protein content revealed three QTLs on chromosome 2, 6 and 9. Four QTLs for amylose content were detected on chromosome 3, 7, 9 and 12. One QTL for texture characteristics were identified on chromosome 3. The DNA markers linked to these QTLs will be used for the MAS for good eating quality.

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The dm-type semi-dwarf mutant DMF-1 is controlled by the semi-dominant gene, Short second internode 1 (Ssi1), located in the region within ca. 20 cM on the long arm of chromosome 1. In order to elucidate the function this gene in relation to the lodging resistance and panicle characters in various genetic backgrounds, these agronomic characters were investigated by using DMF-1, Reimei (sd1 allele), six different F₁₀ lines (DML-1 to -6) with homozygous form of Ssi1, and their parents. The experimental results showed that all the dm-type lines except for DML-6 (DMF-1 × Tan-Ginbozu) whose genotype belonged to the homozygous form of both Ssi1 and d35, significantly increased the lodging resistance, compared to their respective parents. On the other hand, although DMF-1 reduced seed production because of the high ratio of appearance of hull-cracked kernels, such a negative character had been improved in DML-1 (Kinmaze × DMF-1), DML-2 (Nipponbare × DMF-1) and DML-4 (Sasanishiki × DMF-1). Principal component analysis based on fifteen panicle characters also showed that these three F₁₀ lines should be assigned to a group of normal cultivars. These results suggested that it is possible to develop a new semi-dwarf variety for practical use, expressing a high level of lodging resistance associated with the Ssi1 gene in various genetic backgrounds.

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Chilling is one of the major environmental stresses for plants including tomato. A newly established F₁ hybrid cooking tomato cultivar, Lycopersicon esculentum Mill cv. Nitaki-Koma, has been recognized as a promising hybrid to satisfy consumers’ demands in Japan. However, there are few reports on agronomical and physiological characteristics, which are necessary to increase its cultivation. Thus, we evaluated the responses of Nitaki-Koma to chilling temperatures as well as its agronomical characteristics in the field and compared the results with those of its parents. The maternal parent cultivar, Coudoulete (referred to as ‘P1’), had some good agronomical characteristics, germinated faster, and was more tolerant to chilling temperatures than the paternal parent cultivar, Piline (referred to as ‘P2’). P2 had, however, some desirable agronomical characteristics but germinated slowly under chilling conditions and was sensitive to chilling. The F₁ hybrid, Nitaki-Koma, showed the better-parent heterosis in some favorable characteristics, such as the number of clusters/plant, the total number of fruits/plant and the total fruit production/plant. It also showed the mid-parent heterosis in other characteristics, such as average fruit weight, plant height and chilling tolerance. Furthermore, seeds of F₁ germinated faster under chilling conditions than the paternal parent (P2). Therefore, F₁ was considered as a chilling tolerant tomato cultivar good for fruit production. These results further suggest the usefulness of tomato breeding for chilling tolerance by producing the F₁ hybrids by crossing chilling-tolerant cultivars.

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So far, only five microsatellite markers have been developed in common buckwheat (Fagopyrum esculentum). The purpose of the present study was to develop a larger number of microsatellite markers in common buckwheat. By sequencing 2785 clones from the libraries, which were enriched for (CT)_n and (GT)_n repeats using magnetic particles, it was shown that 1483 clones contained microsatellites, of which 352 had unique sequences. Primer pairs were designed for 237 of the microsatellite loci, of which 180 primer pairs each amplified PCR products. Fifty-four primer pairs that each amplified a clear PCR product of the expected size were evaluated for their ability to detect variations in common buckwheat populations and to be utilized in seven related Fagopyrum species. Forty-eight (88.9%) out of the 54 microsatellite markers tested were found to be highly variable (the average number of alleles was 12.2 and the average polymorphism information content (PIC) was 0.79) in a population of cultivated buckwheat. This PIC value was comparatively large when compared with the average values for microsatellite markers reported for other crops. A high rate of successful amplification of common buckwheat microsatellite markers was observed in closely related species; in particular all the 54 loci were successfully amplified in the wild ancestor of cultivated common buckwheat. The microsatellite markers developed in the present study should contribute to the promotion of molecular breeding in common buckwheat.

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We analyzed quantitative trait loci (QTLs) in rice (Oryza sativa L.) for leaf bronzing induced by growing plants in a saline paddy field. The mapping population comprised 98 backcross-inbred lines (BILs) derived from crosses between Nipponbare, the recurrent parent, and Kasalath varieties. Fifteen of the BILs showed obvious leaf bronzing, unlike the parent varieties. We identified two QTLs for leaf bronzing: one (qLb-3) was located close to the R1925 marker on the long-arm of chromosome 3, the other (qLb-11) lay in the interval between the C1350 and C477 markers on the short-arm of chromosome 11. The Kasalath allele of qLb-3 and the Nipponbare allele of qLb-11 were found to promote leaf bronzing. Moreover, it was observed that the two QTLs were epistatic, the Kasalath qLb-3 allele causing obvious leaf bronzing in the presence of the Nipponbare qLb-11 allele. To confirm this interaction, we investigated leaf bronzing in three chromosome segment substitution lines: two of these lines, SL2 and SL61, carried an introgressed Kasalath qLb-3 allele in a Nipponbare background while the third line, SL47, carried introgressed Kasalath alleles of both qLb-3 and qLb-11. Leaf bronzing occurred in the SL2 and SL61 plants, but not in the SL47 plants. We further investigated the interaction between the two QTLs using F₂ plants derived from crosses between the SL47 and SL61. In the F₂ plants, segregants harboring both the Kasalath qLb-3 allele and the Nipponbare qLb-11 allele showed leaf bronzing. These results indicate that leaf bronzing required the epistatic interaction of the Kasalath qLb-3 allele on chromosome 3 and the Nipponbare qLb-11 allele on chromosome 11.

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When breeding crops that utilize nitrogen efficiently, it is important to reveal the regulation of nitrate uptake at the molecular level. We focused on the expression of two nitrate uptake-related genes, NRT2 and NAR2, and nitrate uptake during the nitrate induction. Four rice NRT2s (OsNRT2.1~2.4) and two NAR2s (OsNAR2.1~2.2) were identified in the rice genome database. We analyzed the expression of the genes in the roots and shoots after supply of nitrate with or without ammonium pretreatment by the RT-PCR method. The apparent nitrate uptake was also measured. Differential expression of OsNRT2.1 and OsNRT2.2, which have the same ORF with different 5′- and 3′-UTR, was observed in the seedlings pretreated with ammonium. The apparent nitrate uptake synchronously increased after supply of nitrate with the expression pattern of OsNRT2.1 in roots either with or without ammonium pretreatment. The expression of OsNAR2.1, which is thought to be an activator gene for the function of NRT2, was also coordinated with that of OsNRT2.1. On the other hand, OsNRT2.2 was already expressed after ammonium pretreatment, suggesting that OsNRT2.2 would be transcribed to take up nitrate in flooded soils. The present results indicate that OsNRT2.1, OsNRT2.2 and OsNAR2.1 are candidate genes when breeding for efficient use of nitrate in rice.

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Amphidiploid induction by chromosome-doubling is the most efficient method of utilizing a sterile interspecific hybrid for breeding. Previously, although we successfully obtained an interspecific hybrid between carnation (Dianthus caryophyllus L.) and Dianthus japonicus Thunb., the hybrid was sterile. In the present study, therefore, attempts were mode to induce amphidiploids to restore the fertility by artificial chromosome-doubling, i.e., by applying drops of a colchicine solution on to the shoot tips of greenhouse-grown plants or by treating nodal segments excised from in vitro-grown plants by incubation in a colchicine or amiprophos-methyl (APM) solution. Among 156 hybrid plants regenerated after these treatments, nine were revealed to be tetraploids, in addition to one octoploid and 88 mixoploids. Colchicine dropping treatment at a concentration of 2,000 mg·l⁻¹ for one day led to the highest rate (14%) of tetraploid formation, whereas 10% tetraploid formation was obtained by in vitro shaking culture with 5 and 10 mg·l⁻¹ APM for 24 hrs. The amphidiploids exhibited a larger flower size and later flowering time than the original diploid hybrid, and the fertility of both pollen and seed was restored. These amphidiploids are expected to be used for the breeding of carnation cultivars, which are suitable for growing under the Japanese climatic conditions.

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In order to develop an efficient method of shuttle breeding with rapid generation advancement applicable to the breeding programme of early maturity wheat in the central to southwestern regions of Japan, the possibility of eliminating late heading plants was investigated by combining fall-sown cultivation on Ishigaki Island (24°N), Okinawa, with spring-sown cultivation at Memuro (42°N), Hokkaido. The analysis of heading traits using wheat cultivars and F₂ populations indicated that the earliness of heading in Okinawa and Hokkaido was closely related to both Ppd and Vrn genotypes and the Vrn genotype, respectively. The two hybrid populations segregating for the Ppd or Vrn genes were subjected to rapid generation advancement (F₂: Okinawa, F₃: Hokkaido). The analysis of their photoperiod and vernalization responses showed that the plants with photoperiod sensitivity and winter growth habit were eliminated in Okinawa and those with winter growth habit in Hokkaido. The elimination of photoperiod-sensitive plants significantly decreased the proportion of late heading plants in the F₄ population cultivated in the southwestern region of Japan, whereas it was not influenced by the elimination of the winter type plants. It was confirmed that the earliness of heading in the southwestern region of Japan was closely related to the Ppd genotype and was independent of the Vrn genotype, and also indicated that this system of rapid generation advancement was effective in eliminating the late heading plants with photoperiod sensitivity.

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DNA markers are powerful tools for verifying the varietal identity and genetic homogeneity of F₁ hybrid seeds. F₁ varieties are becoming increasingly prevalent in bunching onion (Allium fistulosum L.) production in Japan because of the high uniformity of agronomic traits. However, bunching onion is an allogamous crop and suffers from severe inbreeding depression when selfed. It is considered that not only open-pollinated varieties but also the parental lines of F₁ hybrids should maintain a certain degree of average heterozygosity and hence genetic heterogeneity. In the present study, the genetic homogeneity of eight bunching onion varieties, including six F₁ hybrids, was evaluated using 14 SSR markers. Two or more polymorphic alleles were detected at all of the SSR loci examined in each variety. The number of alleles detected in the eight varieties ranged from 3 to 7 among the 14 SSR loci, and the polymorphism information content from 0.41 to 0.76. All the varieties examined displayed very low degrees of uniformity at all of these polymorphic loci. Based on these results, it may be impossible to determine an appropriate genotypic identity for any of the existing bunching onion varieties. To facilitate and enhance the accuracy of variety identification, we proposed here an “SSR-tagged breeding” scheme in which the plants homozygous at a few SSR loci would be selected out of a foundation seed field. This scheme may enable to achieve efficient variety identification and purity determination of F₁ seeds not only in bunching onion but also in any allogamous crops exhibiting severe inbreeding depression.

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