We previously reported two rice ammonium transporter (OsAMT) genes, OsAMT1;2 and OsAMT1;3, which show root-specific expression at the vegetative stage. To further understand the functional differences between the two genes, we used the promoter::gusA reporter gene to investigate their detailed expression patterns. We show that in transgenic rice plants carrying individual 4-kb promoter::gusA fusion genes, the expression of the two genes was confined to roots during the entire plant life cycle, suggesting the housekeeping roles of the two transporters in rice. Detailed observation of the above transgenic lines further shows that OsAMT1;3 was preferentially expressed in the apices of seminal and lateral roots under nitrogen-deficient conditions, and was repressed when supplied with ammonium. By contrast, OsAMT1;2 expression was induced most intensely in the root elongation zone under nitrogen-sufficient conditions. These observations suggest that the two genes have distinct roles in nitrogen utilization: OsAMT1;3 appears to function as a nitrogen sensor and OsAMT1;2 as an assimilator. In contrast to 4-kb promoters, gusA-reporter expression was not detected in the roots under both nitrogen-deficient and -sufficient conditions when using individual 2-kb promoter sequences proximal to transcription initiation sites for the two genes, indicating that distal 2-kb promoter sequences are essential for the root-specific expression and conditional responsiveness to nitrogen of OsAMT1;2 and OsAMT1;3 genes.

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The Wx^b gene, one of the alleles at the rice waxy locus, responds to environmental temperatures during seed development and, as a result, the amylose content fluctuates and affects rice quality. Seeds of a low-amylose variety ‘Snow Pearl’ could be visibly distinguished depending on whether they matured in warm or cool temperatures; based on this, we isolated a mutant candidate, namely, the enhanced amylose content (ENA) mutant, which showed enhanced amylose content in the endosperm. The amylose contents of ENA mutants that matured at 20°C and 26°C were 6–7% higher than those of Snow Pearl. As the chain length distributions of amylopectin were almost identical between ENA and Snow Pearl, the mutation was not the amylose-extender mutation, but a new mutation that enhanced amylose content in the rice endosperm. Furthermore, ENA and Snow Pearl had a single nucleotide polymorphism on the third exon of the Wx gene and a single base change at the 5′ splice junction of the first intron specific to the Wx^b gene. Genetic analysis revealed that enhanced amylose content is controlled by 2 recessive genes. Thus, we isolated a rice mutant with enhanced amylose content in the endosperm without affecting the amylopectin structure. As global warming or higher maturation temperatures decrease the amylose content in rice endosperms, the mutant obtained in this study would be useful in breeding programs to diversify grain quality and to understand the genetic and molecular mechanisms of amylose synthesis.

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Root aerenchyma formation is considered to be one of the most important characteristics for adapting to flooding. Using an F₂ population of maize B64 × teosinte Zea nicaraguensis, we earlier found that, in Z. nicaraguensis, quantitative trait loci (QTLs) in two regions of chromosome 1 (Qaer1.02-3 and Qaer1.07) are associated with aerenchyma formation under non-flooding conditions. The objective of this study was to determine the utility of these (and any other) aerenchyma QTLs in marker-assisted maize breeding. For this study, an elite maize Mi29 × teosinte Z. nicaraguensis 214 BC₂F₁ individual population was used for advanced backcross-QTL mapping (AB-QTL). An SSR-based map was constructed using 94 markers, covering 852.7 cM for the ten chromosomes. Mapping revealed that a QTL for aerenchyma formation under non-flooding conditions exhibiting a larger effect was located on chromosome 1 (bin 1.06), the position of which was close to the previously identified Qaer1.07. Also, two QTLs with minor effects were mapped to a different position on chromosomes 1 (bin 1.11) and 5 (bin 5.09). With a few additional backcrosses, it will be possible to obtain near-isogenic lines possessing aerenchyma-forming genes. These materials may be useful in breeding flooding-tolerant maize hybrids.

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Oryza diploid species comprise AA, BB, CC, EE, FF, and GG genome groups. Cultivated rice, O. sativa L. of the AA genome, has two subspecies, indica and japonica. These two subspecies are differentiated in many characteristics, such as the number of nucleolar sites, annual-perennial habitat and polymorphism of plastid DNA. Although many researchers accept that japonica must originate from O. rufipogon, the evolutionary route of indica remains unclear. A 69-bp deletion in ORF100 is useful to discriminate between indica and japonica cultivars and between O. nivara and O. rufipogon; furthermore, PSID sequences of O. sativa–O. rufipogon complex contain hyper-variable sites consisting of C and A mononucleotide repeats, which are useful to classify wild accessions and cultivars into the six subtypes of 6C7A and 7C6A (japonica and O. rufipogon), 6C8A and 7C7A (O. nivara), 8C8A and 9C7A (indica). The 69-bp deletion is shared with O. nivara (6C8A, 7C7A) and indica (8C8A, 9C7A) but the 8C8A subtype is found only in indica. To elucidate variations of plastid DNA in the genus Oryza and cytoplasmic origin of indica, we compared two plastid sequences, ORF100 (676 bp) and PSBZ (629 bp), in 40 accessions of 14 Oryza diploid species. Sequence comparison showed that FF and GG genome species were distantly related to AA genome species. Both BB and CC genome species were related with AA genome species but were mutually differentiated. An evolutionary trace of ORF100 and PSBZ sequences indicated that O. barthii might have a consensus sequence among all AA genome species. Both O. longistaminata and O. glumaepatula were highly related with O. barthii; the O. sativa–O. rufipogon complex showed more various polymorphisms than other AA genome species. The results of this study show that 8C8A and 9C7A subtypes of plastids in indica cultivars are differentiated from 6C8A and 7C7A subtypes in O. nivara, despite sharing the same deletion in ORF100. In addition, 6C7A and 7C6A subtypes in japonica cultivars and O. rufipogon share identical sequences in PSBZ but differentiate at two SNP sites in ORF100.

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We previously showed a major quantitative trait locus (QTL) for the eating quality of cooked rice (Oryza sativa L.) on the long arm of chromosome 2 using a population of doubled haploid lines derived from crosses between ‘Koshihikari’ and ‘Akihikari’. Here, we verified the effect of QTL by developing near-isogenic lines (NILs). We successfully developed six NILs on the genetic background of Akihikari in which different segments of chromosome 2 were introduced from Koshihikari. On the basis of the measurement of traits related to eating quality, we observed significant differences in the stickiness of cooked rice and amylose content (AC) of grains among NILs. The stickiness scores and AC values of the NILs were negatively correlated. Comparison of the introduced segments with stickiness revealed that the candidate genomic region of chromosome 2 that affects stickiness lies between 515 kbp (defined by the simple sequence repeat (SSR) markers RM13658 and RM3730) and 773 kbp (defined by the SSR markers KA43 and RM6933). These results clearly showed that a chromosome 2 segment introduced from Koshihikari increased stickiness and decreased AC in the NILs, and hence verified the result obtained from the doubled haploid lines.

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Viral diseases of the sweet potato (Ipomoea batatas L. (Lam)) are highly prevalent and often cause serious damage to the plants, particularly the severe strain of the sweet potato feathery mottle virus (SPFMV-S) that causes ‘obizyo-sohi’ disease in Japan. To confer viral resistance to SPFMV using current biotechnology, a transgenic sweet potato has been developed by introducing SPFMV-S coat protein (CP) genes, which have shown significant resistance to SPFMV-S; however, ecological distribution in the field of SPFMVs is unclear. This study investigated the ecological distribution of SPFMVs in field-infected sweet potato, and virus resistance nearer to the field was evaluated in transgenic sweet potato. SPFMV was not capable of independent infection; rather, infection by SPFMVs was coincidentally complex under natural conditions. Furthermore, infection was by a dominant strain between complex infecting SPFMVs, and was differentiated by the area of infection. Transgenic sweet potato plants expressing the SPFMV-S CP gene were challenged by graft inoculation with field-infected SPFMVs. All of the transgenic sweet potato plants were highly protected in the long term against SPFMV complex infection compared to control plants. These results suggest that transgenic sweet potato can acquire resistance to SPFMVs in the field.

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Microspore culture is an efficient system to produce haploid and double-haploid plants, and is a useful model system in developmental research. Although many studies have been carried out to understand the mechanism of microspore embryogenesis, the details of molecular events occurring during its developmental process are unclear. During embryogenesis, a marked morphological change occurs just after globular stage embryos. To gain insight into the developmental process of androgenesis in rapeseed (Brassica napus), we isolated the genes expressed differentially during globular stage by suppression subtractive hybridization (SSH). Of 254 ESTs isolated and sequenced, 82 were singletons and the remaining 172 were assembled into 28 groups based on homology to the identical gene, resulting in the identification of 110 non-redundant EST groups, which are potentially up-regulated genes in androgenesis. Survey of the potential function of the genes showed that 96.4% of these genes were homologous to known genes with 42.7% of unclassified proteins and 13.6% of metabolism. To confirm the expression profiles of isolated genes during androgenesis and zygotic embryogenesis, six selected genes were examined by quantitative RT-PCR analysis. All genes were highly expressed in globular and other stages of androgenesis. The expressions of these genes were confirmed in zygotic embryogenesis. These results serve as a starting point for understanding the mechanism of androgenesis and zygotic embryogenesis.

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The Zingiber species comprises rhizomatous plants with special significance due to their multiple uses. Previously, we reported the additional use and suitability of primer pairs of rice microsatellite markers as RAPDs for diversity assessment in representative accessions of diverse taxa in the family Zingiberaceae. The current study aimed to investigate the genetic structure of Z. officinale at a genebank, small-scale subsistent farms and commercial markets, and the genetic relationships of three Zingiber species. A difference among accessions at the inter-specific level was observed and amplification of species-specific bands led to high polymorphism. Genetic variability in Z. officinale was exhibited by the three collection sources; however, some of the accessions from each source tended to cluster. AMOVA also displayed significant divergence among the three collection sources and explained most of the variance (87%) within the collection sources. In general, small-scale local farms displayed higher genetic variability in Z. officinale compared to those from the genebank and markets. Comparative assessment displayed high allelic diversity in ginger from Myanmar as compared to other Asian countries. Genetic resources of Zingiber species, particularly available on farms, can be a useful source to capture and utilize diversity for conservation as well as further improvement in gingers.

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To employ molecular marker-aided selection for developing japonica rice (Oryza sativa L.) cultivars with high yielding potential and good eating quality, we carried out QTL analysis for agronomic and grain traits using 231 recombinant inbred lines (RIL) from a cross between two temperate japonica cultivars, Suweon365 and Chucheongbyeo. A linkage map with 221 loci, including 134 simple sequence repeats (SSRs), 66 amplified fragment length polymorphisms (AFLPs), and 21 miniature inverted repeat transposable elements (MITEs)-anchored markers, was constructed covering 2,227 cM of the twelve rice chromosomes, with an average distance of 11.0 cM between markers. The proportion of polymorphic bands between parents averaged 19.5%, 12.9% and 42.3% for SSRs, AFLPs and MITEs, respectively. Construction of maps without AFLP or MITE markers indicated that expansion of this map is due to the incorporation of AFLP markers, and this result is consistent with previous reports. Twenty-seven QTLs for ten traits were identified over two years, and fourteen were consistently detected in two consecutive years. When the locations of each QTL detected in this study were compared with those in previous QTLs studies, eight QTLs for culm length, spikelets per panicle and, thickness, length and shape of grain, had not been reported in previous mapping studies, indicating potentially novel alleles of japonica rice. Interestingly, a cluster of seven QTLs, including heading date and grain morphology traits, was detected on the long arm of chromosome 7. The markers linked to these QTLs would be valuable in a marker-assisted breeding program for japonica cultivars.

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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.

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Peanut (Arachis hypogaea L.), an important source of oil and protein, is native to South America. Most peanuts grown in Japan are large-grain cultivars that are eaten mainly as a snack. The peanut germplasm was introduced from abroad over a century ago and has been used for breeding. Although many peanut varieties have been developed, the relationship between Japanese varieties and peanut phylogeny remains unclear; therefore, this study assessed the diversity and genetic relationships within the peanut germplasms in Japan using allelic variation in a selected set of 13 SSR markers. We analyzed 201 accessions of A. hypogaea and 13 accessions of Arachis wild species: 13 primer pairs amplified 108 polymorphic alleles in A. hypogaea. The detected alleles were 3–15 at each of the 13 markers, with an average of 8.3 per marker. The phenogram based on the SSR genotypes was obtained. A. hypogaea and A. monticola made a separate group from diploid species; they were classified into 150 genotypes. A. hypogaea and A. monticola were divided further into two groups; the first group consisted mainly of spp. fastigiata accessions; the second group consisted mainly of spp. hypogaea accessions and tetraploid wild peanut A. monticola.

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Primula sieboldii (E. Morren), the ancestor of the Japanese garden Primula, is in danger of extinction in the wild. Genetic diversity is a key component for conservation efforts associated with population management. Genetic diversity in visible traits and several molecular makers were evaluated, respectively. Since it is difficult to determine the degree to which genetic and environmental differences contribute to observed phenotypic variation in natural habitats. A common-garden approach is used. The contribution of genetic and environmental differences to variation in petal shape and area of Primula sieboldii were evaluated. Samples from 108 genets gathered from five natural populations in three regions of Japan were analyzed in a common-garden experiment and also analyzed using eight microsatellite markers. From the results of quantitative evaluation based on image analysis, broad genetic variation in petal traits within populations and low level of population differentiation was found. For all petal shapes, Q_st was smaller than F_st, suggesting that wild populations might be under moderate selective pressure for a specific phenotype. For petal area, Q_st was nearly equal to F_st, suggesting that population differentiation has been caused mainly by genetic drift.

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As a result of many decades of potato breeding in Keszthely, Hungary, numerous varieties resistant to the pathogens attacking potato have been developed using wild Solanum species as resistance sources. In order to simplify the time-consuming breeding process, the present research project involved the development of a SCAR marker correlating with the Ry_sto gene, originating from the Solanum stoloniferun species and conferring extreme resistance to the potato Y virus (PVY), using sequence information from a RAPD marker linked to the gene. The usefulness of the marker was tested on 21 varieties resistant or susceptible to PVY and it was found to provide efficient identification of genotypes carrying the Ry_sto gene. Based on its linkage to a new chromosome-specific anchor marker (Cat-in2), identified by means of the intron-targeting method not previously used in potato research, the Ry_sto gene was localized on chromosome XII.

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We analyzed length polymorphisms of 377 simple sequence repeat (SSR) markers in 87 cultivated or wild soybeans, including Japanese elite cultivars and irreplaceable genetic resources. The average number of SSR markers in the 20 linkage groups was 18.9, with a range of 13 to 25. All SSR markers showed polymorphism on agarose gel electrophoresis, which revealed 1380 alleles for all accessions. The number of alleles at each locus ranged from 2 to 10, with an average of 3.7, and the polymorphic information content of each marker ranged from 0.02 to 0.86, with a mean of 0.44. Cluster analysis separated the accessions into three clusters. Most Japanese cultivars were found in cluster 1, which was further divided into three subclusters that corresponded to the geographic origin of the cultivars. Most foreign cultivars and all wild accessions were present in clusters 2 and 3, respectively. Our data on the polymorphism of these SSR loci should facilitate the genetic mapping of agronomically important traits and the choice of appropriate markers for marker-assisted selection in soybean breeding programs in Japan.

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We investigated the effect of total irradiation dose and dose rate on flower color mutation and nuclear DNA content as an index of radiation damage in chrysanthemum. Chrysanthemum morifolium cv. ‘Taihei’ plants grown by in vitro culture were gamma-irradiated with a total dose of 15, 30 and 60 Gy at a rate of 0.5, 1, 2 and 5 Gy/h. Leaf explants cut from the irradiated plants were tissue cultured, and the regeneration rates and frequency of flower color mutation were investigated. Nuclear DNA content was measured by flow cytometric analysis. The regeneration rate decreased with increase in the total dose and dose rate of irradiation. Mutation frequency did not differ significantly among dose rates, indicating that mutation frequency was independent of dose rate and was dependent mainly on total dose. Comparison of the average of the nuclear DNA content with each treatment revealed that it was influenced by both dose rate and total dose, and that the reduction in nuclear DNA content was less at low dose rates, even when total doses were high. It appears that same mutation frequencies were obtained without large reduction in nuclear DNA content by 0.5 Gy/h, when compared with 2 Gy/h. Consequently, we conclude that gamma ray irradiations of high total doses at low dose rates efficiently induce mutations with less radiation damage in chrysanthemum.

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