The genus Oryza that gave rise to rice has a distinctive phylogenetic position within the Poaceae (Graminae). Recently there has been much progress in understanding the phylogenetic relationships of angiosperms. In this review current understanding of angiosperm and monocot phylogeny is discussed in relation to the emergence of Oryza. The lineage leading to Oryza arose early in the evolution of grasses that evolved after 70 mya. Since grasses arose well after the split of Gondwanaland, Oryza did not originate on Gondwanaland and continental drift did not play a role in Oryza biogeography. A new hypothesis is presented to explain the present day distribution of Oryza species. The role of animals, including birds, in the distribution of Oryza spread from a core centre of Oryza diversity in Southeast Asia and New Guinea is the basis for this hypothesis. Humans are also thought to have played a role in distribution of Oryza species particularly in historic times. Finally the continuing evolution of the genus Oryza is discussed. The wild relatives of rice can provide continuously evolving gene resources for rice improvement only if there is a substantial effort to conserve them in situ.

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α-Tocopherol (Toc) is the tocopherol isoform with the highest vitamin E activity. Since soybean seeds contain a low percentage of α-Toc, increasing the α-Toc content to improve vitamin E activity is a breeding objective. Little is known about the genetic variability of the tocopherol content and composition in soybeans. In the present study, we analyzed the tocopherol composition of 1,109 germplasm accessions of cultivated and wild soybean by high performance liquid chromatography (HPLC) and identified three varieties, Dobrogeance, Dobruoza 14 Pancevo and Keszthelyi Aproszemu Sarga with a high α-Toc content. Dobrogeance showed a similar Toc composition to that of Dobruoza 14 Pancevo with the α-Toc content being four to seven times higher than that of standard varieties and almost the same as that of total Toc. Keszthelyi Aproszemu Sarga showed a markedly increased content of α-Toc, but a significantly lower total Toc content.

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The bolting time of varieties used as leafy vegetables or turnips within the Brassica rapa (syn. campestris) species is important, because it affects the yield and quality of the harvested products. In Brassica rapa, the transition from the vegetative to reproductive phase is influenced by environmental factors, such as day-length and low temperature. In order to analyze the genetic basis of bolting time, an AFLP linkage map was constructed using doubled haploid (DH) lines derived from the F₁ hybrid between A9408 and Homei P09. QTL analysis of bolting time was performed based on the evaluation under two field conditions and three artificial conditions differing in day-length and/or temperature. Out of a total of ten QTLs detected, two QTLs showed a low temperature sensitivity. The most effective QTL, BT1, was found to be the main locus controlling the vernalization response and the allele of Homei P09 was highly responsive to low temperature compared with that of the late parent A9408. These results could be useful for marker-assisted selection and isolation of the genes responsible for bolting time in Brassica rapa.

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Maize (Zea mays L.) breeders commonly assign inbred lines to groups in order to maintain the high level of hybrid vigor obtainable from crosses. It had already been reported that the genetic similarity estimated from 60 SSR loci was effective for the assignment of maize inbred lines derived from European hybrids, which contain mixtures of dent and flint germplasm, to dent or flint groups adapted to the cold regions of Japan. We evaluated a simplified assignment method using a smaller number of SSR loci. Two subsets were chosen from the full set of 60 SSR loci distributed uniformly throughout the maize genome. Set 1 composed of 25 loci and Set 2 composed of 14 loci were chosen based on the difference in allele frequency (0.4 for Set 1 and 0.5 for Set 2) between dent and flint groups. SSR loci of Set 1 and Set 2 carried a total of 176 and 99 alleles among 88 inbred lines, respectively. The numbers of alleles for Set 1 and Set 2 were 38% and 21% of the number of alleles for the full set of SSR loci, respectively. The assignments based on mean genetic similarity estimates using Set 1 SSR loci almost all corresponded to those using the full set of SSR loci. Furthermore, the assignments of several inbred lines based on Set 1 SSR loci were ascertained by testcross data. The results indicated that the assignment using Set 1 SSR loci with a similar accuracy to that of the full set of SSR loci is an efficient method in a maize breeding system.

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Appropriate variation of heading date within the optimum range in a particular region is an important objective in rice breeding programs. Quantitative trait loci (QTLs) conferring genetic variation for heading date were identified among the rice varieties in the northern-limits of rice cultivation, Hokkaido (Japan), Portugal, Hungary and Italy. Based on segregation analyses of heading date, these varieties were categorized into two groups: one included the Hokkaido varieties and the other included the European varieties. Two QTLs controlling heading date were detected on chromosomes 6 and 7 using backcross inbred lines (BILs) derived from a cross between Hayamasari (Hokkaido) and Italica Livorno (Italy). The most effective QTL, qDTH-7 on chromosome 7, accounted for 64.5% of total phenotypic variation. Also, clear relationships between days to heading and the marker genotypes that were located in the region of qDTH-7 were observed in the two BIL populations from the crosses of Hoshinoyume (Hokkaido) with Arroz Da Terra (Portugal) or Dunghung Shali (Hungary). Estimation of the photoperiod sensitivity for these varieties indicated that qDTH-7 is involved in photoperiod sensitivity. These results indicate that qDTH-7 was responsible for distinguishing the varieties in the northern-limits of rice cultivation into the two groups.

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The nodulation programme is regulated systemically in leguminous hosts. This mechanism is referred to as the autoregulation of nodulation. Recently, a gene involved in the regulation has been isolated from the model legume Lotus japonicus (HAR1), soybean (NTS1/GmNARK) and pea (PsSYM29) that encode a receptor-like kinase, similar to the well-characterized Arabidopsis CLAVATA1. Here we report the sequencing and expression analysis of the NTS1/GmNARK gene and its homolog GmCLV1A in a soybean supernodulating cultivar, Sakukei 4, which shows a high yielding ability, especially in fields with low nitrogen fertility. Sequencing of the NTS1/GmNARK gene showed that Sakukei 4 has a nonsense mutation (AAA to TAA) near the transmembrane domain as well as En6500 that is the paternal donor parent of Sakukei 4. This mutation is involved in the supernodulating phenotype in both lines En6500 and Sakukei 4. There were no mutations in the GmCLV1A gene, including a single intron between the wild-type, Enrei and both supernodulating lines. Transcripts of both genes were detected in rhizobium-inoculated or non-inoculated cotyledons (7 day-old), primary leaves (14 day-old), leaves (21 day-old) and leaves (14 days after inoculation) by non-quantitative RT-PCR analysis. In the nodules and leaf buds, including the shoot apical meristem where CLAVATA1 is expressed in Arabidopsis, both transcripts were also detected. We observed that NTS1/GmNARK and its homolog GmCLV1A are transcribed constitutively in all the organs we examined, regardless of the presence of symbiosis, stage of growth and normal or mutant status. It is concluded that may be necessary for a leguminous host to initiate the autoregulation system immediately after germination.

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In rice (Oryza sativa L.) plants, the leaf sheaths of the upper leaves accumulate a large amount of starch before heading, and the accumulated starch is converted to sucrose and translocated to the panicles after heading. To analyze the regulation of sink-source transition, we carried out large-scale monitoring of gene expression by microarray analysis using 8987 rice expressed sequence tags (ESTs), and identified 102 developmentally regulated genes in the leaf sheaths of the first leaves below the flag leaves during the heading period. Identified genes included multiple genes related to starch biosynthesis, cell division and expansion, and photosynthesis. All of them showed early stage-preferential expression, probably reflecting the decrease of starch biosynthesis, end of elongation and decrease of photosynthesis in the leaf sheaths during the heading period, respectively. The expression patterns of the genes for starch biosynthesis enzymes and α-tubulin suggest that the leaf sheaths displayed both accumulating- and consuming-sink functions at the very early stage of the heading period. Northern blot analysis of the genes for starch degradation and sucrose biosynthesis enzymes (α-amylase3D and sucrose phosphate synthase) revealed that the induction of the genes did not occur when the starch amount began to decrease, suggesting that the mechanisms of starch degradation and sucrose re-synthesis in the leaf sheaths during the heading period were different from those in germinating seeds. Furthermore, we identified 18 developmentally regulated genes whose functions in the leaf sheaths are unknown. They included seven genes those were preferentially expressed in the bottom part of the leaf sheaths, implying that they were involved in functions related to the starch metabolism. The results obtained showed the effectiveness of the microarray technique to analyze complex and uncharacterized phenomena.

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Defining seawater tolerance as a plant’s ability to survive under saline culture (seawater: average 500 mM NaCl), we attempted to identify gene sources as donors for this trait. Seawater-tolerance was observed in 42 plant species and spread over many families. In the family Gramineae, the presence of seawater tolerance was not random: a large cluster of seawater tolerance was found in the sub-family Eragrostoideae. In contrast, most grass crops, belonging to different sub-families, were susceptible in seawater culture. Evaluation of 369 native rice varieties and 105 wild rice collections showed that the critical lethality level of salinity in Oryza sativa L. was at around 50 mM (10% dilution of seawater). Two genes, PvUGE1 and PvMET1, were identified in one of the most tolerant Gramineae, seashore paspalum (Paspalum vaginatum). The PvUGE1 gene encodes putative UDP-galactose epimerase and the PvMET1 gene encodes metallothioneine. These had not yet been examined in the category of plants with salt tolerance. Although rice was very sensitive to salinity, both genes enhanced the salt tolerance of rice at the critical salinity level. PvUGE1 was significantly more effective than PvMET1, that is, PvUGE1 is one of the major genes associated with seawater tolerance, while PvMET1 is one of the supporter genes.

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Genetic structure of Asian wheat (Tritium aestivum L.) was investigated by the analysis of two isozymes, using 648 wheat landraces. Gene diversity over all populations was 0.254, and the trend of an eastward decline was observed. By cluster analysis and principal co-ordinate analysis based on genetic distance among populations, thirty-three populations were classified into six clusters, and it was indicated that Asian wheat could be divided into at least three lineages. The first lineage of wheat consisted of populations from Turkey to Sichuan (China), suggesting the spread of wheat to southwest China through the ancient Myanmar route. Wheat populations introduced to China through this route were mostly of the red-grain type, and it was considered that wheat adapted to humid and rather hot condition in southern slope of Himalaya had been selected and introduced to China. The second lineage comprised of populations from the areas along the so-called “Silk Road”. Wheat is commonly cultivated under dry and cold condition in these areas, and was characterized by the frequent distribution of white-grain type. The third lineage contained populations from the coastal area of China and Korea, and genetic relationship with the second lineage was suggested. The result of the present study indicated that Asian wheat is genetically and geographically differentiated, and that different types of genetic resources could be found depending on the growing condition and the lineage of wheat populations.

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Dot-blot analysis of rice cultivars was performed using 14 cultivar-specific sequences identified previously by AFLP and PCR-RF-SSCP analyses as probes. DNA samples prepared from leaves by a simple method without the use of organic solvent were found to be usable for dot-blot analysis. Although more than a single grain of rice was needed as material for direct dot-blotting of genomic DNA, PCR amplification of DNA enabled detection of cultivar-specific signals in a DNA sample from a single grain by dot-blot analysis. All 31 rice cultivars examined could be distinguished from each other by analysis using ten cultivar-specific probes. Dot-blot analysis was also applied to genotyping of recombinant inbred lines (RILs), the results of which corresponded to those by electrophoretic analysis of PCR products amplified with specific primer sequences. The probe sequences deleted in ‘Nipponbare’ were mapped on the rice chromosome by BlastN search using all the Kasalath BAC-end sequences and by analysis of RILs and chromosome segment substitution lines (CSSLs). Dot-blot analysis using cultivar-specific sequences was found to be applicable for the identification of cultivars and genotyping of plants in rice.

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Bunketsu-waito, one of the tillering dwarfs, was reported to be controlled by triplicate genes, d3, d4 and d5. Although a segregation ratio fitting to 15: 1 for bunketsu-waito was observed in the F₂s of the crosses between H-52 (bunketsu-waito) and genetic marker lines No.201 or H-61, the backcross of (H-52 × H-61) F₁ × H-52 showed a segregation ratio of 1: 1 for normal vs. bunketsu-waito and the reciprocal backcross showed a decrease of the segregation ratio for bunketsu-waito from 1: 1. These results demonstrated that bunketsu-waito was controlled by a single recessive gene and suggested that the abnormal segregation of bunketsu-waito in the F₂ of H-52 × H-61 was caused by a gametophyte gene linked to a gene for bunketsu-waito. Furthermore, the decrease of the segregation ratio of bunketsu-waito in the F₂ of H-125 × Kasalath was also observed and was considered to be due to a gametophyte gene, based on the results of reciprocal backcrossing between H-125 and the F₁ of H-125 × Kasalath. Thus, it was assumed that bunketsu-waito was controlled by a single recessive gene, and that the bunketsu-waito gene was mapped between RM 587 and RM 4608 on chromosome 6, based on linkage analysis using SSR markers.

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Ninety-nine accessions of melon (Cucumis melo L.) mainly from East and South Asia were analyzed based on the polymorphism of 210 amplified fragment length polymorphism (AFLP) bands to reveal the genetic structure and phylogenetic relationship in Asian melon. A cluster analysis based on their genetic similarity revealed three major clusters, i.e., a vars. makuwa and conomon group, a small-seed type group and a group of Japanese F₁ cultivars and large-seed type accessions. Most of the East Asian melon accessions classified into the first group were of the small-seed type with a seed length shorter than 9.0 mm. The varieties of C. melo were roughly divided into two groups by a principal co-ordinate analysis based on AFLP data, that is, the group of vars. makuwa and conomon and small-seed type melon and the group of var. reticulatus and large-seed type melon. Indian melon accessions were rich in genetic variation. Melon accessions closely related to vars. makuwa and conomon were found in east India, and they were considered as possible candidates of the prototype of vars. makuwa and conomon.

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In order to gain a better understanding of the nature of nuclear dbf-related (Ndr) protein kinase homologs in monocots, three cDNA clones were isolated from common wheat (Triticum aestivum) by data mining of expressed sequence tag (EST) databases, and their primary structure was determined by reverse transcription-polymerase chain reaction (RT-PCR), 5′-rapid amplification of cDNA ends (RACE), and nucleotide sequencing. Deduced amino acid sequences of the cDNA clones contained all 12 highly conserved subdomains of the eukaryotic Ser/Thr protein kinase, including the ATP-binding site (in subdomain I-II) and the Ser/Thr protein kinase active-site (in subdomain VI). The sequences also contained an insert of 56 amino acids between subdomains VII and VIII, and three conserved Ser/Thr residues, being characteristic of the Ndr family of eukaryotic protein kinases. A sequence comparison among cDNAs from wheat and those from ancestral diploid species (T. boeoticum, Aegilops speltoides and Ae. squarrosa) revealed that at least three homeologous genes were expressed in hexaploid wheat. The results confirmed the usefulness of current EST databases including KOMUGI for gene cloning of wheat, since the use of specific primers designed with information about EST sequences has considerably facilitated the cloning of rare cDNAs such as Ndr.

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The objective of the present study was to develop a functional crop that could contribute to the maintenance and improvement of human health by the introduction of the human lactoferrin (hLF) gene. Lactoferrin is an 80-kDa iron-binding glycoprotein that has been considered to play many biological roles, including the regulation of iron absorption, protection against microbial and virus infection, stimulation of the immune system and cellular growth promotion. We introduced two different constructs containing either the native signal peptide from human lactoferrin (pIG211) or the signal peptide from rice glutelin (pIG200) fused to mature human lactoferrin into Javanica rice cv. Rojolele by using the Agrobacterium-mediated transformation system. The expression of the hLF gene under the control of the maize ubiquitin-1 promoter was detected in all the tissues of the transgenic plants. We found that the transgenic rice plants IG200R produced a considerable amount of recombinant hLF (rhLF) in seeds, accounting for approximately 15% of the total soluble protein (TSP). RhLF was purified from mature seeds by cation-exchange chromatography. Amino acid sequencing confirmed that the N-terminal sequences of rhLF for both constructs were identical with those of native LF from human milk. Rice rhLF showed a slightly smaller molecular weight than the native hLF. Immunofluorescence microscopy revealed that rhLF was located in the intracellular and intercellular regions of endosperm cells. Protein extracts from transgenic rice seeds exhibited an antibacterial activity against Bacillus subtilis ATCC6633. The use of signal peptides and a constitutive ubiquitin-1 promoter for successful production of transgenic Javanica rice expressing a high level of rhLF was examined.

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We have generated estimates of the genomic distribution of newly developed molecular markers, which are P450-based analogues (PBAs), in diploid potato (Solanum tuberosum, 2n = 2x = 24) based on functional genomics. A total of 401 markers, including 111 SSR, 33 RFLP, 87 RFLP-STS, 45 CAPS, 94 RAPD, 15 PBA, 9 AFLP, 3 RGL and 4 ISSR markers, were employed in a parental polymorphism survey. A total of 127 out of 401 markers (172 loci) displayed polymorphisms between parents and were confirmed to show segregation in partial progenies. These selected 127 markers were then tested for their possible use for a whole mapping population. The subsequent results of genetic mapping of the PBAs revealed that they were distributed on at least 8 chromosomes, suggesting that they have a significant potential not only as tools for assessing genetic diversity but also as effective markers to construct more detailed genetic maps of potato, in conjunction with existing identified genetic loci. To integrate the information from existing maps into our mapping study, we performed a comparative analysis between two representative maps (RFLP and SSR-based) with our PBA map. Based on the subsequent results, we predict that our map will be useful as a bridge between the existing genetic maps of potato and will enable to integrate information about different markers.

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A near isogenic line T65-LH2 bred from IR8 by five times of successive backcrossing with Taichung 65 (T65) as recurrent parent was found to harbor a lateness gene ef4(t). In the present study, we investigated the allelic relationship between ef4(t) and two earliness genes, Efx and Ef1, followed by linkage analysis to reveal the chromosomal location of ef4(t). T65-LH2 was crossed with two testers, T65-ER21 and T65-ER1 harboring the earliness genes, Efx and Ef1, respectively. F₁ plants of the two crosses were raised to obtain F₂ seeds. Moreover, the F₁ plants were backcrossed with T65-LH2 as to obtain the B₁F₁ seeds. Heading time in F₂ and B₁F₁ plants as well as parental lines were examined. F₂ and B₁F ₁ plants of the crosses between T65-LH2 and T65-ER21 showed monogenic segregation while F₂ and B₁F ₁ plants obtained from the cross between T65-LH2 and T65-ER1 exhibited digenic segregation. Thus, ef4(t) is allelic to Efx on chromosome 3 and independent of Ef1. Subsequently, linkage analysis of ef4(t) was carried out using three marker genes on chromosome 3, Hg, dl and bc1. T65-LH2 was crossed with the isogenic lines of T65 carrying those marker genes. Heading time and morphological characters in F₂ plants and parental lines were observed. Results showed that ef4(t) was linked to Hg and dl with recombination values estimated at 33.2% and 16.8%, respectively, but segregated independently of bc1.

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