藤本 龍 | ![]() |
フジモト リョウ | |
大学院農学研究科 資源生命科学専攻 | |
准教授 | |
農学関係 |
2018年01月 神戸大学, 平成29年度優秀若手研究賞, 雑種強勢研究
日本国その他の賞
2017年10月 神戸大学農学部同窓会六篠会, 六篠論文賞, PNAS誌に掲載された論文
その他の賞
2017年03月 日本育種学会, 平成28年度日本育種学会奨励賞, アブラナ科植物におけるゲノム多様性および雑種強勢に関する研究
日本国国内学会・会議・シンポジウム等の賞
Heterosis is an important phenomenon for high-yield crop breeding and is utilized for breeding F1 varieties in horticultural crops. However, its molecular mechanism has not been elucidated, and compared to cereals, heterosis is less explored at the molecular level in horticultural crops. In this review, we compiled the new genetic and epigenetic studies on heterosis in horticultural crops. Because of the difficulty of predicting the level of heterosis from the parental genetic distance, molecular approaches are being used to study its molecular basis in horticultural crops. Transcriptome analyses in vegetables have identified photosynthesis-related genes as important in heterosis. Analysis of noncoding RNAs has suggested their involvement in regulating the heterosis of vegetative and fruit tissues. Quantitative trait locus (QTL) analysis has revealed the association of heterozygosity of a specific locus or multiple loci with heterosis of vegetative and fruit tissues. A higher level of DNA methylation was noted in the heterotic F1 of Brassica rapa leafy vegetables, while the roles of other epigenetic modifications such as histone marks have not been explored.
MDPI AG, 2023年03月10日, Horticulturae, 9 (3), 366 - 366研究論文(学術雑誌)
Cabbage (Brassica oleracea var. capitata) Fusarium wilt (CFW) is a disease that poses a critical threat to global cabbage production. Screening for resistant resources in order to support the breeding of resistant cultivars is the most reliable approach to control this disease. CFW is caused by Fusarium oxysporum f. sp. conglutinans (Foc), which consists of two physiological races (race 1 and 2). While many studies have focused on resistance screening, gene mining, and inheritance-based research associated with resistance to Foc race 1, there have been few studies specifically analyzing resistance to Foc race 2, which is a potential threat that can overcome type A resistance. Here, 166 cabbage resources collected from around the world were evaluated for the resistance to both Foc races, with 46.99% and 38.55% of these cabbage lines being resistant to Foc race 1 and race 2, respectively, whereas 33.74% and 48.80% were susceptible to these two respective races. Of these 166 analyzed cabbage lines, 114 (68.67%) were found to be more susceptible to race 2 than to race 1, and 28 of them were resistant to race 1 while susceptible to race 2, underscoring the highly aggressive nature of Foc race 2. To analyze the inheritance of Foc race 2 resistance, segregated populations derived from the resistant parental line 'Badger Inbred 16' and the susceptible one '01-20' were analyzed with a major gene plus polygene mixed genetic model. The results of this analysis revealed Foc race 2-specific resistance to be under the control of two pairs of additive-dominant-epistatic major genes plus multiple additive-dominant-epistatic genes (model E). The heritability of these major genes in the BC1P1, BC1P2, and F2 generations were 32.14%, 72.80%, and 70.64%, respectively. In summary, these results may aid in future gene mining and breeding of novel CFW-resistant cabbage cultivars.
2022年09月04日, Genes, 13 (9), 英語, 国際誌[査読有り]
研究論文(学術雑誌)
Tuberous stem of kohlrabi is an important agronomic trait, however, the molecular basis of tuberization is poorly understood. To elucidate the tuberization mechanism, we conducted a comparative transcriptomic analysis between kohlrabi and broccoli at 10 and 20 days after germination (DAG) as tuberous stem initiated between these time points. A total of 5580 and 2866 differentially expressed transcripts (DETs) were identified between genotypes (kohlrabi vs broccoli) and growth stages (10 DAG vs 20 DAG), respectively, and most of the DETs were down-regulated in kohlrabi. Gene ontology (GO) and KEGG pathway enrichment analyses showed that the DETs between genotypes are involved in cell wall loosening and expansion, cell cycle and division, carbohydrate metabolism, hormone transport, hormone signal transduction, and in several transcription factors. The DETs identified in those categories may directly/indirectly relate to the initiation and development of tuberous stem in kohlrabi. In addition, the expression pattern of the hormone synthesis related DETs coincided with the endogenous IAA, IAAsp, GA, ABA and tZ profiles in kohlrabi and broccoli seedlings, that were revealed in our previous phytohormone analysis. This is the first report on comparative transcriptome analysis for tuberous stem formation in kohlrabi at early growth periods. The resulting data could provide significant insights into the molecular mechanism underlying tuberous stem development in kohlrabi as well as in other tuberous organ forming crops. This article is protected by copyright. All rights reserved.
2022年08月26日, Physiologia plantarum, e13770, 英語, 国際誌研究論文(学術雑誌)
DNA methylation is an epigenetic mark associated with several mechanisms in plants including immunity mechanisms. However, little is known about the regulatory role of DNA methylation in the resistance response of Brassica species against fungal diseases. White rust, caused by the fungus Albugo candida, is one of the most widespread and destructive diseases of all the cultivated Brassica species, particularly Brassica rapa L. and Brassica juncea (L.) Czern and Coss. Here, we investigate whole-genome DNA methylation modifications of B. rapa subsp. perviridis in response to white rust. As a result, 233 and 275 differentially methylated regions (DMRs) in the susceptible cultivar “Misugi” and the resistant cultivar “Nanane” were identified, respectively. In both cultivars, more than half of the DMRs were associated with genes (DMR-genes). Gene expression analysis showed that 13 of these genes were also differentially expressed between control and infected samples. Gene ontology enrichment analysis of DMR genes revealed their involvement in various biological processes including defense mechanisms. DMRs were unevenly distributed around genes in susceptible and resistant cultivars. In “Misugi,” DMRs tended to be located within genes, while in “Nanane,” DMRs tended to be located up and downstream of the genes. However, CG DMRs were predominantly located within genes in both cultivars. Transposable elements also showed association with all three sequence contexts of DMRs but predominantly with CHG and CHH DMRs in both cultivars. Our findings indicate the occurrence of DNA methylation modifications in B. rapa in response to white rust infection and suggest a potential regulatory role of DNA methylation modification in defense mechanisms which could be exploited to improve disease resistance.
Frontiers Media SA, 2022年06月23日, Frontiers in Plant Science, 13研究論文(学術雑誌)
If food and feed production are to keep up with world demand in the face of climate change, continued progress in understanding and utilizing both genetic and epigenetic sources of crop variation is necessary. Progress in plant breeding has traditionally been thought to be due to selection for spontaneous DNA sequence mutations that impart desirable phenotypes. These spontaneous mutations can expand phenotypic diversity, from which breeders can select agronomically useful traits. However, it has become clear that phenotypic diversity can be generated even when the genome sequence is unaltered. Epigenetic gene regulation is a mechanism by which genome expression is regulated without altering the DNA sequence. With the development of high throughput DNA sequencers, it has become possible to analyze the epigenetic state of the whole genome, which is termed the epigenome. These techniques enable us to identify spontaneous epigenetic mutations (epimutations) with high throughput and identify the epimutations that lead to increased phenotypic diversity. These epimutations can create new phenotypes and the causative epimutations can be inherited over generations. There is evidence of selected agronomic traits being conditioned by heritable epimutations, and breeders may have historically selected for epiallele-conditioned agronomic traits. These results imply that not only DNA sequence diversity, but the diversity of epigenetic states can contribute to increased phenotypic diversity. However, since the modes of induction and transmission of epialleles and their stability differ from that of genetic alleles, the importance of inheritance as classically defined also differs. For example, there may be a difference between the types of epigenetic inheritance important to crop breeding and crop production. The former may depend more on longer-term inheritance whereas the latter may simply take advantage of shorter-term phenomena. With the advances in our understanding of epigenetics, epigenetics may bring new perspectives for crop improvement, such as the use of epigenetic variation or epigenome editing in breeding. In this review, we will introduce the role of epigenetic variation in plant breeding, largely focusing on DNA methylation, and conclude by asking to what extent new knowledge of epigenetics in crop breeding has led to documented cases of its successful use.
2022年, Frontiers in plant science, 13, 958350 - 958350, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
Long noncoding RNAs (lncRNAs) play important roles in abiotic and biotic stress responses; however, studies on the mechanism of regulation of lncRNA expression are limited in plants. The present study examined the relationship between lncRNA expression level and two active histone modifications (H3K4me3 and H3K36me3) in Brassica rapa. Both histone marks were enriched in the chromatin regions encoding lncRNAs, especially around the transcription start site. The transcription level of long intergenic noncoding RNAs was positively associated with the level of H3K4me3 and H3K36me3, while this association was not observed in natural antisense RNAs (NATs) and intronic noncoding RNAs. As coordinate expression of mRNAs and paired NATs under biotic stress treatment has been identified, the transcriptional relationship between mRNAs and their paired NATs following Fusarium oxysporum f. sp. conglutinans (Foc) inoculation was examined. A positive association of expression levels between mRNAs and their paired NATs following Foc inoculation was observed. This association held for several defense-response-related genes and their NAT pairs. These results suggest that coordinate expression of mRNAs and paired NATs plays a role in the defense response against Foc.
MDPI AG, 2021年12月23日, Horticulturae, 8 (1), 17 - 17, 英語[査読有り]
研究論文(学術雑誌)
Covalent modifications of histone proteins act as epigenetic regulators of gene expression. We report the distribution of two active histone marks (H3K4me3 and H3K36me3) in 14-day leaves in two lines of
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研究論文(学術雑誌)
In vegetables of Brassica rapa L., Fusarium oxysporum f. sp. rapae (For) or F. oxysporum f. sp. conglutinans (Foc) cause Fusarium yellows. A resistance gene against Foc (FocBr1) has been identified, and deletion of this gene results in susceptibility (focbr1-1). In contrast, a resistance gene against For has not been identified. Inoculation tests showed that lines resistant to Foc were also resistant to For, and lines susceptible to Foc were susceptible to For. However, prediction of disease resistance by a dominant DNA marker on FocBr1 (Bra012688m) was not associated with disease resistance of For in some komatsuna lines using an inoculation test. QTL-seq using four F2 populations derived from For susceptible and resistant lines showed one causative locus on chromosome A03, which covers FocBr1. Comparison of the amino acid sequence of FocBr1 between susceptible and resistant alleles (FocBr1 and FocBo1) showed that six amino acid differences were specific to susceptible lines. The presence and absence of FocBr1 is consistent with For resistance in F2 populations. These results indicate that FocBr1 is essential for For resistance, and changed amino acid sequences result in susceptibility to For. This susceptible allele is termed focbr1-2, and a new DNA marker (focbr1-2m) for detection of the focbr1-2 allele was developed.
MDPI AG, 2021年05月27日, Plants, 10 (6), 1082 - 1082, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
KEY MESSAGE: Fusarium yellows resistant and susceptible lines in Brassica rapa showed different salicylic acid responses; the resistant line showed a similar response to previous reports, but the susceptible line differed. Fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans (Foc) is an important disease. Previous studies showed that genes related to salicylic acid (SA) response were more highly induced following Foc infection in Brassica rapa Fusarium yellows resistant lines than susceptible lines. However, SA-induced genes have not been identified at the whole genome level and it was unclear whether they were up-regulated by Foc inoculation. Transcriptome analysis with and without SA treatment in the B. rapa Fusarium yellows susceptible line 'Misugi' and the resistant line 'Nanane' was performed to obtain insights into the relationship between SA sensitivity/response and Fusarium yellows resistance. 'Nanane's up-regulated genes were related to SA response and down-regulated genes were related to jasmonic acid (JA) or ethylene (ET) response, but differentially expressed genes in 'Misugi' were not. This result suggests that Fusarium yellows resistant and susceptible lines have a different SA response and that an antagonistic transcription between SA and JA/ET responses was found only in a Fusarium yellows resistant line. SA-responsive genes were induced by Foc inoculation in Fusarium yellows resistant (RJKB-T23) and susceptible lines (RJKB-T24). By contrast, 39 SA-induced genes specific to RJKB-T23 might function in the defense response to Foc. In this study, SA-induced genes were identified at the whole genome level, and the possibility, the defense response to Foc observed in a resistant line could be mediated by SA-induced genes, is suggested. These results will be useful for future research concerning the SA importance in Foc or other diseases resistance in B. rapa.
2021年04月, Plant cell reports, 40 (4), 605 - 619, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
Long noncoding RNAs (lncRNAs) are RNA fragments that generally do not code for a protein but are involved in epigenetic gene regulation. In this study, lncRNAs of Brassica rapa were classified into long intergenic noncoding RNAs, natural antisense RNAs, and intronic noncoding RNAs and their expression analyzed in relation to genome-wide 24-nt small interfering RNAs (siRNAs), DNA methylation, and histone H3 lysine 27 trimethylation marks (H3K27me3). More than 65% of the lncRNAs analyzed consisted of one exon, and more than 55% overlapped with inverted repeat regions (IRRs). Overlap of lncRNAs with IRRs or genomic regions encoding for 24-nt siRNAs resulted in increased DNA methylation levels when both were present. LncRNA did not overlap greatly with H3K27me3 marks, but the expression level of intronic noncoding RNAs that did coincide with H3K27me3 marks was higher than without H3K27me3 marks. The Brassica genus comprises important vegetables and oil seed crops grown across the world. B. rapa is a diploid (AA genome) thought to be one of the ancestral species of both B. juncea (AABB genome) and B. napus (AACC) through genome merging (allotetrapolyploidization). Complex genome restructuring and epigenetic alterations are thought to be involved in these allotetrapolyploidization events. Comparison of lncRNAs between B. rapa and B. nigra, B. oleracea, B. juncea, and B. napus showed the highest conservation with B. oleracea. This study presents a comprehensive analysis of the epigenome structure of B. rapa at multi-epigenetic levels (siRNAs, DNA methylation, H3K27me3, and lncRNAs) and identified a suite of candidate lncRNAs that may be epigenetically regulated in the Brassica genus.
2021年, PloS one, 16 (3), e0242530, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
The genus Brassica contains important vegetable crops, which serve as a source of oil seed, condiments, and forages. However, their production is hampered by various diseases such as clubroot and Fusarium wilt, especially in Brassica vegetables. Soil-borne diseases are difficult to manage by traditional methods. Host resistance is an important tool for minimizing disease and many types of resistance (R) genes have been identified. More than 20 major clubroot (CR) disease-related loci have been identified in Brassica vegetables and several CR-resistant genes have been isolated by map-based cloning. Fusarium wilt resistant genes in Brassica vegetables have also been isolated. These isolated R genes encode the toll-interleukin-1 receptor/nucleotide-binding site/leucine-rice-repeat (TIR-NBS-LRR) protein. DNA markers that are linked with disease resistance allele have been successfully applied to improve disease resistance through marker-assisted selection (MAS). In this review, we focused on the recent status of identifying clubroot and Fusarium wilt R genes and the feasibility of using MAS for developing disease resistance cultivars in Brassica vegetables.
2020年06月09日, Plants (Basel, Switzerland), 9 (6), 英語, 国際誌[査読有り]
研究論文(学術雑誌)
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研究論文(学術雑誌)
The genus Brassica includes oil crops, vegetables, condiments, fodder crops, and ornamental plants. Brassica species underwent a whole genome triplication event after speciation between ancestral species of Brassica and closely related genera including Arabidopsis thaliana. Diploid species such as Brassica rapa and Brassica oleracea have three copies of genes orthologous to each A. thaliana gene, although deletion in one or two of the three homologs has occurred in some genes. The floral transition is one of the crucial events in a plant's life history, and time of flowering is an important agricultural trait. There is a variation in flowering time within species of the genus Brassica, and this variation is largely dependent on a difference in vernalization requirements. In Brassica, like in A. thaliana, the key gene of vernalization is FLOWERING LOCUS C (FLC). In Brassica species, the vernalization response including the repression of FLC expression by cold treatment and the enrichment of the repressive histone modification tri-methylated histone H3 lysine 27 (H3K27me3) at the FLC locus is similar to A. thaliana. B. rapa and B. oleracea each have four paralogs of FLC, and the allotetraploid species, Brassica napus, has nine paralogs. The increased number of paralogs makes the role of FLC in vernalization more complicated; in a single plant, paralogs vary in the expression level of FLC before and after vernalization. There is also variation in FLC expression levels between accessions. In this review, we focus on the regulatory circuits of the vernalization response of FLC expression in the genus Brassica.
2020年, Frontiers in plant science, 11, 619417 - 619417, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
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研究論文(学術雑誌)
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研究論文(学術雑誌)
Brassica rapa L. is an important vegetable and oilseed crop. We investigated the distribution of the histone mark tri-methylation of H3K27 (H3K27me3) in B. rapa and its role in the control of gene expression at two stages of development (2-day cotyledons and 14-day leaves) and among paralogs in the triplicated genome. H3K27me3 has a similar distribution in two inbred lines, while there was variation of H3K27me3 sites between tissues. Sites that are specific to 2-day cotyledons have increased transcriptional activity, and low levels of H3K27me3 in the gene body region. In 14-day leaves, levels of H3K27me3 were associated with decreased gene expression. In the triplicated genome, H3K27me3 is associated with paralogs that have tissue-specific expression. Even though B. rapa and Arabidopsis thaliana are not closely related within the Brassicaceae, there is conservation of H3K27me3-marked sites in the two species. Both B. rapa and A. thaliana require vernalization for floral initiation with FLC being the major controlling locus. In all four BrFLC paralogs, low-temperature treatment increases H3K27me3 at the proximal nucleation site reducing BrFLC expression. Following return to normal temperature growth conditions, H3K27me3 spreads along all four BrFLC paralogs providing stable repression of the gene.
2019年10月01日, DNA research : an international journal for rapid publication of reports on genes and genomes, 26 (5), 433 - 443, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
There is a wide variation of flowering time among lines of Brassica rapa L. Most B. rapa leafy (Chinese cabbage etc.) or root (turnip) vegetables require prolonged cold exposure for flowering, known as vernalization. Premature bolting caused by low temperature leads to a reduction in the yield/quality of these B. rapa vegetables. Therefore, high bolting resistance is an important breeding trait, and understanding the molecular mechanism of vernalization is necessary to achieve this goal. In this study, we demonstrated that BrFRIb functions as an activator of BrFLC in B. rapa. We showed a positive correlation between the steady state expression levels of the sum of the BrFLC paralogs and the days to flowering after four weeks of cold treatment, suggesting that this is an indicator of the vernalization requirement. We indicate that BrFLCs are repressed by the accumulation of H3K27me3 and that the spreading of H3K27me3 promotes stable FLC repression. However, there was no clear relationship between the level of H3K27me3 in the BrFLC and the vernalization requirement. We also showed that if there was a high vernalization requirement, the rate of repression of BrFLC1 expression following prolonged cold treatments was lower.
2019年09月25日, Scientific reports, 9 (1), 13843 - 13843, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
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研究論文(学術雑誌)
Brassica rapa L. is an important agricultural crop that requires a period of prolonged cold for flowering. This process is known as vernalization. Studies have shown that long noncoding RNAs (lncRNAs) play important roles in abiotic stress responses and several cold-responsive noncoding RNAs have been suggested to be involved in vernalization. We examined the transcriptome of the Chinese cabbage inbred line (B. rapa L. var. pekinensis) RJKB-T24, and identified 1,444 long intergenic noncoding RNAs (lincRNAs), 551 natural antisense transcripts (NATs), and 93 intronic noncoding RNAs (incRNAs); 549 of the 2,088 lncRNAs significantly altered their expression in response to four weeks of cold treatment. Most differentially expressed lncRNAs did not lead to a change of expression levels in mRNAs covering or near lncRNAs, suggesting that the transcriptional responses to four weeks of cold treatment in lncRNA and mRNA are independent. However, some differentially expressed mRNAs had NATs with expression altered in the same direction. These genes were categorized as having an abiotic stress response, suggesting that the paired-expression may play a role in the transcriptional response to vernalization or cold treatment. We also identified short-term cold treatment induced NATs in BrFLC and BrMAF genes, which are involved in vernalization. The lncRNAs we identified differed from those reported in Arabidopsis thaliana, suggesting the role of lncRNAs in vernalization differ between these two species.
2019年06月26日, Scientific reports, 9 (1), 9302 - 9302, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
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研究論文(学術雑誌)
Epigenetic gene regulation is crucial to plant life and can involve dynamic interactions between various histone modifications, DNA methylation, and small RNAs. Detailed analysis of epigenome information is anticipated to reveal how the DNA sequence of the genome is translated into the plant's phenotype. The aim of this study was to map the DNA methylation state at the whole genome level and to clarify the relationship between DNA methylation and transcription, small RNA expression, and histone H3 lysine 9 di-methylation (H3K9me2) in Brassica rapa. We performed whole genome bisulfite sequencing, small RNA sequencing, and chromatin immunoprecipitation sequencing using H3K9me2 antibody in a Chinese cabbage inbred line, RJKB-T24, and examined the impact of epigenetic states on transcription. Cytosine methylation in DNA was analysed in different sequence contexts (CG, CHG, and CHH) (where H could be A, C, or T) and position (promoter, exon, intron, terminator, interspersed repeat regions), and the H3K9me2 and 24 nucleotide small interfering RNAs (24 nt-siRNA) were overlaid onto the B. rapa reference genome. The epigenome was compared with that of Arabidopsis thaliana and the relationship between the position of DNA methylation and gene expression, and the involvement of 24 nt siRNAs and H3K9me2 are discussed.
2018年10月01日, DNA research : an international journal for rapid publication of reports on genes and genomes, 25 (5), 511 - 520, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
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研究論文(学術雑誌)
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研究論文(学術雑誌)
As climatic changes occur over the coming decades, our scientific understanding of plant responses to environmental cues will become an increasingly important consideration in the breeding of agricultural crops. This review provides a summary of the literature regarding vernalization research in Brassicaceae, covering both the historical origins of vernalization research and current understanding of the molecular mechanisms behind the regulatory pathways involved in vernalization and subsequent inflorescence. We discuss the evolutionarily conserved biology between the model organism Arabidopsis thaliana and the Brassica genus of crop cultivars and contrast the differences between the genera to illustrate the importance of Brassica-specific research into vernalization.
CSIRO, 2018年, Crop and Pasture Science, 69 (1), 30 - 39, 英語[査読有り]
研究論文(国際会議プロシーディングス)
DNA methylation is an epigenetic gene regulatory mechanism that plays an essential role in gene expression, transposon silencing, genome imprinting and plant development. We investigated the influence of DNA methylation on gene expression in Brassica rapa L., to understand whether epigenetic differences exist between inbred lines. Genome-wide DNA methylation was analysed by methylated DNA immunoprecipitation sequencing (MeDIP-seq) of 14-day-old first and second leaves from two inbred lines of Chinese cabbage, one susceptible and one resistant to fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans. MACS (model-based analysis for ChIP-seq) identified DNA methylation peaks in genic regions including 2kb upstream, exon, intron and 2kb downstream. More than 65% of genes showed similar patterns of DNA methylation in the genic regions in the two inbred lines. DNA methylation states of the two inbred lines were compared with their transcriptome. Genes having DNA methylation in the intron and in the 200bp upstream and downstream regions were associated with a lower expression level in both lines. A small number of genes showed a negative correlation between differences in DNA methylation levels and differences in transcriptional levels in the two inbred lines, suggesting that DNA methylation in these genes results in transcriptional suppression.
CSIRO, 2018年, Crop and Pasture Science, 69 (1), 107 - 120, 英語[査読有り]
研究論文(国際会議プロシーディングス)
Epigenetic regulation, covalent modification of DNA and changes in histone proteins are closely linked to plant development and stress response through flexibly altering the chromatin structure to regulate gene expression. In this review, we will illustrate the importance of epigenetic influences by discussing three agriculturally important traits of Brassicaceae. (1) Vernalization, an acceleration of flowering by prolonged cold exposure regulated through epigenetic silencing of a central floral repressor, FLOWERING LOCUS C. This is associated with cold-dependent repressive histone mark accumulation, which confers competency of consequence vegetative-to-reproductive phase transition. (2) Hybrid vigor, in which an F1 hybrid shows superior performance to the parental lines. Combination of distinct epigenomes with different DNA methylation states between parental lines is important for increase in growth rate in a hybrid progeny. This is independent of siRNA-directed DNA methylation but dependent on the chromatin remodeler DDM1. (3) Self-incompatibility, a reproductive mating system to prevent self-fertilization. This is controlled by the S-locus consisting of SP11 and SRK which are responsible for self/non-self recognition. Because self-incompatibility in Brassicaceae is sporophytically controlled, there are dominance relationships between S haplotypes in the stigma and pollen. The dominance relationships in the pollen rely on de novo DNA methylation at the promoter region of a recessive allele, which is triggered by siRNA production from a flanking region of a dominant allele.
Springer Verlag, 2018年01月01日, Plant Cell Reports, 37 (1), 87 - 101, 英語[査読有り]
研究論文(学術雑誌)
Heterosis or hybrid vigor is a phenomenon where hybrid progeny have superior performance compared to their parental inbred lines. This is important in the use of F1 hybrid cultivars in many crops and vegetables. However, the molecular mechanism of heterosis is not clearly understood. Gene interactions between the two genomes such as dominance, overdominance, and epistasis have been suggested to explain the increased biomass and yield. Genetic analyses of F1 hybrids in maize, rice, and canola have defined a large number of quantitative trait loci, which may contribute to heterosis. Recent molecular analyses of transcriptomes together with reference to the epigenome of the parents and hybrids have begun to uncover new facts about the generation of heterosis. These include the identification of gene expression changes in hybrids, which may be important for heterosis, the role of epigenetic processes in heterosis, and the development of stable high yielding lines.
Japanese Society of Breeding, 2018年, Breeding Science, 68 (2), 145 - 158, 英語[査読有り][招待有り]
研究論文(学術雑誌)
Background: Breeding programs often rely on marker-assisted tests or variant calling of next generation sequence (NGS) data to identify regions of genomic introgression arising from the hybridization of two plant species. In this paper we present IntroMap, a bioinformatics pipeline for the screening of candidate plants through the application of signal processing techniques to NGS data, using alignment to a reference genome sequence (annotation is not required) that shares homology with the recurrent parental cultivar, and without the need for de novo assembly of the read data or variant calling. Results: We show the accurate identification of introgressed genomic regions using both in silico simulated genomes, and a hybridized cultivar data set using our pipeline. Additionally we show, through targeted marker-based assays, validation of the IntroMap predicted regions for the hybrid cultivar. Conclusions: This approach can be used to automate the screening of large populations, reducing the time and labor required, and can improve the accuracy of the detection of introgressed regions in comparison to a marker-based approach. In contrast to other approaches that generally rely upon a variant calling step, our method achieves accurate identification of introgressed regions without variant calling, relying solely upon alignment.
BIOMED CENTRAL LTD, 2017年12月, BMC GENETICS, 18, 101, 英語[査読有り]
研究論文(学術雑誌)
Resistant and susceptible lines in Brassica rapa have different immune responses against Fusarium oxysporum inoculation.Fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans (Foc) is an important disease of Brassicaceae; however, the mechanism of how host plants respond to Foc is still unknown. By comparing with and without Foc inoculation in both resistant and susceptible lines of Chinese cabbage (Brassica rapa var. pekinensis), we identified differentially expressed genes (DEGs) between the bulked inoculated (6, 12, 24, and 72 h after inoculation (HAI)) and non-inoculated samples. Most of the DEGs were up-regulated by Foc inoculation. Quantitative real-time RT-PCR showed that most up-regulated genes increased their expression levels from 24 HAI. An independent transcriptome analysis at 24 and 72 HAI was performed in resistant and susceptible lines. GO analysis using up-regulated genes at 24 HAI indicated that Foc inoculation activated systemic acquired resistance (SAR) in resistant lines and tryptophan biosynthetic process and responses to chitin and ethylene in susceptible lines. By contrast, GO analysis using up-regulated genes at 72 HAI showed the overrepresentation of some categories for the defense response in susceptible lines but not in the resistant lines. We also compared DEGs between B. rapa and Arabidopsis thaliana after F. oxysporum inoculation at the same time point, and identified genes related to defense response that were up-regulated in the resistant lines of Chinese cabbage and A. thaliana. Particular genes that changed expression levels overlapped between the two species, suggesting that they are candidates for genes involved in the resistance mechanisms against F. oxysporum.
SPRINGER, 2017年12月, PLANT CELL REPORTS, 36 (12), 1841 - 1854, 英語[査読有り]
研究論文(学術雑誌)
[招待有り]
研究論文(学術雑誌)
The tuberous stem of kohlrabi is an important quantitative trait, which affects its yield and quality. Genetic control of this trait has not yet been unveiled. To identify the QTLs controlling stem swelling of kohlrabi, a BC 1 population of 92 plants was developed from a cross of broccoli DH line GCP04 and kohlrabi var. Seine. A wide range of variation in tuberous stem diameter was observed among the mapping populations. We constructed a genetic map of nine linkage groups (LGs) with different types of markers, spanning a total length of 913.5 cM with an average marker distance of 7.55 cM. Four significant QTLs for radial enlargement of kohlrabi stem, namely, REnBo1, REnBo2, REnBo3, and REnBo4 were detected on C02, C03, C05, and C09, respectively, and accounted for the phenotypic variation of 59% for the stem diameter and 55% for the qualitative grading of tuberous stem in classes. Then, we confirmed the stability of identified QTLs using BC1S1 populations derived from the BC1 plants having heterozygous alleles at the target QTL and homozygous kohlrabi alleles at the remaining QTLs. REnBo1 and REnBo2 using 128 plants of BC(1)68S(1) and 94 plants of BC(1)43S(1), respectively, and REnBo3 and REnBo4 using 152 plants of BC(1)57S(1) were detected at the same positions as the respective QTLs of the BC1 population. Confirmation of QTLs in two successive generations indicates that the QTLs are persistent. The QTLs obtained in this study could be useful in marker-assisted selection of kohlrabi breeding, and to understand the genetic mechanisms of stem swelling and storage organ development in kohlrabi and other Brassica species.
SPRINGER, 2017年09月, MOLECULAR BREEDING, 37 (9), 109, 英語[査読有り]
研究論文(学術雑誌)
[招待有り]
Cabbage (Brassica oleracea L. var. capitata) is an important vegetable worldwide. Most Japanese commercial cultivars of cabbage use an F-1 hybrid seed production system. The purity of F-1 hybrid seeds is important and the assessment of purity based on DNA markers can be highly accurate. In addition, selection of agronomically important traits such as disease resistance based on DNA markers is useful for breeding of cabbage. The aim of this study is to demonstrate the effectiveness of DNA marker-assisted selection in cabbage. In this study we distinguished the parental S haplotypes in 35 F-1 hybrid cultivars by combining several linked DNA markers. Thirty-one highly polymorphic simple sequence repeats (SSR) markers were screened from 175 reported SSR markers, which are useful for assessment of the purity of F-1 hybrid seeds. We examined the relationship between the DNA marker based genotype and the phenotype by an inoculation test of clubroot disease. A co-dominant PCR-RFLP marker was developed for selection of Fusarium yellows resistance and the genotypes using this marker were consistent with inoculation test in all tested samples.
SPRINGER, 2017年01月, EUPHYTICA, 213 (1), 28, 英語[査読有り]
研究論文(学術雑誌)
Hybrid vigor or heterosis refers to the superior performance of F-1 hybrid plants over their parents. Heterosis is particularly important in the production systems of major crops. Recent studies have suggested that epigenetic regulation such as DNA methylation is involved in heterosis, but the molecular mechanism of heterosis is still unclear. To address the epigenetic contribution to heterosis in Arabidopsis thaliana, we used mutant genes that have roles in DNA methylation. Hybrids between C24 and Columbia-0 (Col) without RNA polymerase IV (Pol IV) or methyltransferase I (MET1) function did not reduce the level of biomass heterosis (as evaluated by rosette diameter). Hybrids with a mutation in decrease in dna methylation 1 (ddm1) showed a decreased heterosis level. Vegetative heterosis in the ddm1 mutant hybrid was reduced but not eliminated; a complete reduction could result if there was a change in methylation at all loci critical for generating the level of heterosis, whereas if only a proportion of the loci have methylation changes there may only be a partial reduction in heterosis.
NATL ACAD SCIENCES, 2016年10月, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 113 (43), E6704 - E6711, 英語[査読有り]
研究論文(学術雑誌)
研究論文(国際会議プロシーディングス)
Inter-specific hybrids are a useful source for increasing genetic diversity. Some reproductive barriers before and/or after fertilization prevent production of hybrid plants by inter-specific crossing. Therefore, techniques to overcome the reproductive barrier have been developed, and have contributed to hybridization breeding. In recent studies, identification of molecules involved in plant reproduction has been studied to understand the mechanisms of reproductive barriers. Revealing the molecular mechanisms of reproductive barriers may allow us to overcome reproductive barriers in inter-specific crossing, and to efficiently produce inter-specific hybrids in cross-combinations that cannot be produced through artificial techniques. Inter-specific hybrid plants can potentially serve as an elite material for plant breeding, produced through the merging of genomes of parental species by allopolyploidization. Allopolyploidization provides some benefits, such as heterosis, increased genetic diversity and phenotypic variability, which are caused by dynamic changes of the genome and epigenome. Understanding of allopolyploidization mechanisms is important for practical utilization of inter-specific hybrids as a breeding material. This review discusses the importance of reproductive barriers and the effect of allopolyploidization in crop breeding programs.
JAPANESE SOC BREEDING, 2016年06月, BREEDING SCIENCE, 66 (3), 333 - 349, 英語[査読有り]
研究論文(学術雑誌)
[査読有り]
研究論文(学術雑誌)
Chinese cabbage (Brassica rapa L. var. pekinensis) is an important vegetable in Asia. Most Japanese commercial cultivars of Chinese cabbage use an F1 hybrid seed production system because of the high yielding cultivars produced. An efficient method for predicting hybrid performance in the parental generations is desired, and genetic distance between parental lines might be a good indicator of the level of hybrid vigor in a cross. Information concerning the genetic relationships among parental candidate inbred lines is useful for variety protection. The number of DNA markers available that can be used to assess the purity of inbred lines is limited in B. rapa. The aim of this study is to use DNA markers to assess the genetic distance between inbred lines to examine early developmental and yield heterosis so as to develop methods for selecting the best parental lines for the production of hybrids. We screened highly polymorphic SSR and CAPS markers to assess the genetic uniformity of inbred lines and characterize their genetic relationship. We examined the early size and yield heterosis in 32 F1 hybrids of Chinese cabbage. There was a moderate correlation in mid-parent heterosis between leaf size at 21 days after sowing and harvested biomass but not in best-parent heterosis. In contrast there was no correlation between genetic distance and mid-parent or best-parent heterosis, indicating that genetic distance does not predict the heterosis phenotype.
Elsevier B.V., 2016年03月01日, Plant Gene, 5, 1 - 7, 英語[査読有り]
研究論文(学術雑誌)
Chinese cabbage (Brassica rapa L. var. pekinensis) is an important vegetable in Asia, and most Japanese commercial cultivars of Chinese cabbage use an F1 hybrid seed production system. Self-incompatibility is successfully used for the production of F1 hybrid seeds in B. rapa vegetables to avoid contamination by non-hybrid seeds, and the strength of self-incompatibility is important for harvesting a highly pure F1 seeds. Prediction of agronomically important traits such as disease resistance based on DNA markers is useful. In this dataset, we identified the S haplotypes by DNA markers and evaluated the strength of self-incompatibility in Chinese cabbage inbred lines. The data described the predicted disease resistance to Fusarium yellows or clubroot in 22 Chinese cabbage inbred lines using gene associated or gene linked DNA markers.
Elsevier Inc., 2016年03月01日, Data in Brief, 6 (6), 229 - 237, 英語[査読有り]
研究論文(学術雑誌)
Background: Heterosis or hybrid vigour is a phenomenon in which hybrid progeny exhibit superior performance compared to their parental inbred lines. Most commercial Chinese cabbage cultivars are F-1 hybrids and their level of hybrid vigour is of critical importance and is a key selection criterion in the breeding system. Results: We have characterized the heterotic phenotype of one F-1 hybrid cultivar of Chinese cabbage and its parental lines from early- to late-developmental stages of the plants. Hybrid cotyledons are larger than those of the parents at 4 days after sowing and biomass in the hybrid, determined by the fresh weight of leaves, is greater than that of the larger parent line by approximately 20 % at 14 days after sowing. The final yield of the hybrid harvested at 63 days after sowing is 25 % greater than the yield of the better parent. The larger leaves of the hybrid are a consequence of increased cell size and number of the photosynthetic palisade mesophyll cells and other leaf cells. The accumulation of plant hormones in the F-1 was within the range of the parental levels at both 2 and 10 days after sowing. Two days after sowing, the expression levels of chloroplast-targeted genes in the cotyledon cells were upregulated in the F-1 hybrid relative to their mid parent values. Shutdown of chlorophyll biosynthesis in the cotyledon by norflurazon prevented the increased leaf area in the F-1 hybrid. Conclusions: In the cotyledons of F-1 hybrids, chloroplast-targeted genes were upregulated at 2 days after sowing. The increased activity levels of this group of genes suggested that their differential transcription levels could be important for establishing early heterosis but the increased transcription levels were transient. Inhibition of the photosynthetic process in the cotyledon reduced heterosis in later seedling stages. These observations suggest early developmental events in the germinating seedling of the hybrid may be important for later developmental vigour and yield advantage.
BIOMED CENTRAL LTD, 2016年02月, BMC PLANT BIOLOGY, 16 (1), 45, 英語[査読有り]
研究論文(学術雑誌)
Fusarium oxysporum f.sp. conlutinans (Foc) is a serious root-invading and xylem-colonizing fungus that causes yellowing in Brassica oleracea. To comprehensively understand the interaction between F oxysporum and B. oleracea, composition of the xylem sap proteome of the non-infected and Foc-infected plants was investigated in both resistant and susceptible cultivars using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after in-solution digestion of xylem sap proteins. Whole genome sequencing of Foc was carried out and generated a predicted Foc protein database. The predicted Foc protein database was then combined with the public B. oleracea and B. rapa protein databases downloaded from Uniprot and used for protein identification. About 200 plant proteins were identified in the xylem sap of susceptible and resistant plants. Comparison between the non-infected and Foc-infected samples revealed that Foc infection causes changes to the protein composition in B. oleracea xylem sap where repressed proteins accounted for a greater proportion than those of induced in both the susceptible and resistant reactions. The analysis on the proteins with concentration change 2-fold indicated a large portion of up- and down-regulated proteins were those acting on carbohydrates. Proteins with leucine-rich repeats and legume lectin domains were mainly induced in both resistant and susceptible system, so was the case of thaumatins. Twenty-five Foc proteins were identified in the infected xylem sap and 10 of them were cysteine-containing secreted small proteins that are good candidates for virulence and/or avirulence effectors. The findings of differential response of protein contents in the xylem sap between the non-infected and Foc-infected samples as well as the Foc candidate effectors secreted in xylem provide valuable insights into B. oleracea-Foc interactions.
FRONTIERS MEDIA SA, 2016年02月, FRONTIERS IN PLANT SCIENCE, 7, 31, 英語[査読有り]
研究論文(学術雑誌)
Epigenetic regulation is crucial for the development of plants and for adaptation to a changing environment. Recently, genome-wide profiles of histone modifications have been determined by a combination of chromatin immunoprecipitation (ChIP) and genomic tiling arrays (ChIP on chip) or ChIP and high-throughput sequencing (ChIP-seq) in species including Arabidopsis thaliana, rice and maize. Validation of ChIP analysis by PCR or qPCR using positive and negative regions of histone modification is necessary. In contrast, information about histone modifications is limited in Chinese cabbage, Brassica rapa. The aim of this study was to develop positive and negative control primer sets for H3K4me3 (trimethylation of the 4th lysine of H3), H3K9me2, H3K27me3 and H3K36me3 in B. rapa. The expression and histone modification of four FLC paralogs in B. rapa, before and after vernalization, were examined using the method developed here. After vernalization, expression of all four BrFLC genes was reduced, and accumulation of H3K27me3 was observed in three of them. As with A. thaliana, the vernalization response and stability of FLC repression correlated with the accumulation of H3K27me3. These results suggest that the epigenetic state during vernalization is important for high bolting resistance in B. rapa. The positive and negative control primer sets developed here revealed positive and negative histone modifications in B. rapa that can be used as a control for future studies.
GENETICS SOC JAPAN, 2016年02月, GENES & GENETIC SYSTEMS, 91 (1), 1 - 10, 英語[査読有り]
研究論文(学術雑誌)
We identified the candidate gene conferring yellow wilt resistance (YR) in B. oleracea . This work will facilitate YR breeding programs for B. oleracea and its closely related species. Yellow wilt disease is one of the most serious diseases of cabbage worldwide. Type A resistance to the disease is controlled by a single dominant gene that is used in cabbage breeding. Our previous QTL study identified the FocBo1 locus controlling type A resistance. In this study, the FocBo1 locus was fine-mapped by using 139 recombinant F-2 plants derived from resistant cabbage (AnjuP01) and susceptible broccoli (GCP04) DH lines. As a result, we successfully delimited the location of FocBo1 within 1.00 cM between markers, BoInd 2 and BoInd 11. Analysis of BAC and cosmid sequences corresponding to the FocBo1 locus identified an orthologous gene of Bra012688 that was recently identified as an candidate gene that confers yellows resistance in Chinese cabbage. The candidate gene-specific DNA markers and phenotypes in F-1 cabbage cultivars and their selfed F-2 populations showed a perfect correlation. Our identification of the candidate gene for FocBo1 will assist introduction of fusarium resistance into B. oleracea cultivars and contribute further understanding of interaction between Brassica plants and fusarium.
SPRINGER, 2015年01月, THEORETICAL AND APPLIED GENETICS, 128 (1), 119 - 130, 英語[査読有り]
研究論文(学術雑誌)
Fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans is an important disease of Brassica worldwide. To identify a resistance (R) gene against Fusarium yellows in Chinese cabbage (Brassica rapa var. pekinensis), we analyzed differential expression at the whole genome level between resistant and susceptible inbred lines using RNA sequencing. Four hundred and eighteen genes were significantly differentially expressed, and these were enriched for genes involved in response to stress or stimulus. Seven dominant DNA markers at putative R-genes were identified. Presence and absence of the sequence of the putative R-genes, Bra012688 and Bra012689, correlated with the resistance of six inbred lines and susceptibility of four inbred lines, respectively. In F-2 populations derived from crosses between resistant and susceptible inbred lines, presence of Bra012688 and Bra012689 cosegregated with resistance, suggesting that Bra012688 and Bra012689 are good candidates for fusarium yellows resistance in Chinese cabbage.
SPRINGER, 2014年06月, PLANT MOLECULAR BIOLOGY, 85 (3), 247 - 257, 英語[査読有り]
研究論文(学術雑誌)
[招待有り]
Brassica napus is a leading oilseed crop throughout many parts of the world. It is well adapted to long day photoperiods, however, it does not adapt well to short day subtropical regions. Short duration B. nap us plants were resynthesized through ovary culture from interspecific crosses in which B. rapa cultivars were reciprocally crossed with B. oleracea. From five different combinations, 17 hybrid plants were obtained in both directions. By self-pollinating the F-1 hybrids or introgressing them with cultivated B. napus, resynthesized (RS) F-3 and semi-resynthesized (SRS) F-2 generations were produced, respectively. In field trial in Bangladesh, the RS B. napus plants demonstrated variation in days to first flowering ranging from 29 to 73 days; some of which were similar to cultivated short duration B. napus, but not cultivated short duration B. rapa. The RS and SRS B. napus lines produced 2-4.6 and 1.6-3.7 times higher yields, respectively, as compared to cultivated short duration B. napus. Our developed RS lines may be useful for rapeseed breeding not only for subtropical regions, but also for areas such as Canada and Europe where spring rapeseed production can suffer from late spring frosts. Yield and earliness in RS lines are discussed.
JAPANESE SOC BREEDING, 2014年01月, BREEDING SCIENCE, 63 (5), 495 - 502, 英語[査読有り]
研究論文(学術雑誌)
Throughout the world, clubroot disease is one of the most damaging diseases affecting Brassica oleracea. To develop marker-assisted selection (MAS) that could assist the incorporation of durable clubroot resistance (CR) into cultivars, previous genetic analyses have identified several CR quantitative trait loci (CR-QTL). However, the independent and cumulative effects of each CR locus against various isolates have rarely been tested. Previously, we identified one major CR-QTL and four minor CR-QTL in the F2 plants from broccoli doubled haploid (DH) line x cabbage DH line of B. oleracea. In the present study, to clarify their effectiveness for controlling disease involving various isolates, inoculation testing was conducted in genotypes with various combinations of the CR genes, which were selected using the DNA markers closely associated with each CR-QTL. In exploring the overall disease incidence, it was apparent that a single involvement of the major CR gene located in the PbBo(Anju)1 locus, or accumulation of CR genes in the minor CR-QTL, is not enough to confer sufficient resistance. One major CR gene in the QTL PbBo(Anju)1 locus plus two to three minor CR genes conferred moderate resistance. The genotype in which all of the CR genes locating in the five QTL including PbBo(Anju)1 were accumulated showed the highest resistance, and it was broadly resistant against six isolates. Accumulation of several CR genes by MAS is necessary to conduct CR breeding in B. oleracea. Our developed DNA markers can be used efficiently to make selections of required loci for the acquisition of resistance, and use of these markers will be a powerful tool for CR breeding in B. oleracea.
SPRINGER, 2013年12月, MOLECULAR BREEDING, 32 (4), 889 - 900, 英語[査読有り]
研究論文(学術雑誌)
Hybrid vigour, or heterosis, refers to the increased yield and biomass of hybrid offspring relative to the parents. Although this has been exploited in plants for agriculture and horticulture, the molecular and cellular mechanisms underlying hybrid vigour are largely unknown. Genetic analyses show that there are a large number of quantitative trait loci (QTLs) that contribute to the heterotic phenotype, indicating that it is a complex phenomenon. Gene expression in hybrids is regulated by the interactions of the two parental epigenetic systems and the underlying genomes. Increasing understanding of the interplay of small RNA (sRNA) molecules, DNA methylation, and histone marks provides new opportunities to define the basis of hybrid vigour and to understand why F1 heterosis is not passed on to subsequent generations. We discuss recent findings that suggest the existence of several pathways that alter DNA methylation patterns, which may lead to transcriptional changes resulting in the heterotic phenotype.
ELSEVIER SCIENCE LONDON, 2013年12月, TRENDS IN GENETICS, 29 (12), 684 - 690, 英語[査読有り]
研究論文(学術雑誌)
Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) is possibly the most important disease of Brassica worldwide. To compare chromosomal positions of Xcc resistance loci in Brassica oleracea between the present and published studies and to develop marker assisted selection (MAS) to resistance against Xcc race 1, we constructed a B. oleracea map, including pW, pX an
Scientific Research, 2013年08月, American Journal of Plant Sciences, 4 (8), 11 - 20, 英語[査読有り]
研究論文(学術雑誌)
[査読有り]
Difference in the level of expression of genes is one of the factors contributing to plant phenotype. As well as being under genetic control, gene expression is regulated by epigenetic processes such as DNA methylation and histone modifications. We compared genome-wide gene expression between Arabidopsis thaliana and the related species Arabidopsis lyrata subsp. lyrata and Arabidopsis halleri subsp. gemmifera to understand which genes might contribute to species differences. Genes categorized into response to stress tended to show differential expression between species, suggesting that divergence of expression in these genes contributes to adaptation to environmental conditions following the divergence of species. Regions methylated in A. lyrata were identified from Methylated-DNA immunoprecipitation, and this DNA methylation profile together with transcriptome data revealed that gene body methylation was not associated with differential gene expression between A. thaliana and A. lyrata. The DNA methylation status of four putative imprinted genes of A. lyrata was examined and found to be conserved in vegetative tissues between A. thaliana and A. lyrata in FIS2, HDG3, and HDG9, but not in HDG8. (C) 2012 Elsevier B.V. All rights reserved.
ELSEVIER SCIENCE BV, 2012年09月, GENE, 506 (2), 301 - 309, 英語[査読有り][招待有り]
研究論文(学術雑誌)
Fusarium wilt caused by Fusarium oxysporum f. sp. conglutinans is one of the most important diseases of Brassica crops, resulting in severe reductions in yield and quality. To characterize the inheritance pattern of fusarium resistance, a cross between a susceptible broccoli and a resistant cabbage was subjected to segregation analysis. Results indicated that resistance was controlled by a single dominant allele. This gene was named Foc-Bo1 and mapped to linkage group seven (O7) by both the segregation test and quantitative trait locus (QTL) analysis. The QTL on O7 was detected with a logarithm of odds score (LOD) of 19.5, which was above the threshold value with genome-wide 1% significance level (2.01). A minor QTL was also detected on O4 with a LOD score of 2.06. Inoculation tests indicated that stable expression of fusarium resistance at high temperatures required Foc-Bo1 homozygosity. The association between Foc-Bo1 and the closest simple sequence repeat marker (KBrS003O1N10) was analyzed in three F3 populations. Based on these studies, KBrS003O1N10 represents an effective marker-assisted selection (MAS) tool for breeding fusarium wilt resistance into Brassica oleracea crops. To our knowledge, this is the first paper to map the fusarium-resistance gene in Brassica species and to validate the effectiveness of MAS in improving fusarium resistance in these important plants.
SPRINGER, 2012年08月, MOLECULAR BREEDING, 30 (2), 809 - 818, 英語[査読有り]
研究論文(学術雑誌)
Natural variation is defined as the phenotypic variation caused by spontaneous mutations. In general, mutations are associated with changes of nucleotide sequence, and many mutations in genes that can cause changes in plant development have been identified. Epigenetic change, which does not involve alteration to the nucleotide sequence, can also cause changes in gene activity by changing the structure of chromatin through DNA methylation or histone modifications. Now there is evidence based on induced or spontaneous mutants that epigenetic changes can cause altering plant phenotypes. Epigenetic changes have occurred frequently in plants, and some are heritable or metastable causing variation in epigenetic status within or between species. Therefore, heritable epigenetic variation as well as genetic variation has the potential to drive natural variation.
MDPI AG, 2012年08月, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 13 (8), 9900 - 9922, 英語[査読有り][招待有り]
研究論文(学術雑誌)
An allopolyploid is an individual having two or more complete sets of chromosomes derived from different species. Generation of allopolyploids might be rare because of the need to overcome limitations such as co-existing populations of parental lines, overcoming hybrid incompatibility, gametic non-reduction, and the requirement for chromosome doubling. However, allopolyploids are widely observed among plant species, so allopolyploids have succeeded in overcoming these limitations and may have a selective advantage. As techniques for making allopolyploids are developed, we can compare transcription, genome organization, and epigenetic modifications between synthesized allopolyploids and their direct parental lines or between several generations of allopolyploids. It has been suggested that divergence of transcription caused either genetically or epigenetically, which can contribute to plant phenotype, is important for the adaptation of allopolyploids.
MDPI, 2012年07月, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 13 (7), 8696 - 8721, 英語[査読有り][招待有り]
研究論文(学術雑誌)
Arabidopsis thaliana shows hybrid vigor (heterosis) in progeny of crosses between Columbia-0 and C24 accessions. Hybrid vigor was evident as early as mature seeds and in seedlings 3 d after sowing (DAS). At 3 DAS, genes encoding chloroplast-located proteins were significantly overrepresented (187) among the 724 genes that have greater than midparent values of expression in the hybrid. Many of these genes are involved in chlorophyll biosynthesis and photosynthesis. The rate of photosynthesis was constant per unit leaf area in parents and hybrids. Larger cell sizes in the hybrids were associated with more chloroplasts per cell, more total chlorophyll, and more photosynthesis. The increased transcription of the chloroplast-targeted genes was restricted to the 3-7 DAS period. At 10 DAS, only 118 genes had expression levels different from the expected midparent value in the hybrid, and only 12 of these genes were differentially expressed at 3 DAS. The early increase in activity of genes involved in photosynthesis and the associated phenomena of increases in cell size and number through development, leading to larger leaf areas of all leaves in the hybrid, suggest a central role for increased photosynthesis in the production of the heterotic biomass. In support of this correlation, we found that an inhibitor of photosynthesis eliminated heterosis and that higher light intensities enhanced both photosynthesis and heterosis. In hybrids with low-level heterosis (Landsberg erecta x Columbia-0), chloroplast-targeted genes were not up-regulated and leaf areas were only marginally increased.
NATL ACAD SCIENCES, 2012年05月, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 109 (18), 7109 - 7114, 英語[査読有り][招待有り]
研究論文(学術雑誌)
[査読有り]
The merging of two different genomes occurs during the formation of amphidiploids, and the merged regulatory networks have the potential to generate a new gene expression pattern. We examined the genome-wide gene expression of two newly synthesized amphidiploids between Arabidopsis thaliana and the related species Arabidopsis lyrata subsp. lyrata and Arabidopsis halleri subsp. gemmifera. 1,137 (4.7%) and 1,316 (5.4%) of probesets showed differential gene expression in A. thaliana-A. halleri and A. thaliana-A. lyrata hybrids respectively, compared to the mid parent value and of these, 489 were in common. Genes that differed in expression between the parental lines tended to have an expression level in both hybrids differing from the mid parent value. In contrast to protein coding genes, there is little differential expression of transposons. Genes in the categories of chloroplast-targeted and response to stress were overrepresented in the non-additively expressed genes in both amphidiploids. As these genes have the potential to contribute directly to the plant phenotype, we suggest that rapid changes of gene expression in amphidiploids might be important for producing greater biomass.
SPRINGER, 2011年11月, PLANT MOLECULAR BIOLOGY, 77 (4-5), 419 - 431, 英語[査読有り]
研究論文(学術雑誌)
Arabidopsis thaliana is a quantitative long-day plant with the timing of the floral transition being regulated by both endogenous signals and multiple environmental factors, fwa is a late-flowering mutant, and this phenotype is due to ectopic FWA expression caused by hypomethylation at the FWA locus. The floral transition results in the activation of the floral development process, the key regulators being the floral meristem identity genes, AP1 (APETALA1) and LFY (LEAFY). In this study, we describe inflorescence abnormalities in plants overexpressing the Arabidopsis lyrata FT (AIFT) and A. thaliana FWA (AtFWA) genes simultaneously. The inflorescence abnormality phenotype was present in only a proportion of plants. All plants overexpressing both AIFT and AtFWA flowered earlier than fwa, suggesting that the inflorescence abnormality and earlier flowering time are caused independently. The inflorescence abnormality phenotype was similar to that of the double mutant of ap1 and fly, and AP1 and LFY genes were down-regulated in the abnormal inflorescences. From these results, we suggest that not only does ectopic AtFWA expression inhibit AtFT/AIFT function to delay flowering but that overexpression of AtFWA and AIFT together inhibits AP1 and LFY function to produce abnormal inflorescences. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
ELSEVIER IRELAND LTD, 2011年10月, PLANT SCIENCE, 181 (4), 496 - 503, 英語[査読有り]
研究論文(学術雑誌)
In this review we have analysed two major biological systems involving epigenetic control of gene activity. In the first system we demonstrate the interplay between genetic and epigenetic controls over the transcriptional activity of FLC, a major repressor of flowering in Arabidopsis. FLC is down-regulated by low temperature treatment (vernalisation) releasing the repressor effect on flowering. We discuss the mechanisms of the reduced transcription and the memory of the vernalisation treatment through vegetative development. We also discuss the resetting of the repressed activity level of the PLC gene, following vernalisation, to the default high activity level and show it occurs during both male and female gametogenesis but with different timing in each. In the second part of the review discussed the complex multigenic system which is responsible for the patterns of gene activity which bring about hybrid vigour in crosses between genetically similar but epigenetically distinct parents. The epigenetic systems that we have identified as contributing to the heterotic phenotype are the 24nt siRNAs and their effects on RNA dependent DNA methylation (RdDM) at the target loci leading to changed expression levels. We conclude that it is likely that epigenetic controls are involved in expression systems in many aspects of plant development and plant function. (C) 2011 Elsevier B.V. All rights reserved.
ELSEVIER SCIENCE BV, 2011年08月, BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS, 1809 (8), 427 - 437, 英語[査読有り]
研究論文(学術雑誌)
P>fwa is a late flowering epi-mutant in Arabidopsis thaliana. FWA is silenced by DNA methylation in vegetative tissue but is demethylated in the central cell of the female ovule and continues to be expressed in the endosperm from the maternal copy. FWA is stably silenced in A. thaliana, but in related Arabidopsis species, FWA expression and DNA methylation levels vary in vegetative tissue. In this study, we show that variation in FWA expression in field isolates having identical DNA sequences is associated with changes in DNA methylation and may change over time. Vegetative FWA expression is correlated with decreased methylation at non-CG sites in the region upstream of the transcription start site in species related to A. thaliana and we conclude that methylation of this region is critical for FWA silencing in these species. In A. thaliana, FWA expression is affected by methylation in regions both upstream and downstream of the transcription start site. Ectopic A. thaliana FWA expression causes a late flowering phenotype, but over-expression of Arabidopsis lyrata FWA does not. In A. thaliana, stable silencing of FWA to prevent late flowering may have evolved through the selection of large tandem repeats and spread of the critical methylated region to include these repeats.
WILEY-BLACKWELL, 2011年06月, PLANT JOURNAL, 66 (5), 831 - 843, 英語[査読有り]
研究論文(学術雑誌)
DNA methylation is an important epigenetic modification regulating gene expression and transposon silencing. Although epigenetic regulation is involved in some agricultural traits, there has been relatively little research on epigenetic modifications of genes in Brassica rapa, which includes many important vegetables. In B. rapa, orthologs of DDM1, a chromatin remodeling factor required for maintenance of DNA methylation, have been characterized and DNA hypomethylated knock-down plants by RNAi (ddm1-RNAi plants) have been generated. In this study, we investigated differences of DNA methylation status at the genome-wide level between a wild-type (WT) plant and a ddm1-RNAi plant by methylation-sensitive amplification polymorphism (MSAP) analysis. MSAP analysis detected changes of DNA methylation of many repetitive sequences in the ddm1-RNAi plant. Search for body methylated regions in the WT plant revealed no difference in gene body methylation levels between the WT plant and the ddm1-RNAi plant. These results indicate that repetitive sequences are preferentially methylated by DDM1 genes in B. rapa.
SPRINGER, 2011年01月, PLANT CELL REPORTS, 30 (1), 81 - 88, 英語[査読有り]
研究論文(学術雑誌)
A central question in genomic imprinting is how a specific sequence is recognized as the target for epigenetic marking. In both mammals and plants, imprinted genes are often associated with tandem repeats and transposon-related sequences, but the role of these elements in epigenetic gene silencing remains elusive. FWA is an imprinted gene in Arabidopsis thaliana expressed specifically in the female gametophyte and endosperm. Tissue-specific and imprinted expression of FWA depends on DNA methylation in the FWA promoter, which is comprised of two direct repeats containing a sequence related to a SINE retroelement. Methylation of this element causes epigenetic silencing, but it is not known whether the methylation is targeted to the SINE-related sequence itself or the direct repeat structure is also necessary. Here we show that the repeat structure in the FWA promoter is highly diverse in species within the genus Arabidopsis. Four independent tandem repeat formation events were found in three closely related species. Another related species, A. halleri, did not have a tandem repeat in the FWA promoter. Unexpectedly, even in this species, FWA expression was imprinted and the FWA promoter was methylated. In addition, our expression analysis of FWA gene in vegetative tissues revealed high frequency of intra-specific variation in the expression level. In conclusion, we show that the tandem repeat structure is dispensable for the epigenetic silencing of the FWA gene. Rather, SINE-related sequence is sufficient for imprinting, vegetative silencing, and targeting of DNA methylation. Frequent independent tandem repeat formation events in the FWA promoter led us to propose that they may be a consequence, rather than cause, of the epigenetic control. The possible significance of epigenetic variation in reproductive strategies during evolution is also discussed.
PUBLIC LIBRARY SCIENCE, 2008年04月, PLOS GENETICS, 4 (4), 英語[査読有り]
研究論文(学術雑誌)
A central question in genomic imprinting is how a specific sequence is recognized as the target for epigenetic marking. In both mammals and plants, imprinted genes are often associated with tandem repeats and transposon-related sequences, but the role of these elements in epigenetic gene silencing remains elusive. FWA is an imprinted gene in Arabidopsis thaliana expressed specifically in the female gametophyte and endosperm. Tissue-specific and imprinted expression of FWA depends on DNA methylation in the FWA promoter, which is comprised of two direct repeats containing a sequence related to a SINE retroelement. Methylation of this element causes epigenetic silencing, but it is not known whether the methylation is targeted to the SINE-related sequence itself or the direct repeat structure is also necessary. Here we show that the repeat structure in the FWA promoter is highly diverse in species within the genus Arabidopsis. Four independent tandem repeat formation events were found in three closely related species. Another related species, A. halleri, did not have a tandem repeat in the FWA promoter. Unexpectedly, even in this species, FWA expression was imprinted and the FWA promoter was methylated. In addition, our expression analysis of FWA gene in vegetative tissues revealed high frequency of intra-specific variation in the expression level. In conclusion, we show that the tandem repeat structure is dispensable for the epigenetic silencing of the FWA gene. Rather, SINE-related sequence is sufficient for imprinting, vegetative silencing, and targeting of DNA methylation. Frequent independent tandem repeat formation events in the FWA promoter led us to propose that they may be a consequence, rather than cause, of the epigenetic control. The possible significance of epigenetic variation in reproductive strategies during evolution is also discussed. © 2008 Fujimoto et al.
2008年04月, PLoS Genetics, 4 (4), e1000048, 英語[査読有り]
研究論文(学術雑誌)
DNA methylation and histone modification play important roles in regulating gene expression. The DDM1 gene in Arabidopsis thaliana (AtDDM1) is required for the maintenance of DNA methylation level and histone H3 methylation pattern. We isolated DDM1 homologs of Brassica rapa, BrDDM1a and BrDDM1b, which have 84.4% and 84.1% deduced amino acid sequence identities with AtDDM1, respectively. Both the BrDDM1a and BrDDM1b genes were found to be expressed in vegetative and reproductive tissues. B. rapa ddm1-RNAi transgenic plants with reduced levels of BrDDM1a/BrDDM1b expression showed genome-wide and non-tissue-specific demethylation. These results suggest that the BrDDM1a and BrDDM1b genes are orthologs of AtDDM1 and are required for the maintenance of DNA methylation as is AtDDM1. Despite genome-wide demethylation, developmental abnormalities were not found in the ddm1-RNAi transgenic plants. Dominance relationships of SP11/SCR alleles, the determinant of pollen recognition specificity in Brassica self-incompatibility, in S heterozygotes in B. rapa were not influenced by the low level of the BrDDM1 expression. Transcriptional reactivation of retrotransposon-like sequences observed in the ddm1-RNAi transgenic plants indicates that BrDDM1a and BrDDM1b participate in silencing of retrotransposons. Hypomethylation states of the ddm1-RNAi transgenic plants were inherited by plants of the next generation even by plants which had lost the RNAi construct by segregation. Remethylation was observed in a few progenies. Efficiencies of remethylation in the progenies without the RNAi construct were different between 18S rDNA, BoSTF12a/15a, and BrTto1 sequences.
SPRINGER, 2008年03月, PLANT MOLECULAR BIOLOGY, 66 (5), 463 - 473, 英語[査読有り]
研究論文(学術雑誌)
One of the causes of genome size expansion is considered to be amplification of retrotransposons. We determined nucleotide sequences of 24 PCR products for each of six retrotransposons in Brassica rapa and Brassica oleracea. Phylogenetic trees of these sequences showed species-specific clades. We also sequenced STF7a homologs and Tto1 homologs, 24 PCR products each, in nine diploids and three allopolyploids, and constructed phylogenetic trees. In these phylogenetic trees, species-specific clades of diploid species were also formed, but retrotransposons of allopolyploids were clustered into the clades of their original genomes, indicating that these two retrotransposons amplified after speciation of the nine diploids. Genetic variation in these retrotransposons may have arisen before emergence of allopolyploid species. There was a positive correlation between the genome size and the average number of substitutions of STF7a and Tto1 homologs in at least seven diploids. The implications of these results in the genome evolution of Brassicaceae are herein discussed.
GENETICS SOC JAPAN, 2008年02月, GENES & GENETIC SYSTEMS, 83 (1), 13 - 22, 英語[査読有り]
研究論文(学術雑誌)
Molecular imprinting is the differential expression and/or silencing of alleles according to their parent of origin [1, 2]. Conflicts between parents, or parents and offspring, should cause "arms races," with accelerated evolution of the genes involved in imprinting. This should be detectable in the evolution of imprinting genes' protein sequences and in the promoter regions of imprinted genes. Previous studies, however, found no evidence of more amino acid substitutions in imprinting genes [1, 3]. We have analyzed sequence diversity of the Arabidopsis lyrata Medea (MEA) gene and divergence from the A. thaliana sequence, including the first study of the promoter region. In A. thaliana, MEA is imprinted, with paternal alleles silenced in endosperm cells [4, 5], and also functions in the imprinting machinery [4, 6]; MEA protein binding at the MEA promoter region indicates self-regulated imprinting [7-9]. We find the same paternal MEA allele silencing in A. lyrata endosperm but no evidence for adaptive evolution in the coding region, whereas the 5' flanking region displays high diversity, with distinct haplotypes, suggesting balancing selection in the promoter region.
CELL PRESS, 2007年11月, CURRENT BIOLOGY, 17 (21), 1885 - 1889, 英語[査読有り]
研究論文(学術雑誌)
In self-incompatibility, a number of S haplotypes are maintained by frequency-dependent selection, which results in trans-specific S haplotypes. The region of several kilobases (similar to 40-60 kb) from SP6 to SP2, including self-incompatibility-related genes and some adjacent genes in Brassica rapa, has high nucleotide diversity due to the hitchhiking effect, and therefore we call this region the "S-locus complex." Recombination in the S-locus complex is considered to be suppressed. We sequenced regions of >50 kb of the S-locus complex of three S haplotypes in B. rapa and found higher nucleotide diversity in intergenic regions than in coding regions. Two highly similar regions of >10 kb were found between BrS-8 and BrS-46 Phylogenetic analysis using trans-specific S haplotypes (called interspecific pairs) of B. rapa and B. oleracea suggested that recombination reduced the nucleotide diversity in these two regions and that the genes not involved in self-incompatibility in the S-locus complex and the kinase domain, but not the S domain, of SRK have also experienced recombination. Recombination may reduce hitchhiking diversity in the S-locus complex, whereas the region from the S domain to SP11 would disfavor recombination.
GENETICS, 2007年10月, GENETICS, 177 (2), 949 - 958, 英語[査読有り]
研究論文(学術雑誌)
DNA polymorphism of the Wx gene in glutinous rice cultivars was investigated by PCR-RF-SSCP and heteroduplex cleavage analysis using Brassica petiole extract, and the nucleotide sequence variations were identified. Most japonica-type glutinous rice was found to have a 23-bp duplication in the second exon, which causes loss of the function of granule-bound starch synthase (GBSS) encoded by the Wx gene. Without the 23-bp duplication, there was an insertion of 7,764 bp in the ninth exon of the wx allele of 'Oragamochi'. Expression analysis of the wx allele using RT-PCR and Northern blot analysis revealed that transcripts of the 'Oragamochi' wx allele are about 1-kb shorter and that the deduced amino acid sequence of the transcript lacks a motif important for GBSS. Therefore, this insertion was considered to be the cause of the glutinous trait of 'Oragamochi'. This 7,764-bp insertion had long terminal repeats, a primer binding site, and a polypurine tract, but no sequence homologous with gag and pol, suggesting that it is a non-autonomous element. Furthermore, it had a structure similar to Dasheng and may be a member of Dasheng.
SPRINGER, 2007年07月, THEORETICAL AND APPLIED GENETICS, 115 (2), 217 - 224, 英語[査読有り]
研究論文(学術雑誌)
Brassica napus is an amphidiploid species with the A genome from Brassica rapa and the C genome from Brassica oleracea. Although B. rapa, B. oleracea and artificially synthesized amphidiploids with the AC genome are self-incompatible, B. napus is self-compatible. Six S genotypes were identified in B. napus, five of which had class I S haplotypes from one species and a class II S haplotype from the other species, and mutations causing self-compatibility were identified in three of these S genotypes. The most predominant S genotype (BnS-1;BnS-6), which is that of cv. 'Westar', had a class I S haplotype similar to B. rapa S-47 (BrS-47) and a class II S haplotype similar to B. oleracea S-15 (BoS-15). The stigmas of 'Westar' rejected the pollen grains of both BrS-47 and BoS-15, while reciprocal crossings were compatible. Insertion of a DNA fragment of about 3.6 kb was found in the promoter region of the SP11/SCR allele of BnS-1, and transcripts of SP11/SCR were not detected in 'Westar'. The nucleotide sequence of the SP11 genomic DNA of BnS-6 was 100% identical to that of SP11 of BoS-15. Class I SP11 alleles from one species showed dominance over class II SP11 alleles from the other species in artificially synthesized B. napus lines, suggesting that the non-functional dominant SP11 allele suppressed the expression of the recessive SP11 allele in 'Westar'. Two other S genotypes in B. napus also had non-functional class I S haplotypes together with recessive BnS-6. These observations suggest independent origins of self-compatibility in B. napus.
BLACKWELL PUBLISHING, 2007年05月, PLANT JOURNAL, 50 (3), 391 - 400, 英語[査読有り]
研究論文(学術雑誌)
Self-incompatibility system has been most intensively studied in Brassica because of the advantage as plant materials and the importance as crop species. The determinants of self-recognition specificity in the stigma and the pollen are SRK and SP11/SCR, respectively. The genes of SRK and SP11 have many alleles in a species and are closely linked with each other in the S-locus complex. A set of SRK and SP11 alleles are referred to as S haplotype. Sequence analyses of many alleles of SRK and SP11 in Brassica species revealed pairs of S haplotypes having highly similar SRK and SP11 alleles between different species. The interspecific pair of S haplotypes is considered to be derived from the same ancestral S haplotype and to have maintained the same recognition specificity. The structure of the S-locus complex is highly variable, and this high structural polymorphism is considered to be important for suppressing the recombination between SRK and SP11, which may result in the breakdown of self-incompatibility. The first candidate protein identified as the female S determinant was SLG, which is abundant on the stigma surface, but the function of SLG in self-incompatibility is unclear. The dominance relationships of the SRK alleles are different from those of the SP11 alleles, and dominance order of S haplotypes is nonlinear. Recently, methylation of the promoter region of SP11 has been reported to be responsible for transcriptional suppression of recessive SP11. Several mutations in SP11 and SRK causing self-compatibility have been identified, and other genes controlling self-incompatibility have also been reported. These molecular studies can contribute to the development of the F(1) hybrid breeding using the self-incompatibility system in Brassica crops.
ACADEMIC PRESS LTD-ELSEVIER SCIENCE LTD, 2007年, ADVANCES IN BOTANICAL RESEARCH: INCORPORATING ADVANCES IN PLANT PATHOLOGY, VOL 45, 45, 139 - 154, 英語[査読有り]
論文集(書籍)内論文
DNA methylation is essential for normal development and plays important roles in regulating gene expression in plants. Analysis of the key enzymes catalyzing DNA methylation is important to understand epigenetic phenomena. In this study, three putative methyltransferase genes, BrMET1a, BrMET1b, and BrCMT, were isolated from a genome library of Brassica rapa. Structural conservation of the amino acid sequence between BrMET1a/BrMET1b and AtMET1 and that between BrCMT and AtCMT3 suggests that they may function as DNA methyltransferase. BrMET1a was expressed in vegetative and reproductive organs, while BrMET1b was expressed only in pistils, indicating that these two genes have different functions. BrCMT was expressed especially in stamens at the stage of 2-4 days before anthesis. We isolated three DNA methyltransferase genes in Brassica rapa and indicated differences of expression patterns of these DNA methyltransferase genes and expression levels in different tissues and developmental stages, suggesting that these genes might play important roles in epigenetic gene regulation in B. rapa.
GENETICS SOC JAPAN, 2006年08月, GENES & GENETIC SYSTEMS, 81 (4), 235 - 242, 英語[査読有り]
研究論文(学術雑誌)
Mutations in the S locus of a self-compatible cultivar Yellow Sarson in Brassica rapa, which has a self-compatible class-I S haplotype, S-f2, were investigated. S-28 in Brassica oleracea was found to be a member of an interspecific pair with S-f2 in B. rapa. The original S haplotype of S-f2 was identified to be S-54 in B. rapa. Sequence comparison of alleles in S-f2 with those in S-54 and B. oleracea S-28 revealed insertion of a retrotransposon-like sequence in the first intron of SRK and 89-bp deletion in the promoter region of SP11. No transcripts of SRK and SP11 were detected in S-f2 homozygotes, suggesting that the insertion and the deletion in SRK and SP11, respectively, caused the loss of the function of these genes. Promoter assay using transgenic plants indicated that the SP11 promoter of S-f2 has no activity. Heterozygotes of S-f2 and a normal class-II S haplotype, S-60, in B. rapa were found to be self-compatible. Interestingly, transcription of SP11-60 was revealed to be suppressed in the S-f2/S-60 heterozygotes, suggesting that an untranscribed class-I SP11 allele suppresses the expression of a recessive class-II SP11 allele in the anthers of S heterozygotes. Similar phenomenon was observed in heterozygotes of a self-compatible class-I S haplotype and a self-incompatible class-II S haplotype in B. oleracea.
SPRINGER, 2006年07月, PLANT MOLECULAR BIOLOGY, 61 (4-5), 577 - 587, 英語[査読有り]
研究論文(学術雑誌)
Nuclear so-called fertility-restorer genes reverse the pollen sterility of cytoplasmic male-sterile (CMS) plants caused by disturbed mitochondrial-nuclear interactions. We identified a CMS-associated chimeric mitochondrial gene in an alloplasmic CMS line of Brassica oleracea in the 'mur' system. This novel chimeric gene, orf72, was found in the mitochondrial genome of donor cytoplasm. It was located downstream of normal rps7 and contained part of atp9 (atp9-b). It was expressed specifically on the nuclear background of CMS B. oleracea, partially suppressed in the fertility-restored line and entirely suppressed in the cytoplasmic donor.
OXFORD UNIV PRESS, 2006年04月, PLANT AND CELL PHYSIOLOGY, 47 (4), 549 - 553, 英語[査読有り]
研究論文(学術雑誌)
Self-compatible S-54 homozygotic plants were found in progenies of an F-1 hybrid cultivar in Chinese cabbage. Pollination tests revealed that this self-compatibility is controlled by the S locus and caused by the loss of the recognition function of the stigma. SRK, the gene for the recognition molecule in the stigma, was normally transcribed and translated in the self-compatible plants. The 1034-bp region in the receptor domain of SRK in the self-compatible plants was 100% identical to SLG in S-54, while that in self-incompatible S-54 homozygotic plants was 95.1% identical. These results suggest that the self-compatibility of the S-54 homozygotes is due to amino-acid changes caused by gene conversion from SLG to SRK. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
ELSEVIER SCIENCE BV, 2006年01月, FEBS LETTERS, 580 (2), 425 - 430, 英語[査読有り]
研究論文(学術雑誌)
The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, both of which are encoded in the S locus. The nucleotide sequence analyses of many SRK and SP11/SCR alleles have identified several interspecific pairs of S haplotypes having highly similar sequences between B. oleracea and B. rapa. These interspecific pairs of S haplotypes are considered to be derived from common ancestors and to have maintained the same recognition specificity after speciation. In this study, the genome structures of three interspecific pairs of S haplotypes were compared by sequencing SRK, SP11/SCR, and their flanking regions. Regions between SRK and SP11/SCR in B. oleracea were demonstrated to be much longer than those of B. rapa and several retrotransposon-like sequences were identified in the S locus in B. oleracea. Among the seven retrotransposon-like sequences, six sequences were found to belong to the ty3 gypsy group. The gag sequences of the retrotransposon-like sequences were phylogenetically different from each other. In Southern blot analysis using retrotransposon-like sequences as probes, the B. oleracea genome showed more signals than the B. rapa genome did. These findings suggest a role for the S locus and genome evolution in self-incompatible plant species. Copyright © 2006 by the Genetics Society of America.
2006年, Genetics, 173 (2), 1157 - 1167, 英語[査読有り]
研究論文(学術雑誌)
[査読有り]
研究論文(学術雑誌)
The nucleotide sequence of an 86.4-kb region that includes the SP11, SRK, and SLG genes of Brassica rapa S-60 (a class-II S haplotype) was determined. In the sequenced region, 13 putative genes were found besides SP11-60, SRK-60, and SLG-60. Five of these sequences were isolated as cDNAs, five were homologues of known genes, cDNAs, or ORFs, and three are hypothetical ORFs. Based on their nucleotide sequences, however, some of them are thought to be non-functional. Two regions of colinearity between the class-II S-60 and Brassica class-I S haplotypes were identified, i.e., S flanking region 1 which shows partial colinearity of non-genic sequences and S flanking region 2 which shows a high level of colinearity. The observed colinearity made it possible to compare the order of SP-11, SRK, and SLG genes in the S locus between the five sequenced S haplotypes. It emerged that the order of SRK and SLG in class-II S-60is the reverse of that in the four class-I S haplotypes reported so far, and the order of SP11, SRK and SLG is the opposite of that in the class-I haplotype S-910. The possible gene designated as SAN1 ( S locus Anther-expressed Non-coding RNA like-1), which is located in the region between SP11-60 and SRK-60, has features reminiscent of genes for non-coding RNAs (ncRNAs), but no homologous sequences were found in the databases. This sequence is transcribed in anthers but not in stigmas or leaves. These features of the genomic structure of S-60 are discussed with special reference to the characteristics of class-II S haplotypes.
SPRINGER-VERLAG, 2003年06月, MOLECULAR GENETICS AND GENOMICS, 269 (3), 361 - 369, 英語[査読有り]
研究論文(学術雑誌)
We have identified several interspecific pairs of S haplotypes having highly similar SRK and SP11/SCR sequences between Brassica oleracea and Brassica rapa. The recognition specificities of S haplotypes in these pairs were examined with three different methods. Stigmas of interspecific hybrids between an S-32 homozygote in B. oleracea and an S-60 homozygote in B. rapa, which were produced to avoid the interspecific incompatibility between the two species, showed incompatibility to the pollen of an S-8 homozygote in B. rapa and to the pollen of an S-15 homozygote in B. oleracea, while it showed compatibility to the pollen of other S haplotypes, suggesting B. oleracea S-32 and B. rapa S-60 have the same recognition specificity as B. rapa S-8 and B. oleracea S-15. Pollen grains of transgenic S-60 homozygous plants in B. rapa carrying a transgene of SP11-24 from B. oleracea were incompatible to B. rapa S-36 stigma, indicating that B. oleracea S-24 and B. rapa S-36 have the same recognition specificity. Application of the SP11 protein of B. rapa S-41 and S-47 onto the surface of B. oleracea S-64 stigmas and S-12 stigmas, respectively, resulted in the incompatibility reaction to pollen grains of another S haplotype, but application onto the stigmas of other S haplotypes did not, suggesting that B. oleracea S-64 stigmas and S-12 stigmas recognized the B. rapa SP11-41 and SP11-47 proteins as self SP11 proteins, respectively. Besides having evolutionary implications, finding of many interspecific pairs of S haplotypes can provide insight into the molecular mechanism of self-recognition. Comparing deduced amino-acid sequences of SP11 proteins and SRK proteins in the pairs, regions of SP11 and SRK important for self-recognition are discussed.
KLUWER ACADEMIC PUBL, 2003年06月, PLANT MOLECULAR BIOLOGY, 52 (3), 617 - 626, 英語[査読有り]
研究論文(学術雑誌)
A self-incompatibility system is used for F(1) hybrid breeding in Brassicaceae vegetables. The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen. Nucleotide sequences of SP11 alleles are more highly variable than those of SRK. We analyzed the S haplotype specificity of SP11 DNA by Southern-blot analysis and dot-blot analysis using 16 S haplotypes in Brassica oleracea, and found that DNA fragments of a mature protein region of SP11 cDNA, SP11(m), of eight S haplotypes can detect only the SP11 alleles of the same S haplotypes. This specificity makes these methods useful for S haplotype identification. Therefore, we developed two methods of dot-blot analysis for SP11. One is dot blotting of DNA samples, i.e. plant genomic DNA probed with labeled SP11(m), and the other is dot blotting of SP11(m) DNA fragments probed with labeled DNA samples, i.e. the SP11 coding region labeled by PCR using a template of plant genomic DNA. The former is useful for testing many plant materials. The latter is suitable, if there is no previous information on the S haplotypes of plant materials.
SPRINGER, 2003年05月, THEORETICAL AND APPLIED GENETICS, 106 (8), 1433 - 1437, 英語[査読有り]
研究論文(学術雑誌)
The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, respectively, In the pair of S haplotypes BrS46 (S-46 in B. rapa) and BoS(7) (S-7 in B. oleracea), which have highly similar SRK alleles, the SP11 alleles were found to be similar, with 96.1% identity in the deduced amino acid sequence. Two other pairs of S haplotypes, BrS47 and BoS(12), and BrS8 and BoS(32), having highly similar SRK and SP11 alleles between the two species were also found. The haplotypes in each pair are considered to have been derived from a single S haplotype in the ancestral species. The allotetraploid produced by interspecific hybridization between homozygotes of BrS46 and BoS(15) showed incompatibility with a BoS(7) homozygote and compatibility with other B. oleracea S haplotypes in reciprocal crossings. This result indicates that BrS46 and BoS(7) have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found in such cases in both SRK alleles and SP11 alleles do not after the recognition specificity. DNA blot analysis of SRK, SP11, SLG and other S-locus genes showed different DNA fragment sizes between the interspecific pairs of S haplotypes. A much lower level of sequence similarity was observed outside the genes of SRK and SP11 between BrS46 and BoS(7). These results suggest that the DNA sequences of the regions intervening between the S-locus genes were diversified after or at the time of speciation. This is the first report demonstrating the presence of common S haplotypes in different plant species and presenting definite evidence of the trans-specific evolution of self-incompatibility genes.
BLACKWELL PUBLISHING LTD, 2002年01月, PLANT JOURNAL, 29 (2), 215 - 223, 英語[査読有り]
研究論文(学術雑誌)
[招待有り]
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To adjust the timing of flowering, plants can sense the temperature and day length. Vernalization is a phenomenon where flowering is induced by a long period of low temperature, and has an important role in controlling flowering time. In Arabidopsis thaliana, FLOWERING LOCUS C (FLC) acts as a floral repressor, and transcription of FLC is repressed through the increase of tri-methylation of histone H3 Lysine 27 (H3K27me3) by vernalization. This transcriptional repression is caused by POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) that functions to modify the H3K27me3. Two types of long non-coding RNAs (lncRNA) from the intragenic region of FLC, COOLAIR and COLDAIR, are expressed by vernalization. Notably COLDAIR leads to an increase of H3K27me3 with PRC2 interaction. A detailed understanding of the regulatory mechanism of the FLC will provide a good model of other epigenetically regulated genes.植物は温度や日長を認識することで開花するタイミングを調節している。春化は、植物が長期間の低温に遭遇することにより花成が誘導される現象であり、開花期を制御する重要な役割を担っている。シロイヌナズナ(Arabidopsis thaliana)の開花抑制遺伝子であるFLOWEING LOCUS C(FLC) では、春化によって、転写抑制にかかわるヒストンマークであるヒストンH3の27番目のリジン残基のトリメチル化(H3K27me3)を介した転写抑制が生じる。この遺伝子サイレンシングにはH3K27me3をターゲットとするPOLYCOMB RPRESSIVE COMPLEX 2(PRC2)複合体が関与する。これは動植物で保存された転写制御メカニズムである。FLC 遺伝子座内から転写される長鎖非翻訳RNA(long non-coding RNA: lncRNA)であるCOOLAIR とCOLDAIR は、春化によって誘導される。特に、COLDAIR はPRC2と結合することで、FLC 遺伝子座内のH3K27me3レベルの増加をもたらす。FLC の制御機構が詳細に理解されれば、他のエピジェネティックな制御を受ける遺伝子のモデルになるだろう。
新潟大学農学部, 2014年03月, 新潟大学農学部研究報告, 66 (2), 105 - 110, 英語親候補となる近交系統の作成は、植物の一代雑種(F1)品種の育成過程の最初の段階である。一般的に遺伝的に均一な近交系統の育成には5、6世代以上の自殖が必要であると考えられている。DNAマーカーを利用した近交系の純度検定は信頼性が高いが、Brassica rapa L. では、純度検定に有効なDNAマーカーの報告は少ない。本研究では、F1品種の両親系統間の多型を見分けることができるDNAマーカーを見出すために、321のSSRプライマー対を試した。F2集団では、両親系統のゲノムが分離することから、両親系統の塩基配列の多型を追跡することが可能となる。材料にはハクサイの市販F1品種であるW77のF2個体を用いた。59のDNAマーカーでF2集団の個体間で多型が見られたことから、これらのDNAマーカーは純度検定に有効的であると考えられた。
新潟大学農学部, 2014年03月, 新潟大学農学部研究報告, 66 (2), 111 - 124, 英語近交系統の作成は、植物のF1品種の育成過程の最初の段階である。遺伝的に均一な近交系統の育成には5、6世代以上の自殖が必要であると考えられている。DNAマーカーを利用した近交系の純度検定は信頼性が高いが、B. oleraceaでは、純度検定に有効なDNAマーカーの報告は少ない。本研究では、F1品種の両親系統間の多型を見分けることができるDNA マーカーを見出すために、175のSSRプライマー対を試した。F2集団では、両親系統のゲノムが分離することから、両親系統の塩基配列の多型を追跡することが可能となる。そこで、材料にはキャベツの市販F1品種であるYR錦秋協力152のF2個体を用いた。12のDNAマーカーでF2集団の個体間で多型が見られたことから、これらのマーカーは純度検定に有効的であると考えられた。
新潟大学農学部, 2013年03月, 新潟大学農学部研究報告 = Bulletin of the Faculty of Agriculture, Niigata University, 65 (2), 137 - 147, 英語葯特異的遺伝子やそれらのプロモーター配列の解析は、葯発達に関わる遺伝子の同定や、遺伝子組換えによる雄性不稔植物の作出を目的として、これまで多くなされてきている。本研究では、6つのプロモーター配列の単離を行い、GUS遺伝子に連結してシロイヌナズナに導入することで、葯の発達段階における発現誘導様式を調べた。プロモーターGUSの発現は、様々な花粉発達ステージにおいて観察された。また、塩基配列の解析では葯/花粉特異的遺伝子の発現に必要とされるシス配列が多く含まれていたことから、これらのシス配列が葯特異的に発現誘導していることが示唆された。
新潟大学農学部, 2012年09月, 新潟大学農学部研究報告 = Bulletin of the Faculty of Agriculture, Niigata University, 65 (1), 31 - 37, 英語細胞質雄性不稔性(Cytoplasmic male sterility:CMS)は、ミトコンドリアゲノム上に存在する特有のorfによって引き起こされ、母性遺伝する形質である。多くの場合、核ゲノムにコードされる稔性回復遺伝子(Restorer of fertility:Rf)の存在下で、稔性が回復する。CMSとCMSの維持系統、Rf遺伝子を保持する稔性回復系統を組合せて利用する事で一代雑種育種を行う事ができるため、CMSやその稔性回復は農業上重要な現象である。これまでに同定されているRf遺伝子の多くは、ミトコンドリアに局在するpentatricopeptide repeat(PPR)タンパク質をコードしており、CMS関連ミトコンドリア遺伝子の発現を抑制している。
新潟大学農学部, 2012年03月, 新潟大学農学部研究報告 = Bulletin of the Faculty of Agriculture, Niigata University, 64 (2), 135 - 142, 日本語アブラナ科野菜の自家不和合性の強度は、F1種子の生産において、自殖種子の混入を避けるために重要な要素である。アブラナ科植物の自家不和合性は、1つの複合遺伝子座(S複合座)によって胞子体型に制御されており、Sハプロタイプ間で優劣性が見られる。しかし、自家不和合性の強度と優劣性との関連性はあまり研究されていない。本研究では、Brassica rapa(白菜)のF1品種に由来するSホモ個体を作成し、3つの独立なSハプロタイプの組合せについて自家不和合性の強度を比較した。優劣性の有無にかかわらず、2つのclass IのSハプロタイプ間では自家不和合性の強度に違いが見られなかった。しかし、花粉において優劣性があるclass Iとclass IIのSハプロタイプ間では、class II Sハプロタイプの自家不和合性の強度はclass I Sハプロタイプに比べて弱かった。これらの結果から、Sハプロタイプ間における自家不和合性の強度は優劣性とは独立していることが示唆された。
新潟大学農学部, 2012年03月, 新潟大学農学部研究報告 = Bulletin of the Faculty of Agriculture, Niigata University, 64 (2), 143 - 149, 英語DNA methylation is an important epigenetic modification regulating gene expression. In this study, we investigateddifferences of DNA methylation status between leaves, stamens, and pistils by methylation sensitive amplificationpolymorphism (MSAP) analysis in Brassica rapa. The difference of DNA methylation status was the largest between leavesand stamens/pistils. About half of the differentially methylated sequences were genic regions, but their expression levelsdid not differ between organs. The percentage of genic regions detected by MSAP analysis was higher than that of the totallength of the genic regions in the genome, suggesting that genic regions are differentially methylated between organs in B.rapa.DNA のメチル化は遺伝子の転写制御に重要なエピジェネティックな変化である。本研究では、Brassica rapa の葉、雄しべ、雌しべのDNA のメチル化レベルの違いについてMSAP 法を用いて調べた。DNA のメチル化は葉と雄しべ/ 雌しべで最も違っていた。DNA のメチル化が異なっていた配列の半分は遺伝子領域であったが、遺伝子の発現量は3つの器官で違いが見られなかった。MSAP 法で見出された領域の内、遺伝子領域が占める割合は、ゲノム中に占める遺伝子領域の割合よりも高かったことから、B. rapa において、器官特異的なDNA のメチル化の違いは遺伝子領域に起こりやすい可能性が示された。
新潟大学農学部, 2011年09月, 新潟大学農学部研究報告, 64 (1), 7 - 16, 英語アブラナ科植物の自家不和合性は、S複合座に座乗するSRK(S receptor kinase)とSP11/SCR(S locus protein 11/S locus cysteine rich)のS特異的な相互作用により決定されている。アブラナ科植物の自家不和合性は、胞子体型自家不和合性であることから、花粉の表現型は親の遺伝子型によって決定される。SP11/SCRの対立遺伝子間で優劣性が見られる場合、ヘテロ接合体の花粉では、劣性側のSP11/SCRの発現が抑制される。この劣性側のSP11/SCRの転写抑制は、優勢側のS複合座から、劣性側のSP11/SCRのプロモーター領域と相同性があるsmall RNAが発現し、その結果、劣性側のSP11/SCRのプロモーター領域にDNAのメチル化が誘導されることで起こる可能性が示唆された。
新潟大学農学部, 2011年09月, 新潟大学農学部研究報告, 64 (1), 17 - 26, 日本語遺伝子の発現情報の伝達は塩基配列に依存するジェネティックな遺伝と塩基配列によらず、遺伝子の修飾状態の変化に依存するエピジェネティックな遺伝が存在する.植物のエピジェネティクス研究は、モデル植物であるシロイヌナズナで進んでおり、特に突然変異体を用いた解析がこの分野の進展に大きく貢献している.動物のエピジェネティックな制御に関連する遺伝子の突然変異体は、致死に到ることが多いが、植物の突然変異体では致死に到らず、遺伝学的な解析が可能であることが理由として挙げられる.また、エピジェネティックな遺伝子の発現制御は、遺伝子機能・環境応答・生殖制御などとの関連性が示唆されていることからも育種において重要な研究課題である.本総説では、育種学的観点からエピジェネティックな制御機構の重要性について、トランスポゾン・生殖・環境応答の項目に分けて概説し、またエピジェネティックな現象による重要農業形質の例について最近の話題を交えて紹介する.
日本育種学会, 2010年, 育種学研究, 12 (4), 123 - 131, 日本語[査読有り]
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アブラナ科植物の多くは、近親交配を防ぐために、自己花粉の発芽・柱頭内への花粉管の侵入を特異的に阻害する機構である自家不和合性を有している。アブラナ科植物の自家不和合性は、基本的には多数の複対立遺伝子を持つ1遺伝子によって説明され、花粉と雌しべのS対立遺伝子が一致したときに、柱頭内への花粉管の侵入が阻害される。また、花粉の認識特異性が胞子体の遺伝子型によって決定されるため、胞子体型自家不和合性に分類される。自己花粉が認識されて花粉管の侵入が柱頭上で抑制されることから、柱頭と花粉に自家不和合性の自他認識反応に関与する分子が存在すると考えられた。アブラナ科植物には、主要な野菜であるキャベツ、ブロッコリー、カリフラワーやカブ、ハクサイ、ツケナ類、チンゲンサイ及び、ダイコンが含まれ、自家不和合性を利用した一代雑種品種の採種が商業的に行われている。アブラナ科植物の自家不和合性は、分子生物学的に興味深い現象であると同時に、育種学的にも重要な研究課題である。
日本育種学会, 2008年03月, 育種学研究, 10 (1), 1 - 9, 日本語記事・総説・解説・論説等(その他)
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