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農学部 農学部門動植物資源生命科学領域 |
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論文 【 表示 ／ 非表示 】
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Fujiwara Y., Nishiyama S., Kusumi A., Okamoto-Furuta K., Yamane H., Yonemori K., Tao R.
Plant Physiology 200 ( 1 ) 2026年1月
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Plant Physiology
<jats:p>Spatial gene expression and 2D/3D ultrastructural analyses reveal a unique intercellular sequestration mechanism underlying massive proanthocyanidin accumulation in persimmon fruit.</jats:p>
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Fujiwara Y., Nishiyama S., Onoue N., Kono A., Sato A., Yonemori K., Tao R.
Horticulture Journal 94 ( 2 ) 148 - 161 2025年
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Horticulture Journal
Persimmon fruit synthesizes and accumulates proanthocyanidins (PAs), resulting in its astringent taste. The pollination-constant and non-astringent (PCNA) trait is conferred by recessive alleles at the ASTRINGENCY (AST) locus, which has been exploited to control PA accumulation of persimmon fruit. However, some individuals with recessive homozygosity at the AST marker locus in breeding populations produce mature fruit with astringency, thereby reducing the marker-selection efficiency for the PCNA trait. In this study, we genetically and physiologically characterized the predicted ast-homozygous individuals exhibiting strong astringency in a cross population of ‘Taigetsu’ (non-PCNA) and ‘Kanshu’ (PCNA). The fruit PA content of these astringent individuals was higher than that of the other PCNA fruit, but lower than that of the nonPCNA fruit. These astringent individuals were similar to the other PCNA plants in terms of tannin cell size. Gene expression analysis revealed that PA synthesis-related genes (e.g., DkMYB4, DkMYC1, and DkANR) were expressed at a higher level in the astringent individuals than in the other PCNA fruit during the early fruit development stage. Furthermore, in the presumed ast-homozygous individuals with various PA contents, the expression of cellular function-related genes was highly correlated with PA accumulation. These genes may be important for tannin cell development and PA synthesis, potentially offering a novel approach to further modulate astringency in persimmons.
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Efficient Transient Expression for Functional Analysis in Fruit Using the Tsukuba System Vector
Omori M., Fujiwara Y., Yamane H., Miura K., Tao R.
Horticulture Journal 92 ( 3 ) 261 - 268 2023年
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Horticulture Journal
Evaluating the function of genes expressed in fruit tissues of fruit tree species using a genetic transformation approach is a long process because the trees are generally recalcitrant to genetic transformation and cannot bear fruit during their long juvenile phases. Transient gene expression in fruit enables the functional analysis of genes associated with fruit traits, which may accelerate the study of fruit physiology. Here, by using the recently developed “Tsukuba system”, we successfully established an efficient transient expression system in harvested fruit tissues. The “Tsukuba system” utilizes a combination of the geminiviral replication system and a double terminator, which ensures sufficient levels of transgene expression. We used blueberry fruit as a model to characterize the applicability of this system for transient expression in fruit tissue. The pTKB3-EGFP vector was introduced by agroinfiltration into the fruit tissues of several blueberry cultivars. We found that transient GFP fluorescence in fruit peaked 4–6 days after agroinfiltration. Agrobacterium suspensions were easily injected into soft, mature fruit, and GFP was strongly expressed; however, hard, immature fruit were not penetrable by Agrobacterium suspensions, and GFP was rarely detected. We then tested the applicability of the developed system to other fruit tree species: six families, 17 species, and 26 cultivars. GFP fluorescence was detected in all species, except for Japanese apricot. In blueberry, bilberry, sweet cherry, apricot, and satsuma mandarin, GFP was highly expressed and observed in a large proportion of the flesh. In kiwifruit, hardy kiwifruits, persimmon, peach, apple, European pear, and grape, GFP fluorescence was limited to certain parts of the fruits. Finally, transient VcMYBA1 overexpression in blueberry was tested as a model for gene functional analysis in fruit. Transient VcMYBA1 overexpression induced red pigmentation in the flesh, suggesting that VcMYBA1 expression caused anthocyanin accumulation. This study provides a technical basis for the rapid evaluation of genes expressed in fruit, which will be useful for gene function evaluation studies in fruit crops with long juvenile phases.
DOI： 10.2503/hortj.QH-062
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Fujiwara Y., Nishiyama S., Onoue N., Matsuzaki R., Yonemori K., Tao R.
Acta Horticulturae 1338 ( 1338 ) 269 - 275 2022年4月
担当区分：筆頭著者 掲載種別：研究論文（学術雑誌） 出版者・発行元：Acta Horticulturae
The astringent taste caused by proanthocyanidins (PAs), which still remains at fruit maturation or harvesting stage, is a unique characteristic of persimmon. Pollination-constant and non-astringent (PCNA) type cultivars stop accumulating PAs in the early stage of fruit development, allowing the fruit to be edible without artificial de-astringency treatment. The PCNA trait is conferred by a recessive allele at a single locus, ASTRINGENCY (AST). The AST-locating genomic region (AST region) has been delimited in the polyploid D. kaki genome by comparative mapping, but the AST gene has yet to be identified. Here, we characterized a genomic region of diploid D. oleifera that corresponds to the AST region in D. kaki. Firstly, we obtained mRNA-seq data from siblings from a cross between 'Taiten' (non-PCNA) and 'Kanshu' (PCNA) and their parents at two fruit developmental stages in 2015 and at five stages in 2016. The genetic analysis concluded that there is no nonsynonymous substitution in the genic regions in the AST region. Differential expression analysis between PCNA and non-PCNA detected two differentially expressed genes (FDR <0.05) in the region, whereas their expression patterns were not correlated with PA accumulation during fruit development. We also sequenced small RNA from the same population as the differential expression analysis. Small RNAs were annotated in the D. oleifera genome, and 6 small RNAs in the AST region with significantly different normalized counts (p<0.05) between PCNA and non-PCNA were identified. These small RNAs were found to target 5 genes, whereas there were no effects in their mRNA expression levels. In conclusion, this study identified none of the promising AST candidates with clear difference between the astringent and non-astringent alleles. More detail information of polyploid D. kaki genome is required to obtain comprehensive set of candidate genes.
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メタボローム解析によるカキ甘渋分離後代における果実内代謝物の比較 査読あり
浅草遼太郎, 藤原陽介, 西山総一郎, 尾上典之, 河野淳, 山根久代, 田尾龍太郎
園芸学研究 別冊 24 ( 2 ) 2025年
講演・口頭発表等 【 表示 ／ 非表示 】
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Identification of a single candidate gene responsible for fruit astringency type in hexaploid persimmon through a forward-genetic approach
Yosuke Fujiwara, Soichiro Nishiyama, Noriyuki Onoue, Ryusuke Matsuzaki, Hisayo Yamane, Keizo Yonemori, Ryutaro Tao
International Horticulture Conference (IHC) 2026 2026年8月
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カキのastホモ接合個体 における渋味果のプロアントシアニジン分析
藤原陽介, 西山総一郎, 浅草遼太郎, 尾上典之, 松﨑隆介, 山根久代, 宮下正弘, 田尾龍太郎
園芸学会令和八年度春季大会 2026年3月
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完全甘ガキ形質決定遺伝子候補の機能解析
藤原陽介, 西山総一郎, 山根久代, 田尾龍太郎
園芸学会令和七年度秋季大会 2025年9月
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プロアントシアニジン蓄積制御遺伝子の迅速な機能解析に向けたモデル評価系の予備検討
藤原陽介, 浅草遼太郎, 西山総一郎, 田尾龍太郎
園芸学会令和七年度春季大会 2025年3月
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Haplotype-Phased Assembly and Polymorphism Analysis of the Delimited ASTRINGENCY Locus in Hexaploid Persimmon
Fujiwara, Y, S. Nishiyama, R. Matsuzaki, N. Onoue, R. Tao
Plant and Animal Genome Conference / PAG32 2025年1月
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Young Minds Award for best oral presentation in IHC2026
2026年8月 International Society of Horticulture Science
Yosuke Fujiwara, Soichiro Nishiyama, Noriyuki Onoue, Ryusuke Matsuzaki, Hisayo Yamane, Keizo Yonemori, Ryutaro Tao
受賞区分：国内外の国際的学術賞
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優秀発表賞 ポスター発表部門
2024年11月 園芸学会令和六年度秋季大会
藤原陽介・西山総一郎・尾上典之・松﨑隆介・河野淳・佐藤明彦・米森敬三・田尾龍太郎
受賞区分：国内外の国際的学術賞
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AHC2023 Young Minds Award for best oral presentation
2023年8月 IV Asian Horticultural Congress
Yosuke Fujiwara, Soichiro Nishiyama, Noriyuki Onoue, Keizo Yonemori, Ryutaro Tao
受賞区分：国内外の国際的学術賞
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ISHS Young Minds Award for best poster presentation
2021年9月 7th International Symposium on Persimmon
Yosuke Fujiwara, Soichiro Nishiyama, Noriyuki Onoue, Ryusuke Matsuzaki, Keizo Yonemori, Ryutaro Tao
受賞区分：国内外の国際的学術賞
科研費（文科省・学振・厚労省）獲得実績 【 表示 ／ 非表示 】
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カキ果実におけるプロアントシアニジン蓄積の分子制御機構の解明
研究課題/領域番号：23KJ1313 2023年04月 - 2026年03月
日本学術振興会 科学研究費助成事業 特別研究員奨励費
藤原 陽介
担当区分：研究代表者
カキは果実中に渋味成分であるプロアントシアニジン（PA）を多量に蓄積する。カキ品種はPA蓄積パターンにより完全甘ガキ（PCNA）とnon-PCNAに分類され、日本タイプのPCNAは単一遺伝子座ASTRINGENCY（AST）の潜性アレル（ast）によって賦与される。本研究では、カキ育種への応用を見据え、ASTをはじめとしたカキPA蓄積制御のメカニズム解明を目指している。本年度は、昨年度までに同定したAST候補遺伝子について、astアレルが有する変異様式の調査を行った。また、育種集団で見られる渋味形質を持つ個体について、AST遺伝子座に着目した遺伝解析を行った。
第一の実験では、発達中期のカキ果実で発現するAST候補遺伝子の配列を解析した。150に及ぶ網羅的な品種・系統群で調査したところ、PCNAでは機能型配列の割合は5%以下だった。一方、他のnon-PCNAでは機能型配列の割合は20%以上であった。同様の解析を甘渋性分離後代で行ったところ、同じ傾向が認められた。以上から、AST候補遺伝子における機能型転写産物の割合は、甘渋性の分類と完全に一致していた。また、構築した六倍体ゲノムのハプロタイプ別配列の比較から、変異の原因多型が複数種類存在する可能性を明らかにした。
第二の実験では、 ‘太月’ב甘秋’の後代のうち、強い渋味を示す2個体についてDNA-seqデータを用いた詳細なジェノタイピングを行ったところ、これらはAST遺伝子座においてPCNA遺伝子型を有することが示された。また、果実PA含量は他のPCNAとnon-PCNAの中間であった。したがって、PCNA個体においても、AST以外の遺伝子座の効果によってPA蓄積形質が変化することが示された。今後は、PCNA×PCNAの集団内で遺伝解析を行い、PCNAの遺伝的背景においてPA制御因子を調査する必要がある。
