{"created":"2023-10-18T04:06:30.323797+00:00","id":2000050,"links":{},"metadata":{"_buckets":{"deposit":"a80b0ace-d1e8-4607-9dfc-086ac9b9ab1a"},"_deposit":{"created_by":12,"id":"2000050","owner":"12","owners":[12],"pid":{"revision_id":0,"type":"depid","value":"2000050"},"status":"published"},"_oai":{"id":"oai:obihiro.repo.nii.ac.jp:02000050","sets":["6:8"]},"author_link":[],"control_number":"2000050","item_3_alternative_title_1":{"attribute_name":"その他（別言語等）のタイトル","attribute_value_mlt":[{"subitem_alternative_title":"Studies on resistance to bacterial wilt caused by the Ralstonia solanacearum species complex in potato","subitem_alternative_title_language":"ja"}]},"item_3_date_granted_12":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2023-09-29"}]},"item_3_degree_grantor_10":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_language":"ja","subitem_degreegrantor_name":"帯広畜産大学"}],"subitem_degreegrantor_identifier":[{"subitem_degreegrantor_identifier_name":"10105","subitem_degreegrantor_identifier_scheme":"kakenhi"}]}]},"item_3_degree_name_9":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（農学）","subitem_degreename_language":"ja"},{"subitem_degreename":"Doctor of Agriculture","subitem_degreename_language":"en"}]},"item_3_description_11":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_description":"2023","subitem_description_language":"ja","subitem_description_type":"Other"}]},"item_3_description_18":{"attribute_name":"フォーマット","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_language":"en","subitem_description_type":"Other"}]},"item_3_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"ナス科ナス属であるバレイショ類は栽培種7種と野生種226種に分類される多様な遺伝的背景を持ち，このうち現在世界中で栽培されているのはSolanum tuberosum subsp. tuberosumである。このように多様な遺伝的背景を持つにも関わらず，日本を含めた欧米のバレイショ育種における遺伝子プールの脆弱性が指摘されている。一方で，バレイショは世界中の多様な環境で栽培されており，近縁野生種を育種に利用することで多様な形質を栽培種に導入し各地域の気候や病虫害に対応してきた。しかし，地球温暖化による気候変動が進行するにつれ，バレイショ栽培においてもいくつかの問題が生じることが予測される。その一つとして高温で多発する青枯病による被害がある。これまで，日本を含めた世界各国で青枯病抵抗性育種は行われてきたが，バレイショが4倍体であること，青枯病抵抗性が量的形質遺伝子座(Quantitative trait locus, QTL) により支配されていること，そして青枯病の原因細菌が種複合体(Ralstonia solanacearum species complex, RSSC)でありバレイショヘの病原力が多様であることから，普及性の高い抵抗性品種の育成が難しかった。国内における抵抗性個体の選抜はRSSCに汚染された圃場を利用した生物検定に頼っており，青枯病の発病には環境要因が大きく影響を及ぼすため，抵抗性評価に最低3年は必要となっている点が抵抗性育種の大きな制限要因となっている。このような問題に対応するためには，抵抗性検定方法の改善，RSSCの病原力および抵抗性に対する理解，そして育種における効率的選抜を可能にするDNAマーカーの開発が必要である。\n第1章では，バレイショの青枯病抵抗性に関するin vitro検定法を開発するため，抵抗性系統‘西海35号’および罹病性品種‘Kennebec’をin vitro条件下で培養し，R. pseudosolanacearumのphylotype I/biovar 4の菌株を接種株として両系統の発病度を明瞭に識別できる条件を検討した。その結果，最適な検定条件は，6～8葉期の培養植物に対し，接種する菌濃度は10 2 CFu ml -1，接種後の培養温度は28°Cであった。発病程度の評価部位については，葉よりも茎を評価対象とした方の信頼性が高かった。この方法を用いて，圃場での抵抗性程度が異なる9つの品種を評価した。その結果，圃場での抵抗性が高い品種ほど発病指数が低く，本検定法は制御された環境での青枯病抵抗性の評価に有効であると考えられた。\n第2章では，RSSCの病原力を明らかにするため，国内で採取されたバレイショに被害を及ぼす26菌株(phylotype IおよびIV)について，接種後の培養温度を24°Cないし28°Cとし，抵抗性系統‘西海35号’および罹病性品種‘アイユタカ’に対する発病度をin vitro検定法で評価した。多くの菌株で，‘西海35号’の発病指数が‘アイユタカ’より有意に低かった。病原力は菌株によっても異なり，また接種後の温度によっても大きく異なっていた。また，それぞれの菌株の品種および培養温度に対する発病度を基に，階層および非階層クラスター分析を行ったところ，26菌株は5つの病原力型（病原型A, B, C, D, およびE)に分類された。これはphylotypeを含むこれまでの分類方法とは一致しなかった。したがって，対象地域で安定した抵抗性品種を育成するためには，その地域の主要病原型菌株に対する選抜を行う必要がある。\n第3章では，バレイショにおける青枯病に対する抵抗性のQTL解析を行った。抵抗性2 倍体系統‘10-03-30’(RP) と罹病性2倍体系統‘F1-1’(SP)を交配し，94系統からなる凡集団を育成した。この集団をtwo-way pseudotestcrossとみなし，一塩基多型(Single nucleotide polymorphism, SNP) マーカーを用いて，RPに対しては1,476 SNPsが422座にマップされ，SPに対しては2,663 SNPsが475座にマップされた高密度連鎖地図を構築した。F1集団の抵抗性評価は，R.pseudosolanacearum (phylotype I/biovar 4/race 1/病原型A) を用いたin vitro 検定法により行われた。バレイショの第1, 3, 7, 10,および11番染色体上に5つのQTLs(qBWR-1~-5) が同定され，各QTLの寄与率は9.3~18.4％を示した。抵抗性親はqBWR-2, qBWR-3, およびqBWR-4に抵抗性アレルを持ち，qBWR-1とqBWR-5に罹病性アレルを持っていた。5つのQTLsに抵抗性アレルが集積されることで，抵抗性親と比較して抵抗性程度が向上した。また，第1, 7, および11番染色体におけるQTLsの相互作用による抵抗t生程度の向上効果も確認された。本研究は，ゲノムワイドマーカーを用いてバレイショの青枯病抵抗性に関する新規QTLを同定した初めての研究である。\n第4章では，第3章の結果を拡張するため，同じ2倍体凡集団およびSNPマーカーによるRPとSPの連鎖地図を用いた。In vitro 検定を用いて3種の異なる菌株(R.pseudosolanacearum のphylotype I/biovar 4/病原型A株とphylotype I/biovar 3/病原型C株，およびR.syzygiiのphylotype IV/biovar N2/病原型A株）を接種し，24°cまたは28°Cで培養した後抵抗性を評価した。その結果， 第1, 3, 5, 6, 7, 10, および11番染色体上に5つの主働QTLを含む合計10 QTLsを同定した。主働QTLであるPBWR-3 とPBWR-7 はR.pseudosolanacearum (phylotype I) とR.syzygii (phylotype IV) に対して安定した抵抗性を示したが，同じく主働QTLであるPBWR-6b はR.pseudosolanacearum (phylotype I/biovar 3) に対する菌株特異性を示し，低温でより高い抵抗性を示した。したがって，広範な抵抗性を示すQTL と菌株特異的抵抗性QTLを組み合わせることで，有効な青枯病抵抗性品種を開発できることが示唆された。\n第5章では，前章において同定された菌株特異性を示し，低温でより高い抵抗性を示すQTLであるPBWR-6bのマーカー開発を行った。QTL領域内に位置する候補遺伝子の1つNucellin-like aspartic protease遺伝子の塩基配列を，RPとSPで比較した。その結果，RPの抵抗性アレルに固有の配列を持つAl アレルを同定し，これを特異的に検出する2つの分子マーカー (Rbw6-1とRbw6-2) を開発した。マーカーを用いてRPの系譜を辿ったところ，青枯病抵抗性QTLであるPBWR-6bは，‘インカのめざめ’の交配親であるS.tuberosum subsp. andigena に由来すると推定された。また，107のバレイショ品種系統を調査したところ，‘インカのめざめ’の後代系統でのみRbw6-1 とRbw6-2 が検出されその多くで抵抗性が確認された。2倍体および4倍体の交配集団においてマーカーの有無と抵抗性の程度は一致し，PBWR-6b は顕性遺伝により後代に伝わることが明らかとなった。さらに，Rbw6-2 マーカーを既存のマルチプレックスポリメラーゼ連鎖反応(PCR)法に組み込み，青枯病(PBWR-6b) ，ジャガイモシストセンチュウ(Hl)，ジャガイモY ウイルス(Rychc) ，ジャガイモXウイルス(Rxl) ，および疫病(Rl， 品種‘さやあかね’由来のR2) に対する抵抗性遺伝子が同時検出できるようになったため，青枯病抵抗性系統の迅速な選抜が可能となった。\n以上述べたように，本研究では，新たな青枯病抵抗性の評価方法が開発され，より効率的に抵抗性育種を進めることが可能となった。加えて，国内の青枯病菌が5つの病原力型に分類されたことから，今後は，これら病原力型別に抵抗性を評価することで，国内の幅広い地域で安定した抵抗性を示す品種育成が可能になると考えられる。また，青枯病抵抗性の主働QTLを同定し，その一つであるPBWR-6bの抵抗性アレルの有無を判定するDNAマーカーを開発した。このマーカーを従来のマルチプレックスPCRに組み込んだことで，主要な6つの病虫害抵抗性遺伝子の迅速な選抜技術を開発した。これらの得られた知見と開発した技術は，バレイショにおける青枯病抵抗性育種の効率化に大いに寄与するものと期待される。","subitem_description_language":"ja","subitem_description_type":"Abstract"},{"subitem_description":"Potato and its relatives are classified into 7 cultivated and 226 wild species in the Solanaceae family. Among them, Solanum tuberosum subsp. tuberosum is only species currently cultivated worldwide. The fragility of the genetic diversity is concerned for the gene pools in Western and Japanese potato breeding programs, despite its diverse genetic variability existing in closely related wild species. On the other hand, to grow potatoes under diverse environments, useful traits such as tolerance to biotic and abiotic stresses have been introduced from primitive cultivated and wild potato species. However, climatic changes due to global warming are progressing, which make challenges to continue stable and safe supply of potatoes. One of the significant challenges is against increasing damages caused by bacterial wilt, which occur frequently at high temperatures. However, potatoes are mainly tetraploid, resistance to bacterial wilt is controlled quantitatively, bacterial wilt is caused by a complicated species complex (the Ralstonia solanacearum species complex, RSSC), and the virulence of RSSC varies widely depending on strains and potato varieties, altogether making it difficult to rapidly breed resistant cultivars. Since selection of resistant genotypes to bacterial wilt is conducted in the heavily infested field and affected largely by environmental conditions, the resistance assay takes at least three years in Japan, which is considered a major limiting factor for resistance breeding. To overcome these problems, it is desired to develop a simple and reliable resistance assay method, to understand virulence types of the various RSSC strains and resistance types against different strains, and to develop DNA markers for rapid selection of resistant genotypes.\nIn Chapter 1, to develop an in vitro assay for resistance to bacterial wilt of potatoes, the resistant breeding line 'Saikai 35' and the susceptible variety 'Kennebec' were cultured under in vitro conditions. The inoculation conditions were examined by culturing the phylotype I/biovar 4 strain of R. pseudosolanacearum as inoculum. The optimal conditions were; inoculating plants at the 6-8 leaf stage at a bacterial concentration of 10 2 CFU ml -1 and an incubation temperature of 28 °C. Stems were more reliable as the site for evaluation of disease severity than leaves. This method evaluated nine cultivars with different degrees of resistance in the field. The results showed that varieties with higher resistance in the field had lower disease indices, suggesting that this test method is effective for evaluating bacterial wilt resistance in a controlled environment.\nIn Chapter 2, virulence of various RSSC strains was investigated. Twentysix strains (phylotypes I and IV) of RSSC collected in Japanese potato fields were evaluated by in vitro assays for virulence against the resistant line 'Saikai 35' and the susceptible variety 'Aiyutaka'. The effect of environmental temperature on the virulence of RSSC strains was very diverse, and virulence varied greatly among strains. Hierarchical and non-hierarchical cluster analyses classified the 26 strains into five virulence types (pathotypes A~E), inconsistent with previous classification methods, including phylotypes. Therefore, selection for breeding stable resistant varieties in the target region should be based on virulence types.\nIn Chapter 3, QTL analysis was performed for resistance to potato bacterial wilt. The resistant diploid line'10-03-30'was crossed with the susceptible diploid line'F1-1'to generate an F1 population of 94 genotypes. Resistance evaluation of the F1 population was performed using an inoculation strain of R.pseudosolanacearum (phylotype I/biovar 4/race 1/pathotype A) in an in vitro assay. Five QTLs (qBWR-1 to qBWR-5) were identified on potato chromosomes 1, 3, 7, 10, and 11, with each QTL contributing 9.3 to 18.4%. The resistant parent had resistance alleles at qBWR-2, qBWR-3, and qBWR-4 and diseased alleles at qBWR-1 and qBWR-5. The accumulation of resistance alleles at all five QTLs resulted in increased resistance compared to the resistant parent. The effect of interaction among QTLs at chromosomes 1, 7, and 11 on the degree of resistance was also observed. This is the first study to identify novel QTLs for resistance to bacterial wilt in potatoes using genome-wide markers.\nIn Chapter 4, a further expansion of the QTL analysis performed in Chapter 3 was conducted using the same F1 population and the linkage maps with different RSSC strains and temperature conditions. An in vitro assay was used to inoculate different strains and species (phylotype I/biovar 3, phylotype I/biovar 4, and phylotype IV /biovar N2) and evaluate resistance under controlled conditions at 24 or 28°C. Ten QTLs were identified, including five major QTLs on chromosomes 1, 3, 5, 6, 7, 10, and 11. PBWR-3 and PBWR-7, the major QTLs, showed stable resistance to R.pseudosolanacearum (phylotype I) and R. syzygii (phylotype IV), whereas PBWR-6b, also a major QTL, showed strain-specific and more effective properties at low temperatures against R.pseudosolanacearum (phylotype I/biovar 3). Therefore, it is suggested that effective bacterial wilt resistant cultivars can be developed by combining broad resistance QTL and strain-specific resistance QTL.\nIn Chapter 5, to develop DNA markers for PBWR-6b, the nucleotide sequence of one of the candidate genes located within the QTL region of PBWR-6b was compared between the parents used for QTL analysis. The resistance allele was identified, and resistance allele-specific molecular markers Rbw6-1 and Rbw6-2 were developed for PBWR-6b. PBWR-6b is thought to be derived from S. tuberosum subsp. andigena, the hybrid parent of 'Inca-no-mezame'. Both markers were detected only in the 'Inca-no-mezame' progeny among 107 potato cultivar and breeding lines. The developed resistance allele-specific DNA markers demonstrated that it is possible to select resistant individuals from diploid and tetraploid populations and inferred that the mode of inheritance of PBWR-6b is dominance. Furthermore, Rbw6-2 was incorporated into an existing multiplex polymerase chain reaction (PCR) method to select for resistance to bacterial wilt (PBWR-6b), golden cyst nematode (Hl), Potato virus Y (RYchc), Potato virus X (Rxl), and late blight (R1 and 'Saya-akane' -derived R2). This method is expected to improve the efficiency of breeding for resistance to bacterial wilt in potato.\nIn conclusion, the in vitro assay method developed in this study is reliable and enable to evaluate bacterial wilt resistance more efficiently. Since the RSSC strains in Japan were classified into five virulence types, varieties with stable resistance in a wide range of regions in Japan could be bred by considering these virulence types. Major resistance QTLs were identified, which made possible to develop DNA markers. One of markers diagnostic to a major resistance QTL, PBWR-6b, was incorporated into the existing multiplex PCR, which enabled to simultaneously detect six major disease and pest resistance genes. Based on these findings and the developed technologies, it is expected to breed more efficiently bacterial wilt resistant varieties.","subitem_description_language":"en","subitem_description_type":"Abstract"}]},"item_3_description_8":{"attribute_name":"内容記述","attribute_value_mlt":[{"subitem_description":"博士学位論文","subitem_description_language":"ja","subitem_description_type":"Other"},{"subitem_description":"大学院畜産学研究科","subitem_description_language":"ja","subitem_description_type":"Other"}]},"item_3_dissertation_number_13":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"乙第15号"}]},"item_3_rights_14":{"attribute_name":"権利","attribute_value_mlt":[{"subitem_rights":"波部　一平","subitem_rights_language":"ja"}]},"item_3_subject_20":{"attribute_name":"日本十進分類法","attribute_value_mlt":[{"subitem_subject":"615","subitem_subject_language":"ja","subitem_subject_scheme":"NDC"}]},"item_3_version_type_19":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_970fb48d4fbd8a85","subitem_version_type":"VoR"}]},"item_access_right":{"attribute_name":"アクセス権","attribute_value_mlt":[{"subitem_access_right":"open access","subitem_access_right_uri":"http://purl.org/coar/access_right/c_abf2"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"波部, 一平","creatorNameLang":"ja"},{"creatorName":"HABE, Ippei","creatorNameLang":"en"}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_access","date":[{"dateType":"Available","dateValue":"2023-10-18"}],"filename":"Habe.pdf","filesize":[{"value":"14.7 MB"}],"format":"application/pdf","mimetype":"application/pdf","url":{"label":"全文　Full text","objectType":"fulltext","url":"https://obihiro.repo.nii.ac.jp/record/2000050/files/Habe.pdf"},"version_id":"39c16402-78c3-45b0-be31-1eec151b74d6"},{"accessrole":"open_access","date":[{"dateType":"Available","dateValue":"2023-10-18"}],"filename":"Abstract(Habe).pdf","filesize":[{"value":"174 KB"}],"format":"application/pdf","mimetype":"application/pdf","url":{"label":"全文の要旨　Abstract","objectType":"abstract","url":"https://obihiro.repo.nii.ac.jp/record/2000050/files/Abstract(Habe).pdf"},"version_id":"94047dbf-69ec-4001-bf25-ba281a0677f8"},{"accessrole":"open_access","date":[{"dateType":"Available","dateValue":"2023-10-18"}],"filename":"Evaluation(Habe).pdf","filesize":[{"value":"295 KB"}],"format":"application/pdf","mimetype":"application/pdf","url":{"label":"審査の要旨　Evaluation","objectType":"other","url":"https://obihiro.repo.nii.ac.jp/record/2000050/files/Evaluation(Habe).pdf"},"version_id":"c969d3c9-0a10-43a8-806b-da342e2914ba"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"doctoral thesis","resourceuri":"http://purl.org/coar/resource_type/c_db06"}]},"item_title":"バレイショにおける青枯病抵抗性に関する研究","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"バレイショにおける青枯病抵抗性に関する研究","subitem_title_language":"ja"}]},"item_type_id":"3","owner":"12","path":["8"],"pubdate":{"attribute_name":"PubDate","attribute_value":"2023-10-18"},"publish_date":"2023-10-18","publish_status":"0","recid":"2000050","relation_version_is_last":true,"title":["バレイショにおける青枯病抵抗性に関する研究"],"weko_creator_id":"12","weko_shared_id":-1},"updated":"2023-10-30T00:18:09.658040+00:00"}