JP2022516735A - トマトブラウンルーゴスフルーツウイルスに抵抗性のトマト植物 - Google Patents
トマトブラウンルーゴスフルーツウイルスに抵抗性のトマト植物 Download PDFInfo
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Abstract
Description
a)トバモウイルスに抵抗性であるS.ハブロカイテス植物を選択するステップであり、本発明の抵抗性遺伝子、ゲノム配列又は抵抗性遺伝子座の存在を確認するステップを含むステップと、
b)ステップa)の同定されたゲノム配列又は遺伝子座をS.リコペルシクム植物中に移入し、それによって前記S.リコペルシクム植物にトバモウイルス抵抗性を付与するステップとを含む、方法に関する。移入は、選択されたS.ハブロカイテス植物をS.リコペルシクムと交雑することによって行われ得る。その後、トバモウイルス抵抗性のS.リコペルシクム植物が選択され得る。
TBRFV分離株AE050(サウジアラビア起源)を使用して、病害アッセイを実行した。植物材料としてTBRFVに感染性の植物株である株OT9を使用して、ウイルスを維持した。株OT9に葉液を機械的に接種するために元サンプルから入手した病徴のある葉を使用した。ウイルスを、新たな3週齢の苗に対する毎月の葉液の機械的接種によって全身的に感染させたトマト植物OT9上で維持した。
感染をELISAによって決定した。全ての植物から頂部葉(完全に広がっている)1枚を採集した。葉を、R302 D63N-472型機械(VECTOR aandrijftechniek B.V.、Rotterdam、オランダ)を使用して破砕し、葉液を、PBS-Tween緩衝液2mLを添加することによって採集した。抽出液100μLを、ToMVに対する抗体(供給元Prime Diagnostics、Wageningen、オランダ)と共にELISAに使用した。ELISAの読み取りを、FLUOstar Galaxy装置で405nmの吸収を測定することによって行った。純系の対照植物の1.5倍以上の吸収値を示す植物は、感染している(感染性)と考えられた。
元のLYC4943(S.ハブロカイテス)種子ロットを、抵抗性について分離する。9つの異なるF1ファミリーを、バイオアッセイ用に播種して、完全に抵抗性であり(抵抗性が固定されている)、さらなる戻し交配に使用されることになるF1ファミリーを同定した。4つのF1ファミリーが発芽し、バイオアッセイにおいて試験された。LYC4943植物3(90479-3)で創出されたF1植物を抵抗性表現型について選択して、戻し交配株としてS.リコペルシクム株OT9及びOT1317を選択してマッピング用として2つの集団を創出した。マッピングに使用されたマーカーM1~M42(それぞれ、配列番号19~配列番号102)を表1に挙げる。
TBRFV抵抗性遺伝子座(配列番号1)を含む本発明のトマト植物(S.リコペルシクム)を、Tm0、1及び2系統に対して抵抗性について試験した。TBRFV抵抗性遺伝子座の存在を、マーカーM16、M17及びM33によって決定した。植物が、Tm22遺伝子(Tm0、1及び2系統に対する抵抗性をもたらす公知の遺伝子である)を含有しないことを、さらに確認した。一部の場合において、植物は、Tm1抵抗性遺伝子を含有した。対照として、TBRFV抵抗性遺伝子座を含有しない植物を選択した。
TBRFV抵抗性遺伝子座を含有した植物は全て抵抗性である。植物7及び8は、TBRFV抵抗性遺伝子座を含有しないが、抵抗性であった。その結果を説明し得る理由は、Tm1遺伝子が、ToMV分離株Tm-0に対する抵抗性の原因であるということである。加えて、植物1、2及び3は、Tm1遺伝子を含有しないが、TBRFV抵抗性遺伝子座を含有し、抵抗性であることを示した。
抵抗性表現型は、TBRFV遺伝子型と連鎖しており、ToMV分離株Tm-1に対する抵抗性をもたらす。
抵抗性表現型(ヘテロ接合性、ホモ接合性)は、TBRFV遺伝子型と連鎖しており、ToMV分離株Tm-2に対する抵抗性をもたらす。
TBRFV抵抗性遺伝子座(ヘテロ接合性又はホモ接合性)を含むトマト植物及びこの領域を含有しない植物を、マーカー(M16及びM17)を使用してTBRFVバイオアッセイのために選択した。植物をTBRFVに感染させ、感染性トマト株OT9(感染させていない及び感染させたOT9)を対照として含めた。
2018年4月27日にNature、Protocol Exchange(2018年)、Rachael Workmanら、「High Molecular Weight DNA Extraction from Recalcitrant Plant Species for Third Generation Sequencing」において公開されたプロトコールに従って、ゲノムDNAを、本発明の抵抗性植物(S.リコペルシクム)、即ちTBRFV抵抗性遺伝子座を含む植物から単離した。配列決定ライブラリを、PCR不要の、非多重化、DNA Ligation Sequencing Kit-Promethion(SQK-LSK109)を使用して調製した。単離手順の結果、配列決定のためにOxford Nanoporeシステム(ONT配列決定)で使用される高品質配列決定ライブラリを得た。Promethion Flowcell Packs(3000個細孔/フローセル)バージョンR9.4.1.を配列決定に使用した。
TBRFV抵抗性遺伝子(配列番号115)が実際にTBRFVに対する抵抗性を付与する遺伝子であることを確認するために、ウイルス誘導遺伝子サイレンシング(VIGS)分析を実行した。タバコ茎壊疽ウイルス(TRV)由来のVIGSベクターが、シロイヌナズナ(Arabidopsis thaliana)、ニコチアナベンサミアナ(Nicotiana benthamiana)、リコペルシコンエスクレンタム(Lycopersicon esculentum)などの植物及び他の植物において遺伝子機能を研究するために多く記載されている[例えばHuang C、Qian Y、Li Z、Zhou X.:Virus-induced gene silencing and its application in plant functional genomics.Sci China Life Sci.2012年;55(2):99~108頁を参照のこと]。
Claims (20)
- トバモウイルスに抵抗性のS.リコペルシクム種の植物であって、TBRFV抵抗性タンパク質をコードするTBRFV抵抗性遺伝子を含み、前記タンパク質が配列番号116と少なくとも90%のアミノ酸配列同一性を有する、植物。
- 前記TBRFV抵抗性遺伝子が、配列番号115と少なくとも90%のヌクレオチド配列同一性を有するコード配列を含む、請求項1に記載の植物。
- 配列番号1、配列番号2、配列番号3、配列番号4、配列番号5、配列番号6、配列番号7、配列番号8、配列番号9、配列番号10、配列番号11、配列番号12、配列番号13、配列番号14、配列番号15、配列番号16、配列番号17及び配列番号18からなる群から選択される、又は前記配列番号のいずれかと少なくとも95%の配列同一性を有する、1つ又は複数のゲノム配列を含む、請求項1又は2に記載の植物。
- 配列番号8、配列番号11、配列番号14を含む、請求項1又は2に記載の植物。
- 配列番号8、配列番号9、配列番号10、配列番号11、配列番号12、配列番号13及び配列番号14を含む、請求項1又は2に記載の植物。
- トバモウイルス株Tm0、Tm1及びTm2に抵抗性である、請求項1~5のいずれか一項に記載の植物。
- 前記トバモウイルスが、トマトブラウンルーゴスフルーツウイルス(TBRFV)である、請求項1~6のいずれか一項に記載の植物。
- 前記TBRFV抵抗性遺伝子が、前記植物のゲノムにおいてヘテロ接合性又はホモ接合性で存在する、請求項1~7のいずれか一項に記載の植物。
- 前記1つ又は複数のゲノム配列が、前記植物のゲノム中でヘテロ接合性又はホモ接合性で存在する、請求項3~8のいずれか一項に記載の植物。
- 前記1つ又は複数のゲノム配列が、寄託アクセッション番号NCIMB 43279中に見られるものである、請求項3~9のいずれか一項に記載の植物。
- 前記TBRFV抵抗性遺伝子が、寄託アクセッション番号NCIMB 43279中に見られるものである、請求項1~10のいずれか一項に記載の植物。
- 請求項1~11のいずれか一項に記載の植物に由来する植物、植物部分、組織、細胞及び/又は種子。
- S.リコペルシクム植物においてトバモウイルスに対する抵抗性を提供する抵抗性遺伝子であって、配列番号115と少なくとも90%のヌクレオチド配列同一性を有するコード配列によって表される、抵抗性遺伝子。
- S.リコペルシクム植物においてトバモウイルスに対する抵抗性を提供するゲノム配列であって、配列番号5、配列番号6、配列番号7、配列番号8、配列番号9、配列番号10、配列番号11、配列番号12、配列番号13、配列番号14、配列番号15、配列番号16、配列番号17及び配列番号18からなる群から選択される、又は前記配列番号のいずれかと少なくとも95%の配列同一性を有する、ゲノム配列。
- S.リコペルシクム植物においてトバモウイルスに対する抵抗性を提供する抵抗性遺伝子座であって、配列番号1、配列番号2、配列番号3又は配列番号4、好ましくは配列番号3によって表される、遺伝子座。
- 前記トバモウイルスがTBRFVである、請求項13~15のいずれか一項に記載の抵抗性遺伝子、ゲノム配列又は遺伝子座。
- トバモウイルスに抵抗性であるS.リコペルシクム種の植物を提供する方法であって、
a)トバモウイルスに抵抗性であるS.ハブロカイテス植物を選択するステップであり、請求項13~16のいずれか一項に記載の抵抗性遺伝子、ゲノム配列又は抵抗性遺伝子座、好ましくは前記抵抗性遺伝子の存在を確認するステップを含むステップと、
b)ステップa)の同定された抵抗性遺伝子、ゲノム配列又は遺伝子座を、例えば交雑によってS.リコペルシクム植物中に移入し、それによって前記S.リコペルシクム植物にトバモウイルス抵抗性を付与するステップとを含む、方法。 - ステップb)の後に、トバモウイルスに抵抗性である第1のS.リコペルシクム植物が選択され、トバモウイルスに抵抗性でない第2のS.リコペルシクム植物と交雑され、その後トバモウイルスに抵抗性であるS.リコペルシクム植物を選択する、請求項17に記載の方法。
- ステップa)においてS.ハブロカイテス植物中の前記抵抗性遺伝子、抵抗性を付与するゲノム配列又は抵抗性遺伝子座の前記存在を確認するステップが、配列番号83、配列番号84、配列番号85、配列番号86、配列番号87、配列番号88、配列番号89、配列番号90、配列番号91、配列番号92、配列番号93、及び配列番号94、配列番号105、配列番号106、配列番号107、配列番号108、配列番号109、配列番号110、配列番号111、及び配列番号112、好ましくは配列番号105、配列番号106、配列番号107、配列番号108、配列番号109、配列番号110、配列番号111及び配列番号112からなる群から選択される1つ又は複数のマーカーによって実行される、請求項17又は18に記載の方法。
- S.リコペルシクム植物において請求項13~16のいずれか一項に記載のTBRFV抵抗性遺伝子、抵抗性を付与するゲノム配列又は抵抗性遺伝子座の存在を確認するためのマーカーの使用であって、マーカーが、配列番号83、配列番号84、配列番号85、配列番号86、配列番号87、配列番号88、配列番号89、配列番号90、配列番号91、配列番号92、配列番号93及び配列番号94、配列番号105、配列番号106、配列番号107、配列番号108、配列番号109、配列番号110、配列番号111及び配列番号112、好ましくは配列番号105、配列番号106、配列番号107、配列番号108、配列番号109、配列番号110、配列番号111及び配列番号112からなる群から選択される1つ又は複数である、使用。
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