JP2004113234A - Characteristic base sequence produced in plant gene, and method of using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a single nucleotide polymorphism (SNP) produced in barley gene, and to provide a method for utilizing the same. <P>SOLUTION: Large amounts of cDNA clones originating from kinds of barley:'H. spontaneum', 'Sekishinryoku', and 'Haruna Nijo' are sequenced, and a non-synonymous permutaion SNP in a single nucleotide polymorphism-recognized site (SNP), is found among the kinds. The non-synonymous permutaion SNP has high possibility to participate in a phenotype characteristic to the kind and can thereby be utilized for various uses. For example, an oligonucleotide produced from the region containing the non-synonymous permutaion SNP, can be utilized as a tool for judging the base of the polymorphism existing on the gene of the barley, thereby enabling the genetical identification of the kind, the judgment of the type of the gene, and the like. Further, the oligonucleotide can be used as a probe for isolating a gene or detecting a single nucleotide polymorphism to produce and screen a new transformant. <P>COPYRIGHT: (C)2004,JPO

Description

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上記の表１〜表３９における各項目には、それぞれ以下に示す事項が記載されている。
【００８０】
「ｃｔｇ＿ＩＤ」　　：該当するコンティグの塩基配列が示される配列表の配列番号
「ｓｎｐ＿位置」　：各コンティグ配列上のＳＮＰの位置（多型部位）
「塩基」　　　：各ＳＮＰの位置における塩基の種類について、「はるな」には「はるな二条」のものを、「野生型」には「Ｈ．ｓｐｏｎｔａｎｅｕｍ」のものを、「赤神力」には「赤神力」のものをそれぞれ記載
「アミノ酸」　：各ＳＮＰ（多型部位またはその相補部位）を含むコドンによりコードされるアミノ酸について、「はるな」には「はるな二条」のものを、「野生型」には「Ｈ．ｓｐｏｎｔａｎｅｕｍ」のものを、「赤神力」には「赤神力」のものをそれぞれ記載（読み枠（ｆｒａｍｅ）の決定方法については後述する）
「置換有無」　：上記の「はるな」、「野生型」、「赤神力」に記載されたアミノ酸が同じアミノ酸である場合は、アミノ酸の置換が生じないものと判定して「Ｓ」と記載。一方、少なくとも二品種間でアミノ酸が相違する場合は、アミノ酸の置換が生じ得るものと判定して「Ｒ」と記載
「領域内判定」　：上記「Ｒ」と判定された各ＳＮＰについて、後述の方法により決定した翻訳領域（コード領域）の領域内に存在する場合は「ｉｎ」と記載。一方、領域外に存在する場合は「ｏｕｔ」と記載
「評価」　　　：上記「置換有無」欄において「Ｒ」と判定され、かつ、上記「領域内判定」欄において「ｉｎ」と判定されたＳＮＰをノンサイレント（非同義置換）と判定して「○」を記載。
【００８１】
このように、表１〜表３９の「評価」欄において「○」と判定されたＳＮＰが本発明の非同義置換ＳＮＰに相当するものである。コンティグ（１）〜（５３４）中には合計１９８９個のＳＮＰが見出されたが、このうち９３９個のＳＮＰが非同義置換ＳＮＰと判定された。
【００８２】
なお、各コンティグ（１）〜（５３４）の相補配列側にもオオムギの品種間で一塩基多型が認められるが、必要に応じて、コンティグ側のＳＮＰをコンティグ側一塩基多型（コンティグ側ＳＮＰ）と称し、上記相補配列中のＳＮＰを相補側一塩基多型（相補側ＳＮＰ）と称する。また、コンティグ側ＳＮＰが認められるヌクレオチドを多型部位と称する場合に、二本鎖ＤＮＡにおいて当該多型部位と塩基対を形成するヌクレオチドを相補部位（相補側多型部位）と称する。これら多型部位、相補部位はｃＤＮＡ上で見出されたものであるが、特に断りのない限り、ゲノムＤＮＡ上の対応領域も多型部位、相補部位と総称する。さらに、本発明において、「相補（的）」、「相補部位」、「相補配列」、「相補領域」等の用語は、特に断りのない限り、対象とするＤＮＡ配列（ＤＮＡ領域）と完全にハイブリダイズする関係、部位、配列、領域、すなわち、互いの塩基配列がワトソン−クリック塩基対（Ｗａｔｓｏｎ−Ｃｒｉｃｋ　ｂａｓｅ−ｐａｉｒｉｎｇ）　に完全に従っている関係、部位、配列、領域を指す。
【００８３】
上記の相補側／コンティグ側ＳＮＰの何れか一方はセンス側（遺伝子側）ＳＮＰに相当し、他方はアンチセンス側ＳＮＰに相当する。これらセンス側／アンチセンス側ＳＮＰはいずれも、１）同一品種内では実質的に認められず、かつ、異なる品種間で安定して認められ、また、２）センス側ＳＮＰはＥＳＴ（ｃＤＮＡ断片）中に存在し、非同義置換を生ぜしめるものであり、品種固有の表現型（形質）に関与する可能性が高いことから、様々な用途に利用可能である。
【００８４】
具体的には、例えば、このＳＮＰを品種識別の用途に供することができる。また、このＳＮＰの存在がオオムギの品種間で表現型に相違をもたらす場合（つまり、遺伝子と、当該遺伝子に一塩基多型が生じたアリルとで与える表現型が異なる場合）には、品種識別の用途に加え、例えば、１）この表現型に関与する遺伝子を取得するマーカー（遺伝子取得用マーカー）として、２）戻し交雑の際に反復親（ｒｅｃｕｒｒｅｎｔ　ｐａｒｅｎｔ）と交配すべき候補雑種を選別するマーカー（候補雑種選別用マーカー）として、３）遺伝子導入の成否を判定するマーカー（導入成否判定用マーカー）として、４）戻し交雑や遺伝子工学的手法などにより取得した新品種を選別するマーカー（新品種選別用マーカー）として、使用可能である。
【００８５】
また、上記多型部位またはその相補部位を含んでなる本発明にかかるオリゴヌクレオチドは、例えば、１）多型部位または相補部位における塩基の検定（ＳＮＰタイピング）用のプローブまたはプライマーとして、２）多型部位またはその相補部位を含んでなる遺伝子取得用／遺伝子同定用のプローブまたはプライマーとして、使用可能である。なお、本発明にかかるオリゴヌクレオチドの利用方法および作製方法の詳細については、後述する。
【００８６】
（Ｂ）コンティグ配列の作製法と非同義置換ＳＮＰの検出・同定法
次に、前記してきたコンティグ配列の作製方法について説明する。
【００８７】
まず、オオムギの複数品種（例えば、本発明においては「Ｈ．ｓｐｏｎｔａｎｅｕｍ」、「はるな二条」及び「赤神力」）の組織からそれぞれのｃＤＮＡライブラリーを常法にしたがって製造する。例えば、組織の全ＲＮＡを粗抽出し、全ＲＮＡから全ｍＲＮＡ（ポリ（Ａ）＋ＲＮＡ）を精製し、このｍＲＮＡを鋳型とするファーストストランドｃＤＮＡを合成し、さらに、セカンドストランドｃＤＮＡの合成を行う。次いで、得られた二本鎖ｃＤＮＡを適切なベクターにライゲーションし、このベクターを宿主内にパッケージングしてｃＤＮＡライブラリーを作製する。なお、必要により二本鎖ｃＤＮＡを適当な長さに切断してもよい。
【００８８】
次いで、得られたクローンを抽出し、各ｃＤＮＡクローンのシークエンス解析を行う。各クローンのシークエンス解析で明らかとなったｃＤＮＡの塩基配列に基づいて、相互にオーバーラップする塩基配列を有するか否かでクローンをクラスタリングし、クラスタリングされた一連のクローン群が有する挿入断片（ｃＤＮＡ）のオーバーラップ部分を見つけて繋ぎ合わせて（アセンブリし）、上記コンティグを得るとともに、ＳＮＰの検出が可能なように挿入断片を整列化する（マルチプルアライメントする）。
【００８９】
上記全ＲＮＡが抽出されるオオムギの組織としては、芽、葉を構成する各種組織が好適に使用されるが、特にこれに限定されない。また、オオムギの実生から上記組織を取得する場合、実生の栽培方法やその生育ステージなども特に限定されない。なお、実生の栽培方法に関しては、オオムギを実験材料とする場合の標準法（細胞工学別冊　植物細胞工学シリーズ１４　植物のゲノム研究プロトコール　頁１９３〜１９５　秀潤社（２０００　年）　参照）が特に好適に採用される。
【００９０】
なお、全ＲＮＡの抽出は、一般には、オオムギの芽生え期の芽または葉を構成する組織を用いて行われるが、この理由として、組織の摩砕が比較的容易であり、多糖類などの不純物の混合割合が比較的少なく、また、種子から短期間で育成可能である点が挙げられる。また、オオムギの各品種が有する固有の形質の発現時期にあわせて、当該オオムギの組織から全ＲＮＡを抽出してもよい。
【００９１】
オオムギの組織から全ＲＮＡを抽出する工程〜クローンのシークエンス解析までの各ステップは、一般的に採用される方法、例えば、育種学研究第２巻別１　２８頁（２０００　年）、ゲノム解析ラボマニュアル　ｐ６３−ｐ７７　：シュプリンガー・フェアラーク東京（１９９８年）に記載の方法を採用して容易に行うことができる。なお、解析結果の信用性を確保する目的で、より好ましくは、１）オオムギの品種毎に、全ＲＮＡ抽出用の個体を複数用意する、２）各クローンを複数個ずつシークエンス解析に供する、３）所定以上の長さを有する挿入断片（ｃＤＮＡ）につき５’端および３’端の双方から配列を解析する、ことの少なくとも一つを行うことが好ましい。また、各クローンが有する挿入断片については、いずれも再シークエンシングにより配列の確認を行うことがさらに好ましい。
【００９２】
次いで、各クローンのシークエンス解析により得られた挿入断片の塩基配列を、互いにオーバーラップする配列部分をもとにアセンブリして各コンティグを作製する。あわせて、コンティグ配列を構成するクローンの挿入断片をマルチプルアライメントして、異なるクローン間（品種間）での対応する塩基の相違（ここではＳＮＰ）を検出可能とする。
【００９３】
挿入断片のシークエンス解析におけるベースコーリング、オーバーラップする挿入断片のアセンブリ、及びマルチプルアライメントは、例えば、公知のＳＮＰ検出ソフトウエアや配列編集ソフトウエアを用いて行うことができる。これらソフトウエアにインストールされた代表的なプログラムには、Ｔｒａｃｅ−Ｄｉｆｆ（Ｂｏｎｆｉｅｌｄ．Ｊ．Ｋ．　ｅｔ　ａｌ．：Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓｅａｒｃｈ　２６：ｐ３４０４−３４０８，１９９８）、Ｐｏｌｙｂａｙｅｓ（Ｍａｒｔｈ．Ｇ．Ｔ．　ｅｔ　ａｌ．：Ｎａｔｕｒｅ　Ｇｅｎｅｔ．２３：ｐ４５２−４５６，１９９９）、Ｐｏｌｙｐｈｒｅｄ（Ｎｉｃｋｅｒｓｏｎ．Ｄ．Ａ．：Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓｅａｒｃｈ　２５：ｐ２７４５−２７５１，１９９７）　などがあり、これらは何れも、ベースコーリングツールとしてフェレッド（Ｐｈｒｅｄ）を、アセンブリツールとしてフェラップ（Ｐｈｒａｐ）を採用したフェレッド／フェラップ（Ｐｈｒｅｄ　／Ｐｈｒａｐ）タイプのものである（実験医学　Ｖｏｌ．１８　Ｎｏ．１４（９月号），２０００：ｐ１８９０−１８９２　参照）。
【００９４】
また、ベースコーリング時の各塩基の読み取りエラー確率を一定と仮定し（Ｐｈｒｅｄ　スコア（Ｑｕａｌｉｔｙ　ｖａｌｕｅ）　を一定値とし）、アセンブリツールとしてＰｈｒａｐ　を採用して、互いにオーバーラップする挿入断片をアセンブリすることもできる。
【００９５】
コンティグの作製に際しては、当該コンティグを構成する全クローン間で挿入断片の塩基配列を比較し、（１）塩基の相違が見られた部位では、アセンブリ後の品質精度が最も高い、例えば、Ｐｈｒａｐ　スコア（Ｑｕａｌｉｔｙ　ｖａｌｕｅ）　が最も高い塩基をコンティグ上の代表塩基として選択する処理を行う。（２）ただし、赤神力由来の複数のシークエンスデータ、Ｈ．ｓｐｏｎｔａｎｅｕｍ由来の複数のシークエンスデータ、および、はるな二条由来の複数のシークエンスデータからなる３群のシークエンスデータのうち、少なくとも２群のシークエンスデータ間で塩基の相違が見られる場合には、これら塩基を示すユニバーサルコードをコンティグ上の塩基として採用する。
【００９６】
そして、本発明では、特に信頼性が高いと推定される上記（２）の場合にのみ品種間のＳＮＰと称する。なお、上記（２）の場合には、Ｈ．ｓｐｏｎｔａｎｅｕｍ由来の複数の挿入断片群、赤神力由来の複数の挿入断片群、および、はるな二条由来の複数の挿入断片群から選択される少なくとも２以上の挿入断片群間で、対応する塩基に相違が見られる場合以外にも、Ｈ．ｓｐｏｎｔａｎｅｕｍ由来の単一挿入断片を３’側・５’側双方から読んだシークエンスデータ、赤神力由来の単一挿入断片を３’側・５’側双方から読んだシークエンスデータ、および、はるな二条由来の単一挿入断片を３’側・５’側双方から読んだシークエンスデータから選択される、少なくとも２以上のシークエンスデータ間で、対応する塩基に相違が見られる場合も含まれる。またもちろん、上記オオムギ三品種の何れか由来の単一挿入断片を３’側・５’側双方から読んだシークエンスデータと、他品種の複数の挿入断片のシークエンスデータとの間で、対応する塩基に相違が見られる場合も含まれる。ただし、上記の方法によりコンティグ上でＳＮＰと判定された箇所が三つ以上連続する場合には、信頼性の観点からこれらを本発明のＳＮＰとは取り扱わない。
【００９７】
さらに、上記の方法によりコンティグ上でＳＮＰと判定されたものの中から、非同義置換ＳＮＰを次のようにして選別した。まず、作製した各コンティグについて公知の方法により相同性検索を行い（結果については後述する）、何れかの読み枠（ｆｒａｍｅ）にしたがって翻訳されたアミノ酸配列が、既知タンパク質（既知遺伝子がコードするタンパク質を含む）のアミノ酸配列と一定以上の相同性が認められる場合に、その読み枠を当該コンティグの読み枠と決定した。
【００９８】
次に、その読み枠にしたがって翻訳されたアミノ酸配列において、品種間の塩基の相違に応じてアミノ酸の非同義置換を生ぜしめるＳＮＰ、つまり、前記した表１〜表３９の「置換有無」欄において「Ｒ」と判定された各ＳＮＰについて、翻訳領域（コード領域）内に存在するか否か検討した。具体的には、上記「Ｒ」と判定された各ＳＮＰの位置から５’側上流をさかのぼり、開始コドンよりも先に停止コドンが現れた場合には、そのＳＮＰは翻訳領域外と判定した。残余のＳＮＰは翻訳領域内と判定し、これにより非同義置換ＳＮＰを決定したが、これら各ＳＮＰの上流および下流における開始コドンおよび停止コドンの有無をさらに詳細に検討し、各コンティグの翻訳領域を決定した。その結果を以下の表４０〜表６７に示す。
【００９９】
【表４０】

【０１００】
【表４１】

【０１０１】
【表４２】

【０１０２】
【表４３】

【０１０３】
【表４４】

【０１０４】
【表４５】

【０１０５】
【表４６】

【０１０６】
【表４７】

【０１０７】
【表４８】

【０１０８】
【表４９】

【０１０９】
【表５０】

【０１１０】
【表５１】

【０１１１】
【表５２】

【０１１２】
【表５３】

【０１１３】
【表５４】

【０１１４】
【表５５】

【０１１５】
【表５６】

【０１１６】
【表５７】

【０１１７】
【表５８】

【０１１８】
【表５９】

【０１１９】
【表６０】

【０１２０】
【表６１】

【０１２１】
【表６２】

【０１２２】
【表６３】

【０１２３】
【表６４】

【０１２４】
【表６５】

【０１２５】
【表６６】

【０１２６】
【表６７】

表４０〜表６７中、「ｃｔｇ＿ＩＤ」および「ｓｎｐ＿位置」の欄は、前記の表１〜表３９と同じである。「ｆｒａｍｅ」の欄には、前記したように相同性検索の結果に基づいて各コンティグについて決定した読み枠が記される。ここで、ｆｒａｍｅが「＋」とは、コンティグ配列がセンス鎖と判定された場合であり、「−」とは、その相補配列がセンス鎖と判定された場合である。また、「＋１」「＋２」「＋３」とは、それぞれ、コンティグ配列の５’側第１番目、第２番目、第３番目の塩基から読み枠を設定していくことを意味し、「−１」「−２」「−３」とは、それぞれ、その相補配列の５’側第１番目、第２番目、第３番目の塩基から読み枠を設定していくことを意味する。
【０１２７】
表４０〜表６７中の「Ｑ＿ｓｔａｒｔ」の欄には、各コンティグのセンス鎖（ｆｒａｍｅ「＋」の場合はコンティグ配列、ｆｒａｍｅ「―」の場合はその相補配列）の塩基配列において、相同性検索の結果、既知配列との相同性が認められた領域の開始位置（即ち、当該領域の最も５’側の位置）が示される。したがって、ｆｒａｍｅ「―」の場合、開始位置とは、相補配列において相同性が認められた領域の最も５’側の位置であるが、この場合「Ｑ＿ｓｔａｒｔ」欄には、相補配列上の開始位置について、相補配列の３’側から数えて何番目に位置するかが示される。
【０１２８】
表４０〜表６７中の各コンティグの「翻訳領域」については、非同義置換ＳＮＰと判定されたＳＮＰのセンス鎖上の位置と、決定された読み枠（ｆｒａｍｅ）とに基づいて、開始コドン（ＡＴＧ）と停止コドンとの間に非同義置換ＳＮＰが挟まれる領域を翻訳領域と決定した。表４０〜表６７中には、こうして決定した各コンティグの翻訳領域の「開始位置」および「終了位置」が示される。ただし、非同義置換ＳＮＰの位置から５’側上流に開始コドンが現れない場合、読み枠「＋１」、「−１」のときはセンス鎖５’側１番目の塩基を、「＋２」、「−２」のときはセンス鎖５’側２番目の塩基を、「＋３」、「−３」のときはセンス鎖５’側３番目の塩基を、それぞれ翻訳領域の「開始位置」と判定した。一方、非同義置換ＳＮＰの位置から３’側下流に停止コドンが現れない場合は、決定された読み枠にしたがって翻訳領域が最長となる位置を翻訳領域の「終了位置」と判定した。このように、決定した翻訳領域は、必ずしもタンパク質の全長をコードする領域を示すものではない。
【０１２９】
なお、コンティグのｆｒａｍｅが「―」の場合は、表４０〜表６７中の「開始位置」および「終了位置」には、それぞれの位置について、相補配列の５’側から数えて何番目に位置するかが示される。
【０１３０】
また、コンティグ（８）、コンティグ（１０）、コンティグ（１０１）、及びコンティグ（３８６）については、既知配列との相同性を詳細に比較検討した結果、前記「Ｑ＿ｓｔａｒｔ」欄に記載した開始位置（換言すれば、既知配列との相同性が認められた領域の開始位置）を翻訳領域の「開始位置」とした。
【０１３１】
また、表４０〜表６７中の「翻訳領域の番号」の欄は、上記の方法により翻訳領域と判定された領域が一つのコンティグに複数存在する場合に、各翻訳領域に順番に番号を付したものである（翻訳領域が１つの場合は、その翻訳領域に番号「１」を付している）。
【０１３２】
上記の方法により各コンティグの翻訳領域と判定された領域がコードするアミノ酸配列が、配列表に示される。以下の表６８〜表７８は、それぞれのアミノ酸配列について、配列表中の何れの配列番号に記載されているかを示すものである。
【０１３３】
【表６８】

【０１３４】
【表６９】

【０１３５】
【表７０】

【０１３６】
【表７１】

【０１３７】
【表７２】

【０１３８】
【表７３】

【０１３９】
【表７４】

【０１４０】
【表７５】

【０１４１】
【表７６】

【０１４２】
【表７７】

【０１４３】
【表７８】

表６８〜表７８中、「ｃｔｇ＿ＩＤ」の欄は、前記の表１〜表３９と同じである。「アミノ酸配列」の欄には、各コンティグの翻訳領域がコードするアミノ酸配列を記載した配列表の配列番号が示される。なお、１つのアミノ酸配列について、翻訳領域（コード領域）の塩基配列と並列記載したものと、アミノ酸配列単独で記載したものとの両方が配列表に示される。また、配列表に示されるアミノ酸配列は、前記「ユニバーサルコード」で示される多型部位またはその相補部位の塩基を、以下のａ）〜ｃ）の基準にしたがって選択し、決定したものである。
【０１４４】
ａ）　「はるな」、「野生型」、「赤神力」の３品種のうち、翻訳領域上のすべてのＳＮＰの塩基が決定している品種の各塩基を、各ＳＮＰの塩基として選択する。翻訳領域上のすべてのＳＮＰの塩基が決定している品種が二種類以上存在する場合は、「野生型」＞「赤神力」＞「はるな」の優先順位に従って品種を選択する。
【０１４５】
ｂ）　ただし、翻訳領域内に「アミノ酸→ＳＴＯＰ（停止コドン）」の置換を生じさせるＳＮＰがある場合は、ＳＴＯＰを生じさせる方の塩基を持つ品種は選択せずに、上記優先順位が１つ下の品種の各塩基を、各ＳＮＰの塩基として選択する。
【０１４６】
ｃ）　翻訳領域内に「アミノ酸→ＳＴＯＰ（停止コドン）」の置換を生じさせるＳＮＰがあり、他の品種の塩基も翻訳領域内に「アミノ酸→ＳＴＯＰ（停止コドン）」の置換を生じさせてしまう場合は、翻訳領域上のすべてのＳＮＰの塩基が決定している品種のうち、上記優先順位の高い方の品種の各塩基を、各ＳＮＰの塩基として選択する。
【０１４７】
なお、配列表に示される各アミノ酸配列は、前記した翻訳領域の判定結果に基づくものであり、必ずしもタンパク質の全長を示すものではない。
【０１４８】
上記の表６８〜表７８には、各コンティグについて、「クローン数」と「クローン配列」とがあわせて示される。「クローン数」の欄には、各コンティグを作製・構築するもととなったｃＤＮＡクローンの総数が示される。「クローン配列」の欄には、これらｃＤＮＡクローンの各塩基配列を記載した配列表の配列番号が示される。例えば、コンティグ（１）は、配列番号１６６１〜１６７５に示される各ｃＤＮＡクローンを整列化（マルチプルアラインメント）させることにより得られた配列である。
【０１４９】
（Ｃ）相同性検索結果
前記したように、本発明者らは、作製した各コンティグについて公知の方法により相同性検索を行った。その結果を以下の表７９〜表１１０に示す。
【０１５０】
【表７９】

【０１５１】
【表８０】

【０１５２】
【表８１】

【０１５３】
【表８２】

【０１５４】
【表８３】

【０１５５】
【表８４】

【０１５６】
【表８５】

【０１５７】
【表８６】

【０１５８】
【表８７】

【０１５９】
【表８８】

【０１６０】
【表８９】

【０１６１】
【表９０】

【０１６２】
【表９１】

【０１６３】
【表９２】

【０１６４】
【表９３】

【０１６５】
【表９４】

【０１６６】
【表９５】

【０１６７】
【表９６】

【０１６８】
【表９７】

【０１６９】
【表９８】

【０１７０】
【表９９】

【０１７１】
【表１００】

【０１７２】
【表１０１】

【０１７３】
【表１０２】

【０１７４】
【表１０３】

【０１７５】
【表１０４】

【０１７６】
【表１０５】

【０１７７】
【表１０６】

【０１７８】
【表１０７】

【０１７９】
【表１０８】

【０１８０】
【表１０９】

【０１８１】
【表１１０】

表７９〜表１１０中の「ｃｔｇ＿ＩＤ」の欄は、前記表１〜表３９と同じである。「相同性検索結果」の欄には、各コンティグについて、一定以上の相同性が認められた既知遺伝子または既知タンパク質の名称およびアクセッション番号などが示される。
【０１８２】
（Ｄ）本発明のオリゴヌクレオチド等の利用方法（有用性）
本発明のオリゴヌクレオチドは、前述してきたコンティグ上の非同義置換ＳＮＰの少なくとも一つを含む７ヌクレオチド以上の連続したＤＮＡ配列、またはその相補配列からなるものであり、以下に説明するとおり、種々の有用性を有するものである。
【０１８３】
まず、本発明のオリゴヌクレオチドは、オオムギの品種の相違に起因する上記多型部位またはその相補部位を含むため、当該オリゴヌクレオチドやこのオリゴヌクレオチドを含んでなる組成物は、種々のオオムギ品種間に存在する一塩基多型を検定する目的で利用可能である。また、これによりオオムギの遺伝学的品種識別や、当該オオムギがいずれのタイプ（遺伝子型）の遺伝子を有するかの判定等を迅速かつ簡便に行うことが可能になる。
【０１８４】
例えば、上記オリゴヌクレオチドを基盤（担体）上に固定化してＤＮＡチップ（本発明にかかる組成物の一形態）を構成すれば、オオムギの遺伝子における一塩基多型を検定するオリゴヌクレオチドプローブとして利用できる。この場合、オリゴヌクレオチドの長さは、一塩基多型を検定することができる長さであれば特に制限はないが、７〜５０ヌクレオチド、あるいは１０〜３０ヌクレオチドが好ましく、１５〜２５ヌクレオチドがより好ましい。
【０１８５】
また、上記遺伝子における一塩基多型の検定は、上記多型部位またはその相補部位を含むＤＮＡを用いて行う他にも、該ＤＮＡから得られたＲＮＡを介して間接的に行うこともできる。よって、ＤＮＡチップを用いたハイブリダイズ試験のターゲットには、後述する被検体由来のＤＮＡおよびＲＮＡが使用可能であり、一般的には、該ＤＮＡとしてｃＤＮＡやゲノムＤＮＡが、該ＲＮＡとしてｍＲＮＡやｃＲＮＡが使用される。
【０１８６】
さらに、本発明にかかるオリゴヌクレオチドに相当するＤＮＡ断片を上記被検体から取得し、これを直接的に解析して、被検体の遺伝子における一塩基多型を検定することもできる。具体的には、例えば、配列番号１〜５３４の何れかに表されるＤＮＡ配列からなるオリゴヌクレオチドにおける、前記「ユニバーサルコード」で示された多型部位の少なくとも一つを含む領域、またはその相補領域の少なくとも一方を増幅し得るように設計されたプライマーを用い、被検体由来のＤＮＡを増幅することで、前記被検体の遺伝子に生じた一塩基多型を検定してもよい。
【０１８７】
上記プライマーには、ＰＣＲプライマーのように、前記多型部位の少なくとも一つを含む領域、およびその相補領域の双方を増幅するプライマーに加え、ＲＣＡプライマーのようにそのいずれか一方を増幅するプライマーも含まれる。また、得られた増幅ＤＮＡ断片は、例えば、１）直接シークエンシングして解析する、２）多型部位、および／または、その相補部位の塩基に応じて異なるパターンで切断する制限酵素を用いて消化し、生じた断片の長さを解析する、３）本発明にかかるオリゴヌクレオチドとのハイブリダイズ試験により解析する、などの方法で解析することができる。なお、上記制限酵素には、二本鎖ＤＮＡ上の特定配列を認識して切断する制限酵素（クラスＩＩ制限酵素）に加え、一本鎖ＤＮＡ上の特定配列を認識して切断する制限酵素（クラスＩ制限酵素、およびクラスＩＩ制限酵素の一部）も含まれる。
【０１８８】
なお、遺伝子は通常、その相補配列と結合した二本鎖ＤＮＡ（ｃＤＮＡであってもよい）として存在するから、本発明において「遺伝子における一塩基多型（センス側一塩基多型）を検定する」とは、当該遺伝子の相補配列における一塩基多型（アンチセンス側一塩基多型）の検定を介して、これに相補するセンス側一塩基多型を検定する場合も包含されている。また「一塩基多型を検定する」とは、多型部位または相補部位の塩基の種類まで特定（同定または検出）すること以外に、当該塩基の種類を限定すること（例えば、「少なくともアデニンではない」など）も含む概念である。
【０１８９】
上記の「被検体」とは、本発明にかかるオリゴヌクレオチドと同一あるいは相補的な塩基配列を部分配列または全長とする遺伝子を持つあらゆる生物を指し、オオムギ以外では、他のオオムギ属植物や、オオムギ属と近縁なコムギ連（ＴＲＩＴＩＣＥＡＥ）の植物、例えば、コムギ属（Ｔｒｉｔｉｃｕｍ属）植物、タルホコムギ属（Ａｅｇｉｌｏｐｓ属）植物、ライムギ属（Ｓｅｃａｌｅ属）植物、ドクムギ属（Ｌｏｌｉｕｍ属）植物などが特に好適なものとして挙げられる。なお、本発明では、「Ｗ．Ｄ．Ｃｌａｙｔｏｎ　ａｎｄ　Ｓ．Ａ．Ｒｅｎｖｏｉｚｅ：　ＫＥＷ　ＢＵＬＬＥＴＩＮ　ＡＤＤＩＴＩＯＮＡＬ　ＳＥＲＩＥＳ　ＸＩＩ，　ＧＥＮＥＲＡ　ＧＲＡＭＩＮＵＭ，　Ｇｒａｓｓｅｓｏｆ　ｔｈｅ　Ｗｏｒｌｄ　（第３刷）：ｐ１４６−１５８（１９９９年：キュー王立植物園編）」に記載の分類体系を基本的に採用する。
【０１９０】
さらに、本発明にかかるオリゴヌクレオチドを形質転換体の作製に利用し、有用遺伝子が導入された実用系統を開発することができる。例えば、当該オリゴヌクレオチドをプローブとして、またはプライマーとして若しくは当該オリゴヌクレオチドの領域を得るためのプライマーを用いて有用遺伝子を単離し、次いで、この有用遺伝子を遺伝子操作技術を用いて対象とする細胞に導入して、形質転換体を生産することも可能である。なお、上記オリゴヌクレオチドを遺伝子単離用のプローブとして利用する場合、その長さはプローブとして充分な長さがあればよく、例えば、７〜５００、５１〜５００、あるいは７０〜４００ヌクレオチドが好ましく、９０〜３００ヌクレオチドがより好ましい。
【０１９１】
また、例えば、本発明にかかるオリゴヌクレオチドを部分配列または全長とする遺伝子を有する親植物Ａと、この遺伝子を有さない、あるいはこの遺伝子に一塩基多型（特に表現型に影響を与えるもの）が生じたアリルを有する親植物Ｂとが交配可能な場合に、親植物Ａ・Ｂを交配させて雑種の植物を作出し（作出工程）、次いで、本発明にかかるオリゴヌクレオチドまたはＳＮＰの検定方法を用いて、これら雑種の植物の中から上記遺伝子を持つものを選抜して（選抜工程）、所望する形質転換体（形質転換植物）を生産することができる。なお、上記アリルとは、ある有用遺伝子に少なくとも一つの一塩基多型が生じ、これにより有用形質の発現が抑制される、または消失するものであることがより好ましい。また、上記有用遺伝子の種類は特に限定されるものではないが、通常の育種の場合と同様、例えば、栽培オオムギの祖先野生型オオムギが有する有用遺伝子（環境抵抗性遺伝子など）が特に好適である。
【０１９２】
前述したように、本発明にかかるオリゴヌクレオチドは、塩基の表記が「ユニバーサルコード」で記載されている多型部位のうち、非同義置換を生ぜしめる多型部位の少なくとも一つを含む７ヌクレオチド以上の連続したＤＮＡ配列、またはその相補配列からなるものを特に指す。なお、このオリゴヌクレオチドは、１本鎖として存在する場合に限らず、相補鎖と水素結合し２本鎖として存在するものであってもよい。また、当該オリゴヌクレオチドは、ＲＣＡ法（後述する）によるＳＮＰタイピング用のバドロックプローブや、ベクター配列（発現ベクター配列を含む）などの配列に挿入された状態で存在するものであってもよい。
【０１９３】
上記のオリゴヌクレオチドは、ｃＤＮＡクローンが有する挿入断片を整列化して得られたコンティグまたはその相補配列の一部あるいは全長であり、ｃＤＮＡはもちろんゲノムＤＮＡ上にも同一の配列が存在する可能性が極めて高い。よって、例えば、（１）遺伝子に生じた一塩基多型を検定するプローブまたはプライマー（ＳＮＰタイピング用のプローブまたはプライマー）として、また、（２）遺伝子を取得（単離）するためのプローブまたはプライマーとして利用可能であり、以下の説明では、上記（１）の用途に利用されるものをオリゴヌクレオチドＡ、（２）の用途に利用されるものをオリゴヌクレオチドＢと称する。
【０１９４】
オリゴヌクレオチドＡは、例えば、オオムギのゲノム内に品種間安定的に保存されたＳＮＰを検出する際に使用されるものであり、ａ）オオムギの品種間で遺伝子上にＳＮＰが存在する部位、または、この部位に相補的な部位（相補部位）を少なくとも一つ含み、かつ、ｂ）ＳＮＰを検定するためのハイブリダイズ用プローブまたはプライマーとして適切な長さを有すればよい。これらａ），ｂ）の条件を満たす限りにおいて、オリゴヌクレオチドＡの長さは特に限定されないが、一般には、７〜５０ヌクレオチドの範囲内、あるいは１０〜３０ヌクレオチドの範囲内であることがより好ましく、配列として十分な特異性を示す１５〜２５ヌクレオチドの範囲内であることがさらに好ましい。
【０１９５】
なお、オリゴヌクレオチドＡが、上記多型部位またはその相補部位を２つ以上含んでなる場合には、当該多型部位またはその相補部位の塩基を全て「はるな二条」型、「赤神力」型あるいは「Ｈ．ｓｐｏｎｔａｎｅｕｍ」型の何れか一方に固定して、オリゴヌクレオチドＡの塩基配列を規定すればよい。
【０１９６】
一方、オリゴヌクレオチドＢは、各コンティグあるいはその相補配列を部分配列または全長とする遺伝子およびそのホモログを取得するためのプローブ、あるいはプライマーとして使用される。よって、オリゴヌクレオチドＢは、上記多型部位または相補部位の少なくとも一つを含む７ヌクレオチド以上の長さであれば特に限定されるものではないが、５１〜５００ヌクレオチド、あるいは７０〜４００ヌクレオチドの長さであることがより好ましく、９０〜３００ヌクレオチドの長さであることがさらに好ましい。なお、本発明にかかるオリゴヌクレオチドの中には、上記（１）・（２）の双方の用途に利用されるものもあるので、上記オリゴヌクレオチドＡ・Ｂが塩基配列として同一の場合もある。
【０１９７】
（Ｅ）本発明にかかるオリゴヌクレオチドの作製方法
本発明にかかるオリゴヌクレオチドの製造方法は特に限定されるものではなく、一般的な方法が採用される。例えば、当該オリゴヌクレオチドを、それを有する生物のゲノムＤＮＡ、ゲノムＤＮＡライブラリー、またはｃＤＮＡライブラリーから適切な制限酵素で切り出し、精製すればよい。また、配列番号１〜５３４の何れかに表されるＤＮＡ配列からなるオリゴヌクレオチドにおける、非同義置換ＳＮＰに当たる多型部位の少なくとも一つを含む領域、またはその相補領域の少なくとも一方を増幅し得るように設計されたプライマーを用い、上記生物のゲノムＤＮＡ、ｃＤＮＡを鋳型として増幅反応を行うことで、本発明のオリゴヌクレオチドを単離してもよい。さらに、当該オリゴヌクレオチドを公知の方法により合成することもできる。
【０１９８】
上記オリゴヌクレオチド取得用のプライマーの長さは、ミスマッチによる標的領域以外の増幅が起こらない限りにおいて特に限定されるものではない。例えば、増幅反応がＰＣＲ（Ｐｏｌｙｍｅｒａｓｅ　Ｃｈａｉｎ　Ｒｅａｃｔｉｏｎ）　である場合には、一般に１５〜２５、あるいは１５〜３０ヌクレオチドの範囲内の長さの一組のプライマーセットが用いられる。プライマーの配列についても、特に限定されるものではなく、ＤＮＡ配列上の特異的、特徴的な配列領域をもとに任意に設計すればよい。
【０１９９】
また、ＰＣＲ反応の反応条件も通常の条件に従って行えばよい（Ｓａｉｋｉ，　Ｓｃｉｅｎｃｅ，　２３０，　１３５０−１３５４（１９８５）；ＰＣＲ　テクノロジー、宝酒造（株）１９９０年発行；　等参照）。より具体的には、標的二本鎖ＤＮＡ（鋳型を含む）の熱変性工程は、９０℃〜９６℃、より好ましくは９４℃〜９６℃の温度範囲内で、約１分間〜３分間、より好ましくは約１分間〜２分間行えばよい。また、プライマーのアニーリング工程は、４０℃〜６０℃、より好ましくは５５℃〜６０℃の温度範囲内で、約１分間〜３分間、より好ましくは約１分間〜約２分間行えばよい。さらに、ＤＮＡポリメラーゼによる鎖伸長工程は、７０℃〜７４℃、より好ましくは７２℃〜７４℃の温度範囲内で、約１分間〜４分間、より好ましくは約２分間〜３分間行えばよい。
【０２００】
上記熱変性工程、アニーリング工程、および鎖伸長工程を一サイクルとするＰＣＲ反応のサイクル数も特に限定されないが、通常は２０〜４０サイクル、より好ましくは２５〜３５サイクル行えばよい。その他、使用するＤＮＡポリメラーゼの種類（Ｔａｑ　ポリメラーゼなどの耐熱性ポリメラーゼ）・量、試薬の種類・量、鋳型となるＤＮＡの抽出法（例えばＣ−ＴＡＢ法）などは従来と同様であり、詳細な説明を省略する。なお、上記ＰＣＲ反応の範疇には、ＲＴ−ＰＣＲ（Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｂｅｄ　ＰＣＲ）　反応のような変法も含まれる。また、ＰＣＲ反応では、多型部位を含む二本鎖ＤＮＡ断片が取得されるが、例えば、ＲＣＡ（Ｒｏｌｌｉｎｇ　ｃｉｒｃｌｅ　ａｍｐｌｉｆｉｃａｔｉｏｎ）　法などの単鎖増幅法を用い、適切なプライマー・鋳型（多型部位またはその相補部位を含む環状一本鎖ＤＮＡ）セットで反応を行えば、上記多型部位またはその相補部位の何れか一方を含む一本鎖ＤＮＡ断片（本発明のオリゴヌクレオチド）を取得可能となる（Ｌｉｚａｒｄｉ　ＰＭ　ｅｔ　ａｌ．　Ｎａｔ　Ｇｅｎｅｔ　１９，２２５，１９９８）。
【０２０１】
本発明にかかるオリゴヌクレオチドＡ・Ｂを取得するための上記生物としては、「Ｈ．ｓｐｏｎｔａｎｅｕｍ」、「赤神力」および「はるな二条」などのオオムギ品種や、その他のオオムギ属植物が特に好適であるが、これらの他に、当該オリゴヌクレオチドと同一あるいは相補的な塩基配列を部分配列または全長とする遺伝子を持つあらゆる生物が採用される。オオムギ属植物以外の上記生物としては、オオムギ属と近縁なコムギ連の植物、例えば、コムギ属（Ｔｒｉｔｉｃｕｍ属）　植物、タルホコムギ属（Ａｅｇｉｌｏｐｓ属）　植物、ライムギ属（Ｓｅｃａｌｅ属）　植物、ドクムギ属（Ｌｏｌｉｕｍ属）植物などが好適なものとして挙げられる。
【０２０２】
（Ｆ）本発明のオリゴヌクレオチドを用いたＳＮＰのタイピング法
次に、本発明のオリゴヌクレオチドをＳＮＰのタイピング法に適用する場合について説明する。
【０２０３】
被検体の遺伝子上にある一塩基多型の検定（ＳＮＰのタイピング）は、本発明にかかるオリゴヌクレオチドを用いて行うことができる。この方法は、（Ａ）被検体である生物由来のＤＮＡ（ｃＤＮＡでもよい）から上記オリゴヌクレオチドを単離し、当該オリゴヌクレオチド上の多型部位またはその相補部位における塩基を直接検定する方法（直接法と称する）、および、（Ｂ）本発明にかかるオリゴヌクレオチドＡが有する多型部位または相補部位を用いて、被検体由来の遺伝子上にある一塩基多型を間接的に検定する方法（間接法と称する）、に大別される。つまり、上記（Ａ）・（Ｂ）の方法はいずれも、本発明にかかるオリゴヌクレオチドが有する上記多型部位またはその相補部位の塩基に基づいて、被検体の遺伝子に生じた一塩基多型を検定する方法である。
【０２０４】
なお、上記「被検体」とは、特に好適には、「Ｈ．ｓｐｏｎｔａｎｅｕｍ」、「赤神力」または「はるな二条」などのオオムギ品種や、その他のオオムギ属植物が挙げられるが、本発明にかかるオリゴヌクレオチドと同一あるいは相補的な塩基配列を部分配列または全長とする遺伝子を有する生物であればその種類は特に限定されない。このような遺伝子を持つ可能性が高い生物として、具体的には、例えば、上記のコムギ属植物、タルホコムギ属植物、ライムギ属植物、ドクムギ属植物などのコムギ連の植物が挙げられる。また、交配や遺伝子工学的手法で、これら生物の持つ上記遺伝子が導入された（または導入が試みられた）形質転換体も、上記被検体の範疇である。
【０２０５】
また、遺伝子は通常、その相補配列と結合した二本鎖ＤＮＡ（ｃＤＮＡであってもよい）として存在する。よって、本発明において「遺伝子における一塩基多型を検定する」とは、当該遺伝子の相補配列における一塩基多型の検定を介して、これに相補するセンス側一塩基多型を検定する場合も含むものとする。
【０２０６】
以下、上記直接法についてより具体的に説明する。直接法は、例えば、配列番号１〜５３４の何れかに表されるＤＮＡ配列上にある多型部位の少なくとも一つを含む領域、またはその相補領域の少なくとも一方を増幅し得るように設計されたプライマー（本発明にかかるプライマー）を用いて、被検体由来のＤＮＡ（ゲノムＤＮＡまたはｃＤＮＡ）を増幅して増幅ＤＮＡ断片（本発明にかかるオリゴヌクレオチド）を取得し、次いで、この増幅ＤＮＡ断片を直接的に解析することで遺伝子に生じたＳＮＰを検定する方法である。なお、ここで使用する増幅反応の具体例および条件などは、前記「（Ｅ）本発明にかかるオリゴヌクレオチドの作製方法」にて説明したとおりである。
【０２０７】
直接法の一例として、より具体的には、１）ＭＡＬＤＩ−ＴＯＦ／ＭＳ（Ｍａｔｒｉｘ　Ａｓｓｉｓｔｅｄ　Ｌａｓｅｒ　Ｄｅｓｏｒｐｔｉｏｎ−Ｔｉｍｅ　Ｏｆ　Ｆｌｉｇｈｔ／Ｍａｓｓ　Ｓｐｅｃｔｒｏｍｅｔｒｙ）　法などを利用して、得られた増幅ＤＮＡ断片の質量の違いなどからＳＮＰをタイピングしてもよく、２）この増幅ＤＮＡ断片をシークエンシングして多型部位またはその相補部位の塩基を直接決定してもよく、３）この増幅ＤＮＡ断片を、上記多型部位またはその相補部位の少なくとも一方の塩基の種類に応じて異なるパターンで切断する制限酵素で消化し、生じた断片の長さの相違によりＳＮＰを直接タイピングしてもよい（いわゆるＲＦＬＰ（Ｒｅｓｔｒｉｃｔｉｏｎ　Ｆｒａｇｍｅｎｔ　Ｌｅｎｇｔｈ　Ｐｏｌｙｍｏｒｐｈｏｒｉｓｍ）法）　。なお、３）の方法では、取得される増幅ＤＮＡ断片が一本鎖（ＲＣＡ　法等による）であるか二本鎖であるかに応じて、一本鎖ＤＮＡ上の特定配列を認識して切断する制限酵素（クラスＩ制限酵素、およびクラスＩＩ制限酵素の一部）と、二本鎖ＤＮＡ上の特定配列を認識して切断する制限酵素（クラスＩＩ制限酵素）とを選択すればよい。
【０２０８】
なお、上記２）、３）の方法は極めて一般的な方法であるので、特に、上記１）の方法についてのみ以下に説明する。ＭＡＬＤＩ−ＴＯＦ／ＭＳ法を利用したＳＮＰのタイピングの一例では、上記多型部位を含む領域、またはその相補領域を増幅可能に設計されたプライマーを用い、被検体のゲノムＤＮＡまたはｃＤＮＡを鋳型として増幅反応を行う。次いで、得られた増幅ＤＮＡ断片を鋳型としてプライマー伸長反応を行うことにより、上記多型部位または相補部位を３’端として含む伸長反応生成物を得る。次いで、多型部位または相補部位の塩基が異なることにより生じる伸長反応生成物の質量の相違をマススペクトロメータにより測定し、当該部位の塩基をタイピングする（「ポストシークエンスのゲノム科学（１）　ＳＮＰ遺伝子多型の戦略　第一刷（中山書店）１０６　頁〜１１７　頁」）参照）。なお、複数のＳＮＰを同時に検定する場合には、マルチプレックスな条件で行うことができる。
【０２０９】
一方、上記間接法は、本発明にかかるオリゴヌクレオチドＡを用いたＳＮＰタイピング法であり、具体的には、例えば、１）被検体由来のＤＮＡ（ｃＤＮＡを含む概念）またはＲＮＡを調製する工程、２）オリゴヌクレオチドＡを取得する工程、３）オリゴヌクレオチドＡと、１）で調製したＲＮＡまたはＤＮＡとが完全にハイブリダイズするか否かを調べる工程を含んでなる。そして、両者が完全にハイブリダイズした場合には、被検体の遺伝子が有する多型部位（またはその相補部位）が、オリゴヌクレオチドＡの対応する塩基と相補的な塩基からなると確定され、一方、不完全にハイブリダイズするかハイブリダイズしない場合には、多型部位（またはその相補部位）がオリゴヌクレオチドＡの対応する塩基と非相補的な塩基のいずれかからなると判断される。
【０２１０】
なお、いうまでもないが、オリゴヌクレオチドＡが上記多型部位の少なくとも一つを含むＤＮＡ配列である場合、このＤＮＡ配列は、自身と相補的なゲノムＤＮＡ、ｃＤＮＡと完全にハイブリダイズし、一方、オリゴヌクレオチドＡが該ＤＮＡ配列の相補配列である場合、当該相補配列は、自身と相補的なｍＲＮＡ、ｃＲＮＡ、ゲノムＤＮＡと完全にハイブリダイズする。
【０２１１】
上記被検体由来のＤＮＡとは、このＤＮＡを鋳型としてＰＣＲ法やＲＣＡ法などにより得られる増幅ＤＮＡ断片も含む概念である。また、被検体由来のＤＮＡまたはＲＮＡは、一般には、その生体、すなわち、各種器官、各種組織、細胞、プロトプラスト、スフェロプラスト、カルスなどから抽出したものが用いられるが、場合によっては加工食品（飲料でもよい）、化石など被検体の生体以外から抽出したものが用いられる。
【０２１２】
被検体由来のＤＮＡまたはＲＮＡの調製には、一般的なＤＮＡまたはＲＮＡの抽出法を採用すればよい。また、ＤＮＡまたはＲＮＡ抽出用の組織の種類、被検体の栽培（生育）方法やその生育ステージなども特に限定されるものではない。ＤＮＡまたはＲＮＡとして、ｃＤＮＡまたはｍＲＮＡを調製する場合には、例えば、上記コンティグを作製する際に用いた方法を採用すればよい。また、ｃＲＮＡを調製する場合には、上記ｃＤＮＡを適切なＲＮＡポリメラーゼを用いて転写する一般法を利用すればよい。また、ハイブリダイズ用のターゲットとなるこれらＤＮＡ・ＲＮＡ、および／または、オリゴヌクレオチドＡ（プローブ）は、通常、ビオチン標識、各種蛍光標識などで予め標識してハイブリダイズの状態を確認可能としておくが、ハイブリダイズの状態をインターカレートの挿入、その他の方法により確認する場合には、上記ターゲットおよび／またはプローブを予め標識しておく必要は特にない。
【０２１３】
なお、被検体由来のＤＮＡまたはＲＮＡ（ターゲット）と、オリゴヌクレオチドＡ（プローブ）とをハイブリダイズさせる条件は、オリゴヌクレオチドＡの長さなどを考慮した上で、各方法で採用される一般的な条件に従えばよい（ＤＮＡマイクロアレイ　初版第３刷　ｐ６７−８３（宝酒造株式会社　発行）など参照）。
【０２１４】
また、一般には、基盤（担体）上にオリゴヌクレオチドＡ（プローブ）を固定化してなるＤＮＡチップ（組成物）を製造し、上記ハイブリダイゼーションを行う。なお、本発明において「ＤＮＡチップ」とは、ここでは主として合成したオリゴヌクレオチドをプローブに用いる合成型ＤＮＡチップを意味するが、ＰＣＲ産物等のｃＤＮＡをプローブに用いる貼り付け型ＤＮＡマイクロアレイをも包含する概念である。
【０２１５】
なお、ＤＮＡチップの製造には公知の方法を採用すればよく、オリゴヌクレオチドＡとして合成オリゴヌクレオチドを使用する場合には、フォトリソグラフィー技術と固相法ＤＮＡ合成技術との組み合わせにより基盤上で該オリゴヌクレオチドを合成すればよく、一方、オリゴヌクレオチドＡとしてｃＤＮＡを用いる場合には、アレイ機を用いて基盤上に貼り付ければよい。また、一般的なＤＮＡチップと同様、パーフェクトマッチプローブ（オリゴヌクレオチドＡ）と、該パーフェクトマッチプローブにおいて多型部位（または相補部位）以外で一塩基置換されたミスマッチプローブとを配置してＳＮＰの検定精度をより向上させてもよい。さらに、異なる遺伝子上のＳＮＰを並行して検定するために、複数種のオリゴヌクレオチドＡを同一の基盤上に固定してＤＮＡチップを構成してもよい。
【０２１６】
また、間接法の他の例として、Ｉｎｖａｄｅｒ　法を利用したＳＮＰのタイピングも挙げられる（「ポストシークエンスのゲノム科学（１）　ＳＮＰ遺伝子多型の戦略第一刷（中山書店）９８頁〜１０５　頁」参照）。一例では、コンティグ（１）〜（５３４）の何れかの一部であって多型部位の一つを３’端として含む配列の相補配列をオリゴヌクレオチドＡ（プローブ領域）とし、さらに、このオリゴヌクレオチドＡの５’端側に、ある共通配列（フラップ領域）を連結してアレルプローブを作製する。次いで、当該コンティグの一部であって上記多型部位を３’端（ただし３’端の塩基の種類は任意）として含むインベーダープローブと、アレルプローブとを被検体のゲノムＤＮＡまたはｃＤＮＡにハイブリダイズさせ、次いで、アレルプローブがゲノムＤＮＡまたはｃＤＮＡに完全にマッチ（完全にハイブリダイズ）する場合にのみフラップ領域を切り出す酵素（ｃｌｅｖａｓｅ）　を用いてフラップ領域の切り出しを試みる。次いで、切り出されたフラップ領域と、このフラップ領域に相補結合可能、かつ蛍光色素およびクエンチャーによりラベルされたプローブ（ＦＲＥＴ（Ｆｌｕｏｒｅｓｃｅｎｃｅ　Ｒｅｓｏｎａｎｃｅ　Ｅｎｅｒｇｙ　Ｔｒａｎｓｆｅｒ）プローブ）とをハイブリダイズさせ、上記酵素（ｃｌｅｖａｓｅ）　による蛍光色素の切り出しを行う。多型部位のＳＮＰタイピングは、発生する蛍光量が、上記ゲノムＤＮＡまたはｃＤＮＡとアレルプローブとが完全にハイブリダイズするか否かで異なることを利用して行う。
【０２１７】
さらに、上記間接法には、オリゴヌクレオチドＡと、被検体由来のＤＮＡとのハイブリダイズ（アニーリング）を試み、両者が完全にハイブリダイズしたか否かをＤＮＡの増幅反応により確認する方法が含まれる。これらの方法として、具体的には、例えば、ＴａｑＭａｎ　ＰＣＲ法、ＲＣＡ法などを利用した公知のＳＮＰタイピング法が挙げられる（「ポストシークエンスのゲノム科学（１）　ＳＮＰ遺伝子多型の戦略　第一刷（中山書店）９４頁〜９７頁、１１８　頁〜１２７　頁」参照）。
【０２１８】
ＴａｑＭａｎ　ＰＣＲ法を利用したＳＮＰタイピングの一例では、コンティグ（１）〜（５３４）の何れかの一部であって、多型部位の一つを含む約２０ヌクレオチドからなる配列の相補配列をオリゴヌクレオチドＡとし、このオリゴヌクレオチドＡの５’端、３’端を順に蛍光色素、クエンチャー（消光物質）によりラベルしてＴａｑＭａｎプローブを作製する。なお、この状態のＴａｑＭａｎプローブは、クエンチャーの働きにより蛍光を発しない。次いで、当該コンティグにおける上記多型部位を含む領域およびその相補領域を増幅可能に設計されたＰＣＲプライマーと、Ｔａｑ　ＤＮＡポリメラーゼとを用い、上記ＴａｑＭａｎプローブを被検体のゲノムＤＮＡまたはｃＤＮＡにハイブリダイズさせた状態でＰＣＲ反応を行う。Ｔａｑ　ＤＮＡポリメラーゼは５’ヌクレアーゼ活性を有するので、ＴａｑＭａｎプローブが被検体のゲノムＤＮＡまたはｃＤＮＡに実際にハイブリダイズしていれば、蛍光色素が切り出されて蛍光が観測される。よって、上記多型部位に相補する塩基を適宜選択してＴａｑＭａｎプローブを作製すれば、被検体のゲノムＤＮＡまたはｃＤＮＡにおける多型部位の塩基の種類を、蛍光を発するか否か（すなわち両者が完全にハイブリダイズするか否か）で判定可能となる。
【０２１９】
また、ＲＣＡ法を利用したＳＮＰタイピングの一例では、まず、被検体のゲノムＤＮＡまたはｃＤＮＡ上の多型部位を含む周辺約２０ヌクレオチドずつの配列に対する相補配列が両端部に配され、この２つの相補配列をバックボーンと呼ばれる特定配列により連結した一本鎖プローブ（バドロックプローブ）を作製する。また、バドロックプローブは、その一端（通常３’端）に、上記多型部位の塩基に相補的な塩基が位置するように設計される。次いで、被検体のゲノムＤＮＡまたはｃＤＮＡを鋳型としてバドロックプローブをライゲーションし、適切なプライマーとＤＮＡポリメラーゼとの組み合わせを用いて増幅反応を行う。
【０２２０】
ここで、鋳型となるゲノムＤＮＡまたはｃＤＮＡの多型部位の塩基と、バドロックプローブの一端の塩基とが実際に相補的である場合には、バドロックプローブのライゲーション（ここでは環状化）及び増幅反応が起こり、多型部位の塩基をタイピングすることができる。一方、非相補的である場合には、このライゲーション及び増幅反応が起こらない。なお、ＲＣＡ法は一定温度（通常約６５℃）でのＤＮＡ増幅反応が可能であるので、サンプルの大量解析に特に好適である。また、上記バドロックプローブは、環状化した場合にのみオリゴヌクレオチドＡとなる配列（両端部に配された上記相補配列）を含有するが、このような配列も本発明にかかるオリゴヌクレオチドＡの範疇に含まれる。
【０２２１】
また、オリゴヌクレオチドＡをＰＣＲプライマーの一つとし、当該オリゴヌクレオチドＡと被検体由来のＤＮＡとのハイブリダイズ（アニーリング）、及び、ＰＣＲ法による増幅反応を試み、増幅ＤＮＡ断片が生じるか否か（すなわち両者が完全にハイブリダイズするか否か）でＳＮＰをタイピングする方法（例えば、ＡＳＡ（ａｌｌｅｌｅ−ｓｐｅｃｉｆｉｃ　ａｍｐｌｉｆｉｃａｔｉｏｎ）　法）も上記間接法の一例である。
【０２２２】
間接法で使用されるＳＮＰタイピング用キットはいずれもオリゴヌクレオチドＡを含んでなるため、本発明にかかる組成物に相当する。つまり、本発明にかかる組成物には、１）ＤＮＡチップのようにオリゴヌクレオチドＡが担体に固定化されてなるものも、２）ＴａｑＭａｎ　ＰＣＲ法用、Ｉｎｖａｄｅｒ　法用、ＲＣＡ法用などのＳＮＰタイピング用キットのように、一般にオリゴヌクレオチドＡが固定化されていないものも含まれる。
【０２２３】
すでに説明したように、上記オリゴヌクレオチドＡを用いた方法、およびその他のＳＮＰタイピング法を用いれば、種々のオオムギ属植物（特に、オオムギ品種である「Ｈ．ｓｐｏｎｔａｎｅｕｍ」、「赤神力」および「はるな二条」）について上記多型部位またはその相補部位の塩基を判定することができる。多型部位またはその相補部位における塩基の種類は、オオムギの同一品種内で実質的に同一であり、特定の異なる品種間で相違が見られるので、オオムギの品種識別が可能となる。
【０２２４】
例えば、本発明にかかるＳＮＰタイピング法のいずれかを用いて、「Ｈ．ｓｐｏｎｔａｎｅｕｍ」、「赤神力」および「はるな二条」由来のＤＮＡ上における多型部位、例えば、コンティグ（６）（配列番号６に示すＤＮＡ配列）上の１０８３番目のヌクレオチド（多型部位）に相当する部位の塩基を同定した結果、アデニンの場合には「Ｈ．ｓｐｏｎｔａｎｅｕｍ」と、チミンの場合には「はるな二条」または「赤神力」と判定される。
【０２２５】
なお、オオムギや、オオムギ属植物以外でも同様のＳＮＰが認められる可能性が高い生物（ＳＮＰタイピング用の被検体の例示参照）については、品種識別ができる可能性がある。また、これら多型部位（またはその相補部位）を２つ以上組み合わせて品種識別を行う、あるいは、他の遺伝子マーカーと併せて品種識別を行えば、識別精度がより一層向上する。なお、ＳＮＰなど遺伝子上の多型情報を用いた品種識別は、例えば、オオムギ粒、オオムギ加工食品（デンプンなど）の品質精度の検定（異種オオムギ混入有無の確認）など、肉眼では困難な作業に特に効果が大きい。
【０２２６】
（Ｇ）遺伝子マーカーとしてのＳＮＰの利用
また、遺伝子とそのアリルとで上記多型部位（相補部位の場合もある）における塩基が相違し、その結果、異なる表現型を与える場合には、（１）所望する表現型に関与する遺伝子またはアリルの一方を取得する、（２）当該遺伝子またはアリルの一方を有する植物を選別する、などの目的で、ＳＮＰを遺伝子マーカーとして使用できる。以下、上記（１）・（２）のケースにつき詳細に説明するが、便宜上、多型部位の塩基が「赤神力」型、「はるな二条」型、または「Ｈ．ｓｐｏｎｔａｎｅｕｍ」型の遺伝子の何れか一つを単に「遺伝子」と称し、当該「遺伝子」と多型部位の塩基が異なる（遺伝子型が異なる）ものをその「アリル」と称する。また、遺伝子とそのアリルとでは、遺伝子がより有用な表現型を与える（すなわち有用遺伝子である）と仮定する。
【０２２７】
上記（１）の場合には、一般に、アリルまたは遺伝子の一方のみにハイブリダイズするオリゴヌクレオチドＢをプローブとして使用し、例えば、オオムギのゲノムＤＮＡライブラリーまたはｃＤＮＡライブラリーを公知の方法によりスクリーニングして上記遺伝子（有用遺伝子）を取得（単離）すればよい。あるいは、このようなオリゴヌクレオチドＢをプライマーとする遺伝子増幅法（ＰＣＲ法など）を用いて、例えば、オオムギのゲノムＤＮＡライブラリーまたはｃＤＮＡライブラリーから当該遺伝子を含むクローンを同定し、この遺伝子を取得すればよい。
【０２２８】
単離した上記遺伝子は、公知の遺伝子工学的手法（遺伝子操作技術）により、対象細胞（宿主細胞）内に発現可能に導入されて、当該遺伝子を発現する形質転換体が生産される。上記の遺伝子工学的手法として、具体的には、例えば、プロトプラスト共存培養法やリーフディスク法などアグロバクテリウム属細菌の感染を利用する方法、並びに、ポリエチレングリコール法、エレクトロポレーション法、マイクロインジェクション法、パーティクルガン法、リポソーム法、ＤＮＡ直接導入法、適切なベクター系を用いた導入法、などが挙げられ、上記対象細胞の種類に応じて最適なものを選択すればよい。
【０２２９】
上記対象細胞としては、大腸菌などの原核細胞、または植物細胞などの真核細胞が使用されるが、導入される遺伝子がオオムギ由来であることから、好ましくは植物細胞が、より好ましくはコムギ連の植物細胞が、特に好ましくはオオムギを含むオオムギ属植物の細胞が使用される。また、原核細胞を選択する場合には、上記遺伝子としてｃＤＮＡを選択すればよい。
【０２３０】
なお、上記遺伝子とそのアリルとで与える表現型が実質的に同一の場合や、当該遺伝子のホモログを取得する場合などには、これらを非選択的に単離可能なオリゴヌクレオチドＢを遺伝子取得用のプローブまたはプライマーとしてもよい。また、当該遺伝子、そのアリル、そのホモログは、オオムギ属植物（特に、オオムギ品種である「Ｈ．ｓｐｏｎｔａｎｅｕｍ」、「赤神力」または「はるな二条」）から好適に単離されるが、その他、本発明にかかるオリゴヌクレオチド（より好ましくは各コンティグまたはその相補配列）を部分配列または全長とする遺伝子、あるいはそのホモログを持つ生物からも単離可能である。
【０２３１】
また、上記遺伝子（有用遺伝子）の種類は特に限定されないが、栽培オオムギの祖先野生型オオムギ（Ｈ．ｓｐｏｎｔａｎｅｕｍ）が有する有用遺伝子などが特に好適であり、当該有用遺伝子が導入される上記対象細胞は、栽培オオムギの細胞が特に好適である。
【０２３２】
一方、上記（２）の場合には、すでに説明したＳＮＰタイピング法のいずれかに従って多型部位（または相補部位）の塩基を特定し、これをもとに所望の遺伝子またはアリルの何れか（一般には有用遺伝子である「遺伝子」の方）を備えた植物を選別すればよい。また、このような植物の選抜作業は、例えば、１）上記有用遺伝子を持つ交配親を選抜する際、２）戻し交雑の過程で得られた雑種（形質転換体；形質転換植物）群から、戻し親にさらに交配すべき雑種候補または当該有用遺伝子が安定的に保持された新品種を選別する際、など植物育種の分野において主に行われる。
【０２３３】
以下、交配により形質転換体を生産する具体的な方法につき説明を行う。交配に供される両親（親植物Ａ・Ｂとする）の組み合わせは、一方（親植物Ａ）が上記遺伝子（以下、有用遺伝子と称する）を有し、他方（親植物Ｂ）がこの有用遺伝子を有さない、あるいは当該有用遺伝子のアリルを有する関係にあり、かつ、互いに交配可能な植物種であればよい。
【０２３４】
換言すれば、親植物Ａとしては、本発明にかかるオリゴヌクレオチド（より好ましくは各コンティグまたはその相補配列）を部分配列または全長とする有用遺伝子を持つ植物であれば特に限定されるものではないが、当該有用遺伝子を持つ可能性が高いという観点から、コムギ連の植物がより好ましく、オオムギ属植物（例えば、オオムギ品種である「Ｈ．ｓｐｏｎｔａｎｅｕｍ」、「赤神力」または「はるな二条」）がさらに好ましく、中でも、栽培オオムギにない遺伝子を有する祖先野生型オオムギ（Ｈ．ｓｐｏｎｔａｎｅｕｍ）が特に好ましい。
【０２３５】
一方、親植物Ｂとしては、上記有用遺伝子を持たない、あるいは有用遺伝子のアリルを有し、かつ親植物Ａと交配可能な植物であれば特に限定されるものではないが、コムギ連の植物（親植物Ａの好適な一例）と交配が容易であるという観点から、コムギ連の植物がより好ましく、オオムギを含むオオムギ属植物がさらに好ましい。なお、いうまでもないが、親植物Ａ・Ｂは互いに異なる品種である。また、コムギ連に属する異種植物間の交配は、オオムギ属植物、コムギ属植物、ライムギ属などの間で知られている（最新バイオテクノロジー全書（１）　穀物・いも・牧草類の増殖と育種　１１２　頁　農業図書（株）１９９２年発行　参照）。
【０２３６】
さらに、親植物Ａが「Ｈ．ｓｐｏｎｔａｎｅｕｍ（祖先野生型オオムギ）」で、親植物Ｂが「はるな二条」型、または「赤神力」型の多型部位を持つ栽培オオムギである組み合わせは、交雑が容易であり、かつ、雑種後代が不稔となる虞もないので特に好適である（同種異品種間の交雑のため）。また、栽培品種の遺伝的背景下でのみ良好に発現する、祖先野生型植物の遺伝子の存在も知られているので、祖先野生型オオムギと栽培オオムギとを交雑する意義は極めて大きい。
【０２３７】
また、上記「交配」が戻し交雑の場合、上記の親植物Ａ・Ｂは、順に、一回親・反復親（戻し親）の関係にある植物であってもよく、あるいは、順に、戻し交雑の結果得られた雑種と、この雑種を戻し交雑する反復親との関係であってもよい。
【０２３８】
上記「交配」の目的は、親植物Ｂの遺伝学的背景を持ち、かつ、親植物Ａが有する有用遺伝子が導入された実用系統の選別・確立を行う点にある。換言すれば、親植物Ｂの劣悪形質が排除され、親植物Ａの有用特性のみが導入された実用系統の選別・確立を行う点にある。よって、交配の過程では、親植物Ａ・Ｂの交配により作出された雑種の植物群から、当該有用遺伝子を有する雑種を選抜する工程（選抜工程）が適宜行われる。
【０２３９】
上記の選抜工程では、上記有用遺伝子が、本発明にかかるオリゴヌクレオチドを部分配列または全長として有することを利用する。例えば、親植物Ｂが上記有用遺伝子のアリルを有する場合には、すでに説明したＳＮＰタイピング法の何れかを用いて、得られた雑種の植物群から好適な雑種を選抜すればよい。このような遺伝子マーカーを用いた選抜方法は、上記雑種を育成して表現型を実際に確認する必要がなく、例えば、当該雑種の種子を用いた選抜が可能なので、迅速かつ簡便であるという利点を有する。
【０２４０】
また、親植物Ｂが上記有用遺伝子やそのアリルを有しない場合には、上記ＳＮＰタイピング法を用いる以外に、上記雑種由来のＤＮＡまたはＲＮＡと、本発明にかかるオリゴヌクレオチド（ＳＮＰ検出用のオリゴヌクレオチドＡ以外も含む）とがハイブリダイズするか否かで有用遺伝子の導入を確認することもできる。
【０２４１】
なお、上記の選抜工程で選抜される雑種は、例えば、１）戻し交雑の過程で、戻し親にさらに交配すべき雑種候補や、２）上記有用遺伝子が安定的に保持された新品種、に相当するが、これらの雑種は、実質的に、親植物Ａから上記有用遺伝子のみを受け継いでいる（親植物Ａの劣悪形質は受け継がない）ことが望まれる。したがって、本発明で述べたＳＮＰ以外の遺伝子マーカー（他の遺伝子上にあるＳＮＰでもよい）も用いて、親植物Ａ・Ｂの持つ様々な染色体部分を同定可能な分子マップ（染色体マップ）を作製し、このマップ情報を基に、得られた雑種群（染色体置換系統群）から上記有用遺伝子のみが実質的に導入された雑種を選別することが特に好ましい。なお、分子マップは、例えば、親植物Ａ・Ｂの倍加半数体系統群から作製すればよい。
【０２４２】
また、上記「形質転換体あるいは形質転換植物」とは、交配や遺伝子工学的手法を用いて作出され、上記有用遺伝子を有する雑種を指す。また、形質転換体、形質転換植物の範疇には、生物個体；根、茎、葉、生殖器官（花器官および種子を含む）などの各種器官；各種組織；細胞；などが含まれ、さらにはプロトプラスト、スフェロプラスト、誘導カルス、再生個体およびその子孫、なども含まれるものとする。
【０２４３】
【実施例】
以下、本発明を実施例により詳細に説明する。なお、本発明は、以下の実施例の記載に限定されるものではない。
【０２４４】
〔実施例１：オオムギからのｍＲＮＡの抽出、ｃＤＮＡライブラリーの作製〕オオムギとして「赤神力」、「はるな二条」、および「Ｈ．ｓｐｏｎｔａｎｅｕｍ（ＯＵＨ６０２）」（野生型オオムギ）を用いた。これらオオムギの種子は、細胞工学別冊　植物細胞工学シリーズ１４　植物のゲノム研究プロトコール　１９３　〜１９５　頁（秀潤社（２０００　年）　参照）の記載に従って発芽・生育させ、ＲＮＡ抽出用の材料を得た。なお、「はるな二条」については、発芽時の芽、幼苗の葉身、および止葉期の上位３葉を材料とし、「赤神力」については、栄養成長期の葉身を材料とした。また、「Ｈ．ｓｐｏｎｔａｎｅｕｍ（ＯＵＨ６０２）」については、止葉期の上位３葉を材料とした。
【０２４５】
次いで、セパソールＲＮＡ（Ｓｅｐａｓｏｌ　ＲＮＡ）（ナカライテスク社製）を用い、キットの説明書記載の方法に従って、上記材料から全ＲＮＡを抽出した。さらに、ｍＲＮＡ精製キット（ｍＲＮＡ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ）（ａｍｅｒｓｈａｍ　ｐｈａｒｍａｃｉａ　ｂｉｏｔｅｃｈ社製）　を用い、キットの説明書記載の方法に従って、全ＲＮＡからｍＲＮＡのみを抽出した。
【０２４６】
ｃＤＮＡライブラリーの作製は、λＺＡＰ　−ｃＤＮＡ合成キット（Ｓｔｒａｔａｇｅｎｅ　社製）　を用い、以下に示す変更点（ａ）及び（ｂ）を除いて「ゲノム解析ラボマニュアル　ｐ６３−ｐ７５　：シュプリンガー・フェアラーク東京（１９９８年発行）」に記載された方法に従って行った。
【０２４７】
（ａ）ｃＤＮＡ末端の平滑化ステップにおける２回目の遠心分離工程（ゲノム解析ラボマニュアル　ｐ７１　参照）では、ｃＤＮＡを含んだ水層に、酢酸ナトリウム溶液（３Ｍ）２０μｌと１００％エタノール４００μＬとを加えてボルテックスで十分に混ぜた後、−２０℃で一晩静置（オーバーナイト）し、次いで、４℃、１５，０００ｒｐｍ　の条件で６０分間遠心分離するように条件を変更した。
【０２４８】
（ｂ）ＸｈｏＩによる切断ステップ（ゲノム解析ラボマニュアル　ｐ７２参照）にて得られた反応液は、−２０℃で一晩静置後に、４０℃、１５，０００ｒｐｍの条件で６０分間遠心分離を行った。その後、上澄みを捨てて完全にペレットを乾かした後、これをＳＴＥ（１×）１４μｌに溶解させ、さらに、カラムローディングダイ（ｃｏｌｕｍｎ　ｌｏａｄｉｎｇ　ｄｙｅ）３．５μＬを加えてベクターへのライゲーションステップに供した。
【０２４９】
なお、作製したｃＤＮＡライブラリーのタイターは１０^６ｐｆｕ　以上であり、また、インサートチェックレディー（Ｉｎｓｅｒｔ　Ｃｈｅｃｋ　−Ｒｅａｄｙ−）（ＴＯＹＯＢＯ　社製）　を用い、キット記載の方法により測定した、クローン内の平均インサート（ｃＤＮＡ挿入断片）サイズは、平均約１．５ｋｂ、最大６ｋｂ以上とみられた。
【０２５０】
〔実施例２：ｃＤＮＡ挿入断片の解析〕
マスエキサイション（Ｍａｓｓ　ｅｘｃｉｓｉｏｎ）法（ＺＡＰ−ｃＤＮＡ　Ｇｉｇａｐａｃｋ　ＩＩＩ（Ｓｔｒａｔａｇｅｎｅ社製）Ｇｏｌｄ　ｃｌｏｎｉｎｇ　ｋｉｔ　インストラクションマニュアル参照）により、ｃＤＮＡライブラリーから切り出した各クローンをＬＢ培地で一晩培養し、プラスミドＤＮＡを抽出した。
【０２５１】
次いで、上記プラスミドＤＮＡが有するｃＤＮＡ挿入断片について、５’末端側および３’末端側の双方からシークエンス解析を行った。シークエンス解析には、ＡＢＩ　ＰＲＩＳＭ　ＴＭ３７００遺伝子分析機（ＡＢＩ　ＰＲＩＳＭ　ＴＭ　３７００　Ｇｅｎｅｔｉｃ　Ａｎａｌｙｚｅｒ）（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ社製）　を用いた。さらに、シークエンス解析の結果から各クローンの重複程度を調べるために、クローンのクラスタリングを行った。あわせて、各クローンが有する挿入断片をアセンブリして、各コンティグ（１）〜（５３４）を作製した。また、これらの挿入断片をマルチプルアライメントして、異なるクローン間でのＳＮＰを検出した。
【０２５２】
クローンのクラスタリング、挿入断片のアセンブリ、及びＳＮＰ検出用のソフトウエアとしては、ｄｙｎａｃｌｕｓｔ　ＳＮＰ　ｅｘｐｌｏｒｅｒ（ｄｙｎａｃｌｕｓｔ　ｖｅｒ．　４追加モジュール：株式会社　ダイナコム製）を使用した。より詳細には、当該ソフトウエア内蔵のｂｌａｓｔｎプログラムを用い、Ｅｘｐｅｃｔ　Ｖａｌｕｅを１ｅ^？１０　に設定して、クローンのクラスタリングを行った。また、ベースコーリング時の各塩基の読み取りエラー確率を一定とし（Ｐｈｒｅｄ　スコア＝１５（デフォルト値））、アセンブリツールとしてＰｈｒａｐ　を採用して、表１１１及び表１１２に示すパラメータ設定に従い挿入断片をアセンブリした。
【０２５３】
【表１１１】

【０２５４】
【表１１２】

【０２５５】
【発明の効果】
本発明に係るオリゴヌクレオチドは、以上のように、配列番号１〜５３４の何れかに表されるＤＮＡ配列からなるオリゴヌクレオチドの一部または全長であって、ユニバーサルコードで示された多型部位のうち、非同義置換を生ぜしめる多型部位の少なくとも一つを含む７ヌクレオチド以上の連続したＤＮＡ配列、またはその相補配列からなる構成である。
【０２５６】
上記のオリゴヌクレオチドは、オオムギの品種の相違に起因する上記多型部位またはその相補部位を含むため、例えば、種々のオオムギ品種間に存在する一塩基多型を検定する目的で利用可能である。また、オオムギの遺伝学的品種識別や、当該オオムギがいずれのタイプの遺伝子を有するかの判定等が可能になるという効果を奏する。
【０２５７】
さらに、上記のオリゴヌクレオチドをプローブまたはプライマーとして有用遺伝子を単離し、この有用遺伝子を遺伝子操作技術を用いて対象とする細胞に導入する方法、あるいは、親植物を交配させて得られた雑種の植物のうち有用遺伝子を持つものを、上記のオリゴヌクレオチドを用いて選抜する方法により、所望する形質転換体を生産することができるという効果を奏する。
【０２５８】
【配列表】[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to barley genes. More specifically, by clarifying the differences in the nucleotide sequences of the genes between barley varieties, and by clarifying the characteristics of the nucleotide sequences between the barley gene varieties, and utilizing the results for testing and identification of barley varieties, And so on, so that plant varieties such as plants can be efficiently improved.
[0002]
The characteristics of the nucleotide sequence of the barley gene of the present invention among varieties relate to single nucleotide polymorphism (SNP) related to the difference in barley varieties, and the present invention is characterized by such a gene. The present invention relates to a nucleotide for utilizing the method and a method using the same.
[0003]
[Prior art]
Grain plants such as wheat and rice are plants that have been cultivated since ancient times as food sources for humankind. In Japan, wheat has been used as a general term for wheat and barley as a winter crop of the Poaceae family, but in other countries, it is classified into wheat, barley, rye, and oat.
[0004]
Barley is a plant belonging to the genus Barde (Hordeum), and its place of origin is said to be Central Asia. Barley has been divided into two types: barley and naked, and barley, based on the streaking of the ears, the bareness of the grains, and the degree of brittleness of the cobs. Is coming. In general, the term "barley" refers to the six-row type barley, and the two-row type barley is also called beer wheat.
[0005]
The most common use of barley is for feed. Most of the barley for feed is used for grain, but in the Middle East and inland of Australia, barley is also used as glazing for cultivation on pasture and feed. As a special use form, Cyprus selects and cultivates shedding lines, and uses grassland that regenerates naturally from seeds that fall off after maturity. Breeding targets for feed barley are yield-related traits such as disease resistance and lodging resistance, and breeding related to quality is scarce. Barley is used mainly for food in the Asian region, and even in the Himalaya area, six-row naked ham is still used as a staple food. In these areas, native species are mainly cultivated, and due to the high altitude and dryness, the occurrence of diseases is small and the yield is relatively high. In addition, in the case of powdered food, the appearance of grains is hardly selected, so that those having extremely various variations in ear shape and grain color are also used.
[0006]
In Japan, edible barley is of six-row type. In particular, the eddy barley is a semi-dwarf barley whose morphology is close to that of rice grains, mainly in western Japan. A typical example of such a naked mussel is a variety “Akagami power” that has high yield, is moisture-resistant, and is resistant to pests. This variety was cultivated very much around 1955, and is frequently used as a breeding mother in the current breed improvement field. For example, “Yunagi-Nakadaka”, which was designated as a recommended variety in 1983, has a high yield, is resistant to moisture, and has relatively strong resistance to pests. This variety is “Akami Riki” and “Kagawa Naked. It is an excellent variety produced by crossing with No. 1.
[0007]
On the other hand, beer barley generally requires: 1) its appearance is pale yellow, glossy, and its grains are large and uniform; 2) its germination power is strong and uniform (germination rate is 95% or more). 3) a high starch value and an appropriate protein content; 4) a strong enzymatic activity; and 5) an appropriate content of β-glucan, polyphenol and the like. At present, the varieties considered to be the best as beer barley satisfying these requirements are "Haruna Nijo". Haruna Nijo boasts the world's highest quality beer barley and is widely used as a breeding mother in many countries. This variety is a fixed variety bred by crossing “Seijo No. 15” with a cross line of “G65” and “K-3”, and serves as an index of the quality of beer barley. However, there are drawbacks in that they are inferior in shedding performance, lodging resistance and raw wheat yield, and have low resistance to stripe wilt, Fusarium head blight and powdery mildew. Therefore, crossing with resistant varieties is often performed. For example, the disease-resistant “Nishino Gold” is backcrossed between “Kishiishi Port 3”, which is a stripe wilt resistant variety, and “Azuma Golden”. And crossed with "Haruna Nijo", which is the main cultivar, twice. This is an example of a procedure for introducing a single gene resistant to stripe dwarf disease, which took 20 years.
[0008]
In recent years, the required quality is high, as well as the yield, and the quality has been standardized, and new varieties have been cultivated and spread based on agreements with consumers. Various crops are being improved by gene transfer. However, since barley is difficult to redifferentiate from protoplasts and has a genome size more than 10 times that of rice, it is a groundbreaking and essential technology, especially for crops where genetic analysis is stagnant. One.
[0009]
Since all barley chromosome numbers can be crossed with each other at 2n = 14, "Amagagi Nijo,""HarunaNijo,""NarasakiNijo,""EggplantNijo,""ToneNijo," and "Myogi Nijo." , "Komaki Nijo", "Kinuka Nijo", "Takaho Golden" and many other varieties have been produced by the cross breeding method.
[0010]
However, the conventional breeding method is a method in which a plurality of useful traits are newly combined by crossing and selecting individuals having useful traits in a complementary manner. Is introduced at the same time, so that it takes many generations of time and trial and error to remove unnecessary traits and fix only good traits. Furthermore, since it is not possible to predict what combinations will appear, a large number of individuals must be cultivated, and a vast area is required for breeding.
[0011]
On the other hand, in recent breeding methods using genetic engineering, it is not necessary to repeat crossing, so that it is possible to improve crops suitable for the purpose in a shorter time than before. For example, a transformed plant can be prepared by cloning a gene relating to a specific trait and introducing it into a plant. Theoretically, the relationship between the function of the gene to be introduced and the phenotype is clear, only the desired trait can be introduced, and the variety can be systematically improved according to the purpose.
[0012]
Breeding using genetic markers is also one of breeding using genetic engineering. Gene markers are DNA bands that are linked to useful genes and always appear when the trait is expressed. Techniques for obtaining a gene marker include the RFLP method, the RAPD method, the AFLP method, the PCR-RFLP method, and the like. For example, in the RAPD method, DNA is amplified by performing PCR using DNA having an arbitrary sequence as a primer. In order to detect the DNA band pattern, the amplified DNA is stained and then electrophoresed on an agar gel to analyze the DNA band pattern. Then, a specific polymorphic band is investigated, and if the band moves in the same manner as a useful trait, it can be used as a gene marker. If selection is performed using genetic markers in the cross breeding method, it is possible to reduce the labor required for breeding and to speed up breeding.
[0013]
The above-mentioned genetic markers are used to look at genetic polymorphisms between varieties having different traits, and are also referred to as first-generation genetic polymorphism markers. It takes enormous effort to find each of these genetic markers. In addition, the number of gene markers present in the genome is limited to tens of thousands, and information obtained genetically is limited.
[0014]
As a 21st century polymorphism marker, single nucleotide polymorphism (hereinafter, also simply referred to as SNP) has attracted attention. The SNP is one in which one base is replaced by another base between varieties, and it is considered that there are tens of thousands to millions of SNPs in the genome. Despite the large number of SNPs, the SNP is very easy to judge, and the result can be converted into a digitized signal of (0, 1), so that information processing is easy. In addition, since high-speed and large-volume SNP typing technology is being put into practical use and automation of typing is in the horizons, SNPs are attracting attention as extremely useful and easy-to-use polymorphic markers.
[0015]
Not all SNPs in the genome are necessarily important, and may affect the expression levels of SNPs (cSNPs (coding SNPs)) involving amino acid changes in the translated regions of proteins, and the expression levels of genes such as promoter regions and introns. SNPs in the regulatory region exerted (rSNP (regulatory SNP)) and the like are extremely likely to be accompanied by phenotypic changes and are considered important SNPs. In addition, SNPs (ssNPs (silent SNPs)) that do not involve amino acid changes in the translation regions of proteins and SNPs (gSNPs (genome SNPs)) that are not associated with structural genes are less relevant to the phenotype. It is considered of low importance. Therefore, if attention is paid only to cSNP and rSNP among many SNPs, it becomes extremely useful as a polymorphic marker between varieties.
[0016]
In order to obtain an overview of the relationship between genes expressed in barley and their useful traits, barley having useful traits, such as Haruna Nijo and Akagami Power, which are used as breeding mothers, have It is efficient to investigate SNPs at sites related to structural genes among barley varieties. Thus, if cSNPs and rSNPs that are stably recognized among barley varieties can be found, it is expected to be useful for barley gene mapping and breeding improvement. For example, introduction of unnecessary traits in the cross breeding method Can be used as markers to select promising individuals by selecting varieties with low numbers.
[0017]
With a set of SNPs obtained from analysis of all expressed genes, it is easily presumed that some of the SNPs are related to useful traits. Therefore, this set of SNPs can be used as a marker used for breed improvement.
[0018]
However, since the genome size of barley (Hordeum) is about 5,000 Mbp, which is more than 10 times that of rice, and its relationship with structural genes has not been clarified, barley SNP analysis should be performed based on the results of genome analysis. Is in a difficult situation.
[0019]
A part of the EST sequence data that the present inventors have clarified in a barley EST project described later has already been registered in a data bank (see Non-Patent Document 1 below).
[0020]
[Non-patent document 1]
Telecommunication line "National Institute of Genetics Life Information and DDBJ Research Center Japan DNA Data Bank" (accession number: AV938080, etc.)
[0021]
[Problems to be solved by the invention]
The present invention has been made in view of the above problems, and specifically relates to “Haruna Nijo” and “Akagami Power”, which are used as breeding mothers, and to a structural gene between varieties with wild barley. The purpose is to clarify the features of the nucleotide sequence of the gene at the site.
[0022]
Further, the present invention clarifies the characteristics of the nucleotide sequence of the gene between the varieties by using a single nucleotide polymorphism (SNP) of the gene, and discloses the usefulness of the oligonucleotide including the site of the SNP. It is another object of the present invention to provide a method for using the SNP widely as a polymorphic marker between varieties. Furthermore, an object of the present invention is to disclose a breeding method using the SNP.
[0023]
[Means for Solving the Problems]
Since the genome size of barley (Hordeum vulgare) is about 5,000 Mbp, which is more than 10 times that of rice, and its relationship with structural genes has not been clarified, barley SNP analysis cannot be performed based on the results of genome analysis. Although it is a difficult situation, the present inventors have studied the analysis of SNP based on the results of an EST (expressed sequence tag) project using a cDNA sequence from a barley cDNA library. Among them, it was found that many SNPs among varieties were stably recognized.
[0024]
That is, in the EST project of barley (Hordeum vulgare), the present inventors cloned cDNA synthesized by using mRNAs extracted from tissues of a plurality of varieties as a template, and then succeeded in a series of clone groups having overlapping inserted fragments. And analyzed so as to be a sequence as one clone (hereinafter, the sequence assembled in this manner is collectively referred to as “CONTIG”). However, it was found that even if the analysis was proceeded correctly, there was a place where one kind of base could not be determined, and this was confirmed to be a difference in base type (SNP) between barley varieties. Since this contig sequence was prepared from a cDNA library, it corresponds to a region (part or all of an exon) actually expressed in the genome of barley, and among the SNPs, cSNP Is very high. The present inventors further analyzed the obtained contig sequence, and in particular, selected single nucleotide polymorphisms that cause nonsynonymous substitutions, and found important SNPs among barley varieties.
[0025]
That is, the present invention relates to a polymorphic site in which a non-synonymous substitution occurs in the DNA sequence represented by any one of SEQ ID NOs: 1 to 534, among the polymorphic sites in which the notation of a base is described by “universal code”. An oligonucleotide consisting of a continuous DNA sequence of 7 nucleotides or more containing at least one of the above, or a complementary sequence thereof, and a single nucleotide polymorphism generated in a gene comprising at least one of the oligonucleotides is assayed or identified. To a composition for:
[0026]
In addition, the present invention relates to a polymorphic site in which a non-synonymous substitution is caused among the polymorphic sites in which the notation of a base is described in the “universal code” in the DNA sequence represented by any of SEQ ID NOs: 1 to 534. Or a method for assaying or identifying a single nucleotide polymorphism generated in a gene of a subject based on at least one of its complementary sites.
[0027]
Furthermore, the present invention provides a DNA sequence represented by any one of SEQ ID NOs: 1 to 534, wherein, among the polymorphic sites in which the notation of a base is described by a “universal code”, a polymorphic site causing non-synonymous substitution Or a method for producing a transformant comprising a step of isolating a gene containing at least one of its complementary sites, and a step of introducing the obtained gene into cells of interest using genetic engineering techniques, and The present invention relates to a transformant produced by the method.
[0028]
In addition, the present invention relates to a polymorphic site in which a non-synonymous substitution is caused among the polymorphic sites in which the notation of a base is described in the “universal code” in the DNA sequence represented by any of SEQ ID NOs: 1 to 534. Or a parent plant A having a gene containing at least one of its complementary sites and a parent plant B not having this gene or having an allele in which a single nucleotide polymorphism has occurred in this gene, is hybridized. And the step of producing a plant having the gene from the hybrid plant using at least one of the oligonucleotides according to claim 1 or the assay method according to claim 3. And a step of selecting using the transformant, and a transformant produced by the method.
[0029]
Then, the present invention generates non-synonymous substitutions among the polymorphic sites in which the bases are described in the “universal code” in the polynucleotide consisting of the DNA sequence represented by any one of SEQ ID NOs: 1 to 534. The present invention relates to a primer designed to amplify a region containing at least one polymorphic site to be amplified or at least one of its complementary regions.
[0030]
The term “polymorphic site causing non-synonymous substitution” in the present invention refers to a polymorphic site corresponding to any of the following (a) or (b).
(A) The amino acid encoded by the codon containing the polymorphic site is replaced with another amino acid, or the codon containing the polymorphic site is changed to a stop codon according to the difference in the base at the polymorphic site. Different polymorphic sites.
(B) an amino acid encoded by a codon containing the complementary site is replaced by another amino acid, or a codon containing the complementary site is changed to a stop codon, depending on the difference in bases at the complementary site of the polymorphic site The polymorphic site that will be
[0031]
That is, (a) above is the case where the DNA sequence described in the sequence listing is a sense strand and the polymorphic site itself causes non-synonymous substitution of amino acids. On the other hand, the above (b) is the case where the complementary sequence is a sense strand and the complementary site of the polymorphic site causes non-synonymous substitution of amino acids.
[0032]
The “universal code” in the present invention refers to four types of A, G, C and T at the site where the above-mentioned “contig” (CONTIG) could not be determined as a specific type of base as a result of assembly. Is a code for expressing one of two or more kinds of bases with one letter, and each letter is defined as follows.
[0033]
R = A or G:
Y = C or T:
M = A or C:
K = G or T:
S = G or C:
W = A or T:
H = A or T or C:
B = G or T or C:
V = G or A or C:
D = G or A or T:
N = A or C or G or T.
[0034]
In the present specification (including the sequence listing), differences between bases in “Haruna Nijo”, “Red God Power”, and wild-type barley are expressed by the “universal code” defined above.
[0035]
In the present invention, the term "polymorphism" means that a base present at a corresponding position on DNA (on cDNA or on genomic DNA) is between varieties of barley, and more specifically, in the present invention, "Haruna nijo (Hordeum)" vulgare var.), "Akami Riki (Hordeum vulgare var. one cultivar name)", and wild type barley. In other words, the term “polymorphism” refers to a polymorphism observed between a specific gene and its allele (or between the complementary sequences thereof). In particular, in the present invention, stably between barley varieties. It refers to each of the conserved single nucleotide polymorphisms in the DNA sequence.
[0036]
The “polymorphism” in the present invention includes barley varieties “Haruna Nijo” and “Akagami Power”, and “wild-type barley (Hordeum vulgare ssp. Spontanum (Okayama University Institute of Resources and Biological Sciences, Barley and Wild Plant Resources Research Center registration number)”. OUH602)) (hereinafter, referred to as H. spontanium). "Haruna Nijo" is a cultivar widely spread as a Nijo barley for brewing beer, and "Akagami Riki" is a cultivar known as edible Rojo barley. "H. spontanum" is a wild barley (ancestor wild type) which is regarded as an ancestral type of barley belonging to Nijo barley native to West Asia. In other words, these three varieties of barley are of the same genus and homogenous but have genetic differences, and this difference in genes characterizes the useful traits of "Haruna Nijo" and "Akagami Power". More specifically, for example, due to the difference in bases at the polymorphic site, an environmental resistance gene (such as a disease and insect resistance gene or a poor environmental resistance gene) is expressed in “H. spontanum”, and “Haruna Nijo” or “Red It is possible that the allele is expressed in the "god power". In addition, it is possible that a gene involved in good brewing characteristics is expressed in "Haruna Nijo", a gene specific to "Akagami power" is expressed, and its allele is expressed in "H. spontanium".
[0037]
BEST MODE FOR CARRYING OUT THE INVENTION
As described in detail below, the present inventors prepared cDNA libraries based on mRNAs extracted from the tissues of "Haruna Nijo", "Akagami Riki", and "H. spontanum" of barley, respectively. Thereafter, sequence analysis of tens of thousands of cDNA clones was performed. Also, clustering and assembly of each clone were performed based on the results of the sequence analysis, and 1313 contig sequences were obtained this time. Furthermore, based on the contig sequences obtained in this manner, single nucleotide polymorphisms (SNPs) in which base differences are observed between barley varieties are detected and identified. The resulting 939 single nucleotide polymorphisms (cSNPs) were selected.
[0038]
Therefore, these 939 non-synonymous substitution-producing SNPs (hereinafter, referred to as “non-synonymously substituted SNPs”) and oligonucleotides synthesized from a region containing at least one of these non-synonymously substituted SNPs are used in the practice of the present invention. The form will be described below.
[0039]
(A) Non-synonymous substituted SNPs found among barley varieties
SEQ ID Nos. 1 to 534 in the sequence listing show contig sequences in which the 939 non-synonymous substituted SNPs were found. For convenience of description, these 534 contig arrays are hereinafter referred to as contig (1) to contig (534), respectively.
[0040]
The locations where the bases in each of the contigs (1) to (534) are indicated by the aforementioned “universal code” are sites of single nucleotide polymorphism (SNP) found among barley varieties, and are not synonymous. Not only the replacement SNP but also other SNPs (rSNP, sSNP, etc.) are indicated by “universal codes” in each of the contigs (1) to (534). Tables 1 to 39 below show which SNPs correspond to the non-synonymous substitution SNPs.
[0041]
[Table 1]

[0042]
[Table 2]

[0043]
[Table 3]

[0044]
[Table 4]

[0045]
[Table 5]

[0046]
[Table 6]

[0047]
[Table 7]

[0048]
[Table 8]

[0049]
[Table 9]

[0050]
[Table 10]

[0051]
[Table 11]

[0052]
[Table 12]

[0053]
[Table 13]

[0054]
[Table 14]

[0055]
[Table 15]

[0056]
[Table 16]

[0057]
[Table 17]

[0058]
[Table 18]

[0059]
[Table 19]

[0060]
[Table 20]

[0061]
[Table 21]

[0062]
[Table 22]

[0063]
[Table 23]

[0064]
[Table 24]

[0065]
[Table 25]

[0066]
[Table 26]

[0067]
[Table 27]

[0068]
[Table 28]

[0069]
[Table 29]

[0070]
[Table 30]

[0071]
[Table 31]

[0072]
[Table 32]

[0073]
[Table 33]

[0074]
[Table 34]

[0075]
[Table 35]

[0076]
[Table 36]

[0077]
[Table 37]

[0078]
[Table 38]

[0079]
[Table 39]

The items shown in Tables 1 to 39 above are described below.
[0080]
"Ctg_ID": SEQ ID NO: in the sequence listing showing the base sequence of the corresponding contig
"Snp_position": SNP position on each contig sequence (polymorphism site)
“Base”: Regarding the type of base at the position of each SNP, “Haruna” refers to “Haruna Nijo”, “wild type” refers to “H. spontanium”, and “Akagami power” refers to “red”. `` God '' is listed
“Amino acid”: For amino acids encoded by codons containing each SNP (polymorphic site or its complementary site), “Haruna” refers to “Haruna Nijo” and “wild type” refers to “H. spontanium”. And “Red God Power” are described in “Red God Power” respectively. (The method for determining the reading frame will be described later.)
“Substitution”: When the amino acids described in the above “Haruna”, “Wild type” and “Akagami power” are the same amino acid, it is determined that no amino acid substitution occurs, and described as “S”. On the other hand, when amino acids differ between at least two varieties, it is determined that amino acid substitution may occur, and the result is described as "R".
"Intra-region determination": If each of the SNPs determined to be "R" exists in the region of the translation region (code region) determined by the method described later, "in" is described. On the other hand, if it exists outside the area, it is described as "out"
“Evaluation”: The SNP determined to be “R” in the “presence / absence of substitution” column and “in” in the “determination within region” column is determined to be non-silent (non-synonymous substitution) and “○” Is described.
[0081]
As described above, the SNP determined to be “に お い て” in the “Evaluation” column of Tables 1 to 39 corresponds to the non-synonymous substituted SNP of the present invention. A total of 1989 SNPs were found in contigs (1) to (534), of which 939 were determined to be non-synonymous substituted SNPs.
[0082]
A single nucleotide polymorphism is also observed between the barley varieties on the complementary sequence side of each of the contigs (1) to (534). However, if necessary, the contig SNP may be replaced with the contig single nucleotide polymorphism (contig side). SNP), and the SNP in the complementary sequence is referred to as a complementary single nucleotide polymorphism (complementary SNP). When a nucleotide at which a contig-side SNP is recognized is referred to as a polymorphic site, a nucleotide forming a base pair with the polymorphic site in the double-stranded DNA is referred to as a complementary site (complementary-side polymorphic site). These polymorphic sites and complementary sites are found on the cDNA, but corresponding regions on genomic DNA are collectively referred to as polymorphic sites and complementary sites unless otherwise specified. Further, in the present invention, terms such as “complementary (complementary)”, “complementary site”, “complementary sequence”, “complementary region” and the like, unless otherwise specified, completely refer to a target DNA sequence (DNA region). Hybridizing relationships, sites, sequences, and regions, that is, relationships, sites, sequences, and regions in which the base sequences of each other completely follow Watson-Crick base-pairing.
[0083]
One of the complementary side / contig side SNP corresponds to the sense side (gene side) SNP, and the other corresponds to the antisense side SNP. Each of these sense / antisense SNPs is 1) substantially not found in the same cultivar, and is stably found in different cultivars. 2) The sense SNP is EST (cDNA fragment) It is present in non-synonymous substitutions and is likely to be involved in phenotypes (traits) specific to varieties, and thus can be used for various purposes.
[0084]
Specifically, for example, this SNP can be used for a type identification purpose. When the presence of the SNP causes a difference in phenotype between barley varieties (that is, when a phenotype given by a gene and an allele having a single nucleotide polymorphism in the gene are different), cultivar identification is performed. In addition to the uses described above, for example, 1) as a marker for acquiring a gene involved in this phenotype (gene acquisition marker), 2) a candidate hybrid to be crossed with a recurrent parent at the time of backcrossing is selected. As a marker (marker for selecting candidate hybrids) 3) As a marker for judging the success or failure of gene introduction (marker for judging the success or failure of introduction) 4) A marker for selecting new varieties obtained by backcrossing or genetic engineering techniques (New It can be used as a variety selection marker).
[0085]
In addition, the oligonucleotide according to the present invention comprising the above-mentioned polymorphic site or its complementary site includes, for example, 1) a probe or a primer for a base assay (SNP typing) at the polymorphic site or the complementary site; It can be used as a probe or primer for gene acquisition / gene identification comprising a type site or its complementary site. In addition, the details of the method of using and producing the oligonucleotide according to the present invention will be described later.
[0086]
(B) Method for producing contig sequence and method for detecting / identifying non-synonymous substituted SNP
Next, a method for producing the contig array described above will be described.
[0087]
First, cDNA libraries are produced from tissues of a plurality of barley varieties (for example, in the present invention, "H. spontanum", "Haruna Nijo" and "Akagami Power") in accordance with a conventional method. For example, total RNA of tissue is roughly extracted, total mRNA (poly (A) + RNA) is purified from total RNA, first-strand cDNA is synthesized using this mRNA as a template, and second-strand cDNA is synthesized. Next, the obtained double-stranded cDNA is ligated to an appropriate vector, and the vector is packaged in a host to prepare a cDNA library. If necessary, the double-stranded cDNA may be cut into an appropriate length.
[0088]
Next, the obtained clone is extracted, and the sequence analysis of each cDNA clone is performed. Based on the nucleotide sequence of the cDNA revealed by the sequence analysis of each clone, the clones are clustered based on whether or not they have mutually overlapping nucleotide sequences, and an insert fragment (cDNA) of a series of clustered clones is obtained. The overlapping portions are found and joined (assembled) to obtain the above contig, and the inserted fragments are aligned (multiple alignment) so that SNP can be detected.
[0089]
As the barley tissue from which the total RNA is extracted, various tissues constituting buds and leaves are suitably used, but not particularly limited thereto. When the above-mentioned tissue is obtained from barley seedlings, the method of cultivating the seedlings and the stage of their growth are not particularly limited. Regarding the seedling cultivation method, a standard method using barley as an experimental material (see Cell Engineering Separate Volume, Plant Cell Engineering Series 14, Plant Genome Research Protocol, pages 193 to 195, Shujunsha (2000)) is particularly preferable. Adopted.
[0090]
In addition, the extraction of total RNA is generally performed using tissues constituting the buds or leaves of the barley at the seedling stage, because the tissues are relatively easy to grind and impurities such as polysaccharides are removed. Is relatively low, and can be grown from seed in a short period of time. Further, the total RNA may be extracted from the barley tissue in accordance with the time of expression of the unique trait of each barley variety.
[0091]
The steps from the step of extracting total RNA from barley tissue to the sequence analysis of clones are carried out by generally employed methods, for example, Breeding Science Research Vol. 2, page 128 (2000), Genome Analysis Lab Manual p63-p77: It can be easily carried out by employing the method described in Springer Verlag Tokyo (1998). In order to ensure the reliability of the analysis results, more preferably, 1) prepare a plurality of individuals for total RNA extraction for each barley variety, and 2) subject each clone to a plurality of sequence analysis. It is preferable to perform at least one of analyzing the sequence from both the 5 'end and the 3' end of the insert fragment (cDNA) having a predetermined length or more. Further, it is more preferable to confirm the sequence of each of the inserted fragments of each clone by resequencing.
[0092]
Next, the base sequences of the inserted fragments obtained by the sequence analysis of each clone are assembled based on the sequence portions overlapping each other to prepare each contig. At the same time, the inserted fragments of the clones constituting the contig sequence are subjected to multiple alignment, so that the corresponding base difference (SNP) between different clones (between varieties) can be detected.
[0093]
Base calling, assembly of overlapping inserts, and multiple alignment in sequence analysis of inserts can be performed using, for example, known SNP detection software or sequence editing software. Representative programs installed in these software include Trace-Diff (Bonfield. JK. Et al .: Nucleic Acids Research 26: p3404-3408, 1998) and Polybayes (Marth. GT et al.). : Nature Genet. 23: p452-456, 1999), Polyphred (Nickerson. DA: Nucleic Acids Research 25: p2745-2751, 1997), and all of them are ferred (Phred) as a base calling tool. ) Is of the ferred / ferrap (Phred / Prap) type employing ferrap (Prap) as an assembly tool (experimental medicine). Vol.18 No.14 (September issue), 2000: pp. 1890-1892).
[0094]
Further, assuming that the reading error probability of each base at the time of base calling is constant (Phred score (Quality value) is a constant value), it is also possible to employ Phrap as an assembly tool to assemble insert fragments overlapping each other. it can.
[0095]
When a contig is prepared, the base sequence of the inserted fragment is compared between all the clones constituting the contig. (1) At the site where a base difference is found, the quality accuracy after assembly is the highest, for example, Phrap score A process of selecting a base having the highest (Quality value) as a representative base on the contig is performed. (2) However, a plurality of sequence data derived from Akagi power; If there is a difference in bases between at least two groups of sequence data among three groups of sequence data consisting of a plurality of sequence data derived from Spontaneum and a plurality of sequence data derived from Haruna Nijo, these bases are indicated. Use the universal code as the base on the contig.
[0096]
In the present invention, the SNP between varieties is referred to only in the case of (2) above, which is estimated to have particularly high reliability. In the case of the above (2), H. Differences in corresponding bases among at least two or more insert groups selected from a plurality of insert fragments derived from Spontaneum, a plurality of insert fragments derived from Akashiniki, and a plurality of insert fragments derived from Haruna Nijo. In addition to being seen, Sequence data read from both the 3 'and 5' sides of a single insert fragment derived from Spontaneum, sequence data read from both the 3 'and 5' sides of a single insert fragment derived from Akashiniki, and Haruna Nijo This also includes the case where a difference is found in the corresponding base between at least two or more sequence data selected from the sequence data read from both the 3 ′ side and the 5 ′ side of the single insert fragment. In addition, of course, the corresponding base between the sequence data obtained by reading a single insert fragment from any of the above three barley varieties from both the 3 ′ side and the 5 ′ side and the sequence data of a plurality of insert fragments of other varieties. In some cases. However, when three or more locations determined as SNPs on the contig by the above method continue, these are not treated as SNPs of the present invention from the viewpoint of reliability.
[0097]
Further, non-synonymous substituted SNPs were selected from those determined as SNPs on the contig by the above method as follows. First, a homology search was performed on each of the prepared contigs by a known method (the results will be described later), and the amino acid sequence translated according to any of the open reading frames (frames) is converted to a known protein (protein encoded by a known gene). ), The reading frame was determined to be the reading frame of the contig.
[0098]
Next, in the amino acid sequence translated according to the reading frame, SNPs that cause non-synonymous substitution of amino acids according to the difference in bases between cultivars, that is, in the “Substitution presence / absence” column of Tables 1 to 39 described above Each SNP determined to be "R" was examined whether it was present in the translation region (coding region). Specifically, when the stop codon appears before the start codon from the position 5 'upstream from the position of each SNP determined to be "R", the SNP was determined to be outside the translation region. The remaining SNPs were determined to be within the translation region, and non-synonymous replacement SNPs were determined. The presence or absence of start and stop codons upstream and downstream of each of these SNPs was examined in more detail, and the translation region of each contig was determined. Were determined. The results are shown in Tables 40 to 67 below.
[0099]
[Table 40]

[0100]
[Table 41]

[0101]
[Table 42]

[0102]
[Table 43]

[0103]
[Table 44]

[0104]
[Table 45]

[0105]
[Table 46]

[0106]
[Table 47]

[0107]
[Table 48]

[0108]
[Table 49]

[0109]
[Table 50]

[0110]
[Table 51]

[0111]
[Table 52]

[0112]
[Table 53]

[0113]
[Table 54]

[0114]
[Table 55]

[0115]
[Table 56]

[0116]
[Table 57]

[0117]
[Table 58]

[0118]
[Table 59]

[0119]
[Table 60]

[0120]
[Table 61]

[0121]
[Table 62]

[0122]
[Table 63]

[0123]
[Table 64]

[0124]
[Table 65]

[0125]
[Table 66]

[0126]
[Table 67]

In Tables 40 to 67, the columns of “ctg_ID” and “snp_position” are the same as those in Tables 1 to 39 described above. In the “frame” column, the reading frame determined for each contig based on the result of the homology search as described above is described. Here, when the frame is “+”, the contig sequence is determined to be the sense strand, and when “−”, the complementary sequence is determined to be the sense strand. “+1”, “+2”, and “+3” mean that reading frames are set from the first, second and third bases on the 5 ′ side of the contig sequence, respectively, and “−” “1”, “−2”, and “−3” mean that reading frames are set from the first, second, and third bases on the 5 ′ side of the complementary sequence, respectively.
[0127]
In the column of “Q_start” in Tables 40 to 67, the homology search is performed in the base sequence of the sense strand of each contig (contig sequence for frame “+”, complementary sequence for frame “−”). As a result, the start position of the region in which homology with the known sequence was recognized (that is, the position at the 5'-most side of the region) is shown. Therefore, in the case of frame "-", the start position is the position at the 5'-most side of the region where homology is recognized in the complementary sequence. In this case, the "Q_start" column contains the start position on the complementary sequence. Indicates the position of the 3 ′ position of the complementary sequence counted from the 3 ′ side.
[0128]
The “translation region” of each contig in Tables 40 to 67 is determined based on the position on the sense strand of the SNP determined to be a non-synonymous substituted SNP and the determined reading frame (frame) based on the determined reading frame. The region where the nonsynonymous substitution SNP was sandwiched between ATG) and the stop codon was determined as the translation region. Tables 40 to 67 show the “start position” and “end position” of the translation region of each contig determined in this manner. However, when no start codon appears 5 ′ upstream from the position of the nonsynonymous substitution SNP, the reading frame “+1”, and in the case of “−1”, the first base on the 5 ′ side of the sense strand is changed to “+2”, “ In the case of "-2", the second base on the 5 'side of the sense strand was determined to be "+3", and in the case of "-3", the third base of the 5' side of the sense strand was determined to be the "start position" of the translation region. . On the other hand, when a stop codon does not appear 3 ′ downstream from the position of the non-synonymous substitution SNP, the position where the translation region is the longest according to the determined reading frame is determined as the “end position” of the translation region. Thus, the determined translation region does not always indicate a region encoding the full length of the protein.
[0129]
When the frame of the contig is “-”, “start position” and “end position” in Tables 40 to 67 indicate the position of each position counted from the 5 ′ side of the complementary sequence. Is shown.
[0130]
As for the contig (8), the contig (10), the contig (101), and the contig (386), the homology with the known sequence was compared and examined in detail, and as a result, the start position ( In other words, the start position of the region in which homology with the known sequence was recognized) was defined as the "start position" of the translation region.
[0131]
In the column of “translation region number” in Tables 40 to 67, when a plurality of regions determined to be translation regions by the above method exist in one contig, numbers are sequentially assigned to the respective translation regions. (If there is one translation region, the translation region is numbered “1”).
[0132]
The amino acid sequence encoded by the region determined to be the translation region of each contig by the above method is shown in the sequence listing. The following Tables 68 to 78 show, for each amino acid sequence, which SEQ ID No. in the Sequence Listing is described.
[0133]
[Table 68]

[0134]
[Table 69]

[0135]
[Table 70]

[0136]
[Table 71]

[0137]
[Table 72]

[0138]
[Table 73]

[0139]
[Table 74]

[0140]
[Table 75]

[0141]
[Table 76]

[0142]
[Table 77]

[0143]
[Table 78]

In Tables 68 to 78, the column of “ctg_ID” is the same as Tables 1 to 39 described above. The column of “amino acid sequence” shows the sequence number of a sequence listing describing the amino acid sequence encoded by the translation region of each contig. In addition, for one amino acid sequence, both the one described in parallel with the base sequence of the translation region (coding region) and the one described alone in the amino acid sequence are shown in the sequence listing. The amino acid sequence shown in the sequence listing is determined by selecting bases at the polymorphic site represented by the “universal code” or its complementary site in accordance with the following criteria a) to c).
[0144]
a) Among the three varieties of “Haruna”, “Wild type”, and “Akagami power”, each base of a variety in which the bases of all SNPs on the translation region are determined is selected as the base of each SNP. When there are two or more varieties in which the bases of all SNPs in the translation region are determined, the varieties are selected according to the priority order of “wild type”> “Akagami power”> “Haruna”.
[0145]
b) However, if there is a SNP that causes the substitution of “amino acid → STOP (stop codon)” in the translation region, the varieties having the base that causes STOP are not selected, and one of the above priorities is selected. Each base of the lower variety is selected as a base for each SNP.
[0146]
c) There is an SNP that causes a substitution of “amino acid → STOP (stop codon)” in the translation region, and bases of other varieties also cause a substitution of “amino acid → STOP (stop codon)” in the translation region. In this case, among the varieties in which the bases of all the SNPs in the translation region have been determined, the bases of the varieties having the higher priority are selected as the bases of the respective SNPs.
[0147]
Each amino acid sequence shown in the sequence listing is based on the result of the above-described determination of the translation region, and does not necessarily indicate the full length of the protein.
[0148]
In the above Tables 68 to 78, the “number of clones” and the “clone sequence” are also shown for each contig. The column “number of clones” shows the total number of cDNA clones from which each contig was prepared and constructed. The column of "clone sequence" shows the sequence number of a sequence listing describing the nucleotide sequences of these cDNA clones. For example, contig (1) is a sequence obtained by aligning (multiple aligning) the cDNA clones shown in SEQ ID NOs: 1661 to 1675.
[0149]
(C) Homology search results
As described above, the present inventors performed a homology search on each of the prepared contigs by a known method. The results are shown in Tables 79 to 110 below.
[0150]
[Table 79]

[0151]
[Table 80]

[0152]
[Table 81]

[0153]
[Table 82]

[0154]
[Table 83]

[0155]
[Table 84]

[0156]
[Table 85]

[0157]
[Table 86]

[0158]
[Table 87]

[0159]
[Table 88]

[0160]
[Table 89]

[0161]
[Table 90]

[0162]
[Table 91]

[0163]
[Table 92]

[0164]
[Table 93]

[0165]
[Table 94]

[0166]
[Table 95]

[0167]
[Table 96]

[0168]
[Table 97]

[0169]
[Table 98]

[0170]
[Table 99]

[0171]
[Table 100]

[0172]
[Table 101]

[0173]
[Table 102]

[0174]
[Table 103]

[0175]
[Table 104]

[0176]
[Table 105]

[0177]
[Table 106]

[0178]
[Table 107]

[0179]
[Table 108]

[0180]
[Table 109]

[0181]
[Table 110]

The columns of “ctg_ID” in Tables 79 to 110 are the same as those in Tables 1 to 39. In the column of “Homology search result”, the names and accession numbers of known genes or known proteins having a certain degree of homology or more are shown for each contig.
[0182]
(D) Method of using the oligonucleotide of the present invention (utility)
The oligonucleotide of the present invention comprises a continuous DNA sequence of 7 nucleotides or more containing at least one of the nonsynonymous substituted SNPs on the contig described above, or a complementary sequence thereof. It has utility.
[0183]
First, since the oligonucleotide of the present invention contains the polymorphic site or its complementary site caused by a difference in barley varieties, the oligonucleotide or a composition comprising this oligonucleotide is used between various barley varieties. It can be used to test for existing single nucleotide polymorphisms. In addition, this makes it possible to quickly and easily identify genetic varieties of barley, determine which type (genotype) the barley has a gene, and the like.
[0184]
For example, if the above oligonucleotide is immobilized on a base (carrier) to constitute a DNA chip (an embodiment of the composition according to the present invention), it can be used as an oligonucleotide probe for testing a single nucleotide polymorphism in a barley gene. . In this case, the length of the oligonucleotide is not particularly limited as long as it can be tested for single nucleotide polymorphism, but is preferably 7 to 50 nucleotides, or 10 to 30 nucleotides, more preferably 15 to 25 nucleotides. preferable.
[0185]
In addition, the single nucleotide polymorphism test in the gene can be performed indirectly via RNA obtained from the DNA, in addition to using a DNA containing the polymorphic site or its complementary site. Therefore, as a target of a hybridization test using a DNA chip, DNA and RNA derived from a subject described later can be used. Generally, cDNA or genomic DNA is used as the DNA, and mRNA or cRNA is used as the RNA. Is used.
[0186]
Furthermore, a DNA fragment corresponding to the oligonucleotide according to the present invention can be obtained from the above-mentioned subject, and this can be directly analyzed to test a single nucleotide polymorphism in the gene of the subject. Specifically, for example, in an oligonucleotide consisting of the DNA sequence represented by any one of SEQ ID NOs: 1 to 534, a region containing at least one of the polymorphic sites represented by the “universal code”, or a complement thereof. A single nucleotide polymorphism generated in the gene of the subject may be assayed by amplifying DNA from the subject using a primer designed to amplify at least one of the regions.
[0187]
The primer includes, in addition to a primer that amplifies both a region containing at least one of the polymorphic sites and a complementary region thereof, such as a PCR primer, and a primer that amplifies one of them, such as an RCA primer. included. In addition, the obtained amplified DNA fragment is analyzed, for example, by 1) direct sequencing and analysis, 2) a restriction enzyme that cuts in a different pattern depending on the base of the polymorphic site and / or its complementary site. It can be analyzed by a method such as analyzing the length of the fragment produced by digestion and 3) analyzing by a hybridization test with the oligonucleotide according to the present invention. The restriction enzymes include a restriction enzyme that recognizes and cuts a specific sequence on double-stranded DNA (class II restriction enzyme) and a restriction enzyme that recognizes and cuts a specific sequence on single-stranded DNA (class II restriction enzyme). Class I restriction enzymes, and part of class II restriction enzymes).
[0188]
In addition, since a gene usually exists as a double-stranded DNA (may be a cDNA) bound to its complementary sequence, in the present invention, "test for single nucleotide polymorphism (sense-side single nucleotide polymorphism) in a gene""" Also encompasses a case where a complementary single nucleotide polymorphism is detected through a single nucleotide polymorphism (antisense single nucleotide polymorphism) test in a complementary sequence of the gene. In addition, "assay a single nucleotide polymorphism" refers to not only identifying (identifying or detecting) the type of base at the polymorphic site or the complementary site but also limiting the type of the base (for example, "at least for adenine. No ").
[0189]
The above-mentioned "subject" refers to any organism having a gene having a partial sequence or a full-length base sequence identical or complementary to the oligonucleotide according to the present invention, and other than barley, other barley genus plants and barley Particularly preferred are plants of the wheat genus (TRITICAE) that are closely related to the genus, for example, plants of the genus Triticum, plants of the genus Targi (Aegilops), plants of the genus Rye (genus Secale), and plants of the genus Wheat (Lolium). It is mentioned as a thing. In the present invention, "WD Clayton and SA Renovize: KE BULLETIN ADDITIONAL SERIES XII, GENERA GRAINUM, Grasesof the World (third edition), p. Basically adopt the classification system described in.
[0190]
Furthermore, a practical strain into which a useful gene has been introduced can be developed by using the oligonucleotide according to the present invention for producing a transformant. For example, a useful gene is isolated using the oligonucleotide as a probe or as a primer or using a primer for obtaining a region of the oligonucleotide, and then the useful gene is introduced into target cells using genetic engineering techniques. Thus, a transformant can be produced. When the oligonucleotide is used as a probe for gene isolation, its length may be any length as long as the probe is sufficient, for example, preferably 7 to 500, 51 to 500, or 70 to 400 nucleotides, 90-300 nucleotides are more preferred.
[0191]
Further, for example, a parent plant A having a gene having a partial sequence or full length of the oligonucleotide according to the present invention, and a single nucleotide polymorphism (especially affecting phenotype) having no such gene or this gene When the parent plant B having the allele resulting from the above can be crossed, the parent plants A and B are crossed to produce a hybrid plant (production step), and then the oligonucleotide or SNP assay method of the present invention The desired transformant (transformed plant) can be produced by selecting a plant having the above gene from these hybrid plants (selection step). The above allele is more preferably one in which at least one single nucleotide polymorphism occurs in a certain useful gene, whereby the expression of a useful trait is suppressed or eliminated. Further, the kind of the useful gene is not particularly limited, but useful genes (such as an environmental resistance gene) possessed by, for example, wild barley ancestors of the cultivated barley are particularly suitable as in the case of normal breeding. .
[0192]
As described above, the oligonucleotide according to the present invention has at least 7 nucleotides containing at least one of the polymorphic sites that cause non-synonymous substitution among the polymorphic sites in which the notation of the base is described in the “universal code”. In particular, those consisting of a continuous DNA sequence or its complementary sequence. In addition, this oligonucleotide is not limited to the case where it is present as a single strand, and may be a double strand which forms a hydrogen bond with a complementary strand. Further, the oligonucleotide may be present in a state of being inserted into a sequence such as a badlock probe for SNP typing by the RCA method (described later) or a vector sequence (including an expression vector sequence).
[0193]
The above oligonucleotide is a contig obtained by aligning the insert fragments of the cDNA clone or a part or the full length of a complementary sequence thereof, and it is highly possible that the same sequence exists not only in cDNA but also in genomic DNA. high. Therefore, for example, (1) a probe or a primer for detecting a single nucleotide polymorphism generated in a gene (a probe or a primer for SNP typing), and (2) a probe or a primer for obtaining (isolating) a gene In the following description, the one used for the above (1) is referred to as oligonucleotide A, and the one used for the above (2) is referred to as oligonucleotide B.
[0194]
Oligonucleotide A is used, for example, when detecting SNPs stably conserved between varieties in the barley genome, and a) a site where the SNP is present on the gene between barley varieties, or It suffices to include at least one site complementary to this site (complementary site), and b) to have an appropriate length as a hybridization probe or primer for assaying SNP. The length of the oligonucleotide A is not particularly limited as long as the conditions of a) and b) are satisfied, but generally it is more preferably in the range of 7 to 50 nucleotides or in the range of 10 to 30 nucleotides. More preferably, it is within the range of 15 to 25 nucleotides showing sufficient specificity as a sequence.
[0195]
When the oligonucleotide A comprises two or more of the above-mentioned polymorphic site or its complementary site, all the bases of the polymorphic site or its complementary site are “Haruna Nijo” type, “Akagami power” type or The nucleotide sequence of the oligonucleotide A may be defined by immobilizing it on one of the “H. spontanium” types.
[0196]
On the other hand, the oligonucleotide B is used as a probe or a primer for obtaining a gene having a partial sequence or full length of each contig or its complementary sequence and a homolog thereof. Therefore, the oligonucleotide B is not particularly limited as long as it has a length of at least 7 nucleotides including at least one of the polymorphic site or the complementary site, but has a length of 51 to 500 nucleotides or 70 to 400 nucleotides. More preferably, the length is 90 to 300 nucleotides. In addition, since some of the oligonucleotides according to the present invention are used for both purposes (1) and (2), the oligonucleotides A and B may have the same base sequence in some cases.
[0197]
(E) Method for producing oligonucleotide according to the present invention
The method for producing the oligonucleotide according to the present invention is not particularly limited, and a general method is employed. For example, the oligonucleotide may be cut out from a genomic DNA, genomic DNA library, or cDNA library of an organism having the oligonucleotide with an appropriate restriction enzyme and purified. Further, in an oligonucleotide comprising a DNA sequence represented by any one of SEQ ID NOs: 1 to 534, a region containing at least one polymorphic site corresponding to a non-synonymous substituted SNP or at least one of its complementary regions can be amplified. The oligonucleotide of the present invention may be isolated by performing an amplification reaction using the genomic DNA and cDNA of the above organism as a template using the primers designed in the above. Further, the oligonucleotide can be synthesized by a known method.
[0198]
The length of the primer for obtaining the oligonucleotide is not particularly limited as long as amplification other than the target region due to mismatch does not occur. For example, when the amplification reaction is a PCR (Polymerase Chain Reaction), a set of primers having a length in the range of 15 to 25 or 15 to 30 nucleotides is generally used. The sequence of the primer is also not particularly limited, and may be arbitrarily designed based on a specific and characteristic sequence region on the DNA sequence.
[0199]
The reaction conditions of the PCR reaction may be performed in accordance with ordinary conditions (see Saiki, Science, 230, 1350-1354 (1985); PCR Technology, published by Takara Shuzo Co., Ltd., 1990). More specifically, the heat denaturation step of the target double-stranded DNA (including the template) is carried out in a temperature range of 90 ° C to 96 ° C, more preferably 94 ° C to 96 ° C for about 1 minute to 3 minutes. Preferably, it may be performed for about 1 to 2 minutes. Also, the annealing step of the primer may be performed within a temperature range of 40 ° C. to 60 ° C., more preferably 55 ° C. to 60 ° C., for about 1 minute to 3 minutes, more preferably about 1 minute to about 2 minutes. Further, the chain elongation step by DNA polymerase may be performed within a temperature range of 70 ° C. to 74 ° C., more preferably 72 ° C. to 74 ° C., for about 1 minute to 4 minutes, more preferably about 2 minutes to 3 minutes.
[0200]
Although the number of cycles of the PCR reaction including the heat denaturation step, the annealing step, and the chain extension step as one cycle is not particularly limited, it is usually 20 to 40 cycles, more preferably 25 to 35 cycles. In addition, the type and amount of the DNA polymerase to be used (a heat-resistant polymerase such as Taq polymerase), the type and amount of the reagent, and the extraction method of the template DNA (for example, the C-TAB method) are the same as those in the related art. Description is omitted. Note that the category of the PCR reaction also includes a modified method such as an RT-PCR (Reverse Transscribed PCR) reaction. In the PCR reaction, a double-stranded DNA fragment containing a polymorphic site is obtained. For example, by using a single-stranded amplification method such as RCA (Rolling circuit amplification), an appropriate primer / template (polymorphic site or If the reaction is carried out with a set of circular single-stranded DNA containing the complementary site, a single-stranded DNA fragment (oligonucleotide of the present invention) containing either the above-mentioned polymorphic site or its complementary site can be obtained ( Lizardi PM et al. Nat Genet 19, 225, 1998).
[0201]
As the organism for obtaining the oligonucleotides A and B according to the present invention, barley varieties such as "H. spontanum", "Akagami power" and "Haruna Nijo", and other barley genus plants are particularly suitable. However, in addition to these, any organism having a gene having a partial sequence or full length of a nucleotide sequence identical or complementary to the oligonucleotide is used. Examples of the above-mentioned organisms other than the barley genus plants include plants belonging to the genus Wheat which are closely related to the genus Barley, such as the genus Wheat (Triticum), the genus Tarho wheat (Aegilops), the rye (Secale), and the genus Wheat ( Lolium) plants are preferred.
[0202]
(F) SNP typing method using the oligonucleotide of the present invention
Next, the case where the oligonucleotide of the present invention is applied to the SNP typing method will be described.
[0203]
Assay of single nucleotide polymorphisms on the gene of the subject (typing of SNP) can be performed using the oligonucleotide according to the present invention. This method comprises a method of (A) isolating the above-mentioned oligonucleotide from DNA (may be cDNA) derived from a living organism as a subject and directly assaying a base at a polymorphic site or a complementary site thereof on the oligonucleotide (direct method) And (B) a method of indirectly assaying a single nucleotide polymorphism on a gene derived from a subject using a polymorphic site or a complementary site of oligonucleotide A according to the present invention (indirect method). ). That is, any of the above methods (A) and (B) converts the single nucleotide polymorphism generated in the gene of the subject based on the base at the polymorphic site or its complementary site of the oligonucleotide according to the present invention. This is a test method.
[0204]
The “subject” particularly preferably includes barley varieties such as “H. spontanum”, “Akagami power” or “Haruna Nijo”, and other barley genus plants. The type of the organism is not particularly limited as long as the organism has a gene having a partial sequence or full length of a nucleotide sequence identical or complementary to the oligonucleotide. Specific examples of organisms having a high possibility of having such a gene include wheat plants such as the above-described plants of the genus Wheat, plants of the genus Talho, rye, and genus Wheat. In addition, transformants in which the above genes of these organisms have been introduced (or attempted to be introduced) by crossing or genetic engineering techniques are also included in the subject.
[0205]
Further, the gene usually exists as a double-stranded DNA (may be a cDNA) bound to its complementary sequence. Therefore, in the present invention, "testing for a single nucleotide polymorphism in a gene" refers to a case where a sense-side single nucleotide polymorphism complementary to the gene is assayed through a single nucleotide polymorphism in a complementary sequence of the gene. Shall be included.
[0206]
Hereinafter, the direct method will be described more specifically. The direct method is designed, for example, to be able to amplify a region containing at least one polymorphic site on the DNA sequence represented by any of SEQ ID NOs: 1 to 534, or at least one of its complementary regions. Using a primer (primer according to the present invention), DNA (genomic DNA or cDNA) derived from a subject is amplified to obtain an amplified DNA fragment (oligonucleotide according to the present invention). This is a method for testing SNPs generated in a gene by performing a statistical analysis. The specific examples and conditions of the amplification reaction used here are as described in "(E) Method for producing oligonucleotide according to the present invention".
[0207]
As an example of the direct method, more specifically, 1) differences in the mass of amplified DNA fragments obtained by using a Matrix Assisted Laser Desorption-Time of Flight / Mass Spectrometry (MALDI-TOF / MS) method, and the like. 2) The amplified DNA fragment may be sequenced to directly determine the base at the polymorphic site or its complementary site, and 3) the amplified DNA fragment may be sequenced at the polymorphic site or The SNP may be digested with a restriction enzyme that cuts in a different pattern depending on the type of at least one base of the complementary site, and the SNP may be directly typed by the difference in length of the generated fragment (so-called RFLP (Restriction Fragment Length Polymorphor). ism) method). In the method 3), a specific sequence on a single-stranded DNA is recognized and cleaved depending on whether the obtained amplified DNA fragment is single-stranded (by the RCA method or the like) or double-stranded. Restriction enzymes (class I restriction enzymes and part of class II restriction enzymes) and restriction enzymes (class II restriction enzymes) that recognize and cut a specific sequence on double-stranded DNA may be selected.
[0208]
Since the methods 2) and 3) are extremely general methods, only the method 1) will be described below. In one example of SNP typing using the MALDI-TOF / MS method, a region containing the above-mentioned polymorphic site, or a primer designed to amplify the complementary region thereof, is amplified using a genomic DNA or cDNA of a subject as a template. Perform the reaction. Next, a primer extension reaction is performed using the obtained amplified DNA fragment as a template to obtain an extension reaction product containing the polymorphic site or the complementary site as a 3 ′ end. Next, the difference in the mass of the extension reaction product caused by the difference in the base at the polymorphic site or the complementary site is measured by a mass spectrometer, and the base at the site is typed (see “Genome science of post-sequence (1) SNP gene”). Strategy of polymorphism, 1st printing (Nakayama Shoten), pp. 106-117 ”)). When a plurality of SNPs are simultaneously tested, the tests can be performed under multiplex conditions.
[0209]
On the other hand, the indirect method is an SNP typing method using the oligonucleotide A according to the present invention. Specifically, for example, 1) a step of preparing a DNA (concept including cDNA) or RNA derived from a subject, 2) a step of obtaining oligonucleotide A, and 3) a step of checking whether or not oligonucleotide A completely hybridizes with the RNA or DNA prepared in 1). When both hybridize completely, it is determined that the polymorphic site (or its complementary site) of the gene of the subject consists of a base complementary to the corresponding base of oligonucleotide A, while In the case of complete hybridization or non-hybridization, it is determined that the polymorphic site (or its complementary site) is composed of any of the bases that are not complementary to the corresponding bases of oligonucleotide A.
[0210]
Needless to say, when the oligonucleotide A is a DNA sequence containing at least one of the above polymorphic sites, this DNA sequence completely hybridizes with genomic DNA and cDNA complementary to itself, When the oligonucleotide A is a complementary sequence to the DNA sequence, the complementary sequence completely hybridizes to mRNA, cRNA, and genomic DNA complementary to itself.
[0211]
The term "DNA derived from the subject" is a concept including an amplified DNA fragment obtained by a PCR method or an RCA method using this DNA as a template. In general, DNA or RNA derived from a subject is extracted from the organism, that is, various organs, various tissues, cells, protoplasts, spheroplasts, calli, and the like. Beverages), fossils and the like extracted from other than the living body of the subject are used.
[0212]
For the preparation of DNA or RNA derived from a subject, a general DNA or RNA extraction method may be employed. Further, the type of tissue for DNA or RNA extraction, the method of growing (growing) the subject, and the growth stage thereof are not particularly limited. When preparing cDNA or mRNA as DNA or RNA, for example, the method used for preparing the above contig may be employed. In addition, when preparing cRNA, a general method of transcribing the above cDNA using an appropriate RNA polymerase may be used. In addition, these DNA / RNA and / or oligonucleotide A (probe) serving as a target for hybridization are usually labeled in advance with a biotin label, various fluorescent labels, or the like so that the state of hybridization can be confirmed. When the state of hybridization is confirmed by intercalation insertion or other methods, it is not particularly necessary to label the target and / or probe in advance.
[0213]
The conditions for hybridizing the DNA or RNA (target) derived from the subject with the oligonucleotide A (probe) are generally used in each method in consideration of the length of the oligonucleotide A and the like. The conditions may be followed (see, for example, DNA Microarray First Edition, Third Printing, p. 67-83 (issued by Takara Shuzo Co., Ltd.)).
[0214]
In general, a DNA chip (composition) in which oligonucleotide A (probe) is immobilized on a substrate (carrier) is produced, and the above hybridization is performed. In the present invention, the term "DNA chip" as used herein means a synthetic DNA chip mainly using a synthesized oligonucleotide as a probe, but also includes an attached DNA microarray using a cDNA such as a PCR product as a probe. It is a concept.
[0215]
It is to be noted that a known method may be employed for the production of the DNA chip, and when a synthetic oligonucleotide is used as the oligonucleotide A, the oligonucleotide may be formed on a substrate by a combination of a photolithography technique and a solid-phase DNA synthesis technique. It is sufficient to synthesize nucleotides. On the other hand, when cDNA is used as oligonucleotide A, it may be attached to a substrate using an array machine. In addition, as in a general DNA chip, an SNP assay is performed by arranging a perfect match probe (oligonucleotide A) and a mismatch probe having a single base substitution at a site other than a polymorphic site (or a complementary site) in the perfect match probe. Accuracy may be further improved. Furthermore, in order to test SNPs on different genes in parallel, a DNA chip may be constructed by immobilizing a plurality of types of oligonucleotides A on the same base.
[0216]
Another example of the indirect method is SNP typing using the Invader method ("Genome science of post-sequence (1) SNP gene polymorphism strategy first printing (Nakayama Shoten) pages 98 to 105"). reference). In one example, the oligonucleotide A (probe region) is a complementary sequence of a part of any of the contigs (1) to (534) and containing one of the polymorphism sites as the 3 ′ end, and An allele probe is prepared by ligating a common sequence (flap region) to the 5 'end of nucleotide A. Next, an invader probe that is a part of the contig and contains the polymorphic site at the 3 ′ end (however, the type of base at the 3 ′ end is arbitrary) and an allele probe are hybridized to the genomic DNA or cDNA of the subject. Then, an attempt is made to cut out the flap region using an enzyme (clevase) that cuts out the flap region only when the allele probe completely matches (completely hybridizes) to the genomic DNA or cDNA. Next, the excised flap region is hybridized with a probe (FRET (Fluorescence Resonance Energy Transfer) probe) capable of complementary binding to the flap region and labeled with a fluorescent dye and a quencher, and is hybridized with the enzyme (clevase). The fluorescent dye is cut out. SNP typing of a polymorphic site is performed by utilizing the fact that the amount of generated fluorescence differs depending on whether or not the genomic DNA or cDNA and the allele probe completely hybridize.
[0217]
Furthermore, the indirect method includes a method in which hybridization (annealing) of oligonucleotide A and DNA derived from a subject is attempted, and whether or not both have completely hybridized is confirmed by a DNA amplification reaction. . Specific examples of these methods include known SNP typing methods using, for example, the TaqMan PCR method, the RCA method, and the like (“Genome science of post-sequence (1) Strategy of SNP gene polymorphism, first print ( Nakayama Shoten) pages 94-97, pages 118-127 ").
[0218]
In one example of SNP typing using the TaqMan PCR method, a complementary sequence of a sequence consisting of about 20 nucleotides, which is a part of any of contigs (1) to (534) and includes one of the polymorphic sites, is used as an oligonucleotide. A, and the 5 ′ end and 3 ′ end of this oligonucleotide A are labeled with a fluorescent dye and a quencher (quencher) in this order to prepare a TaqMan probe. In this state, the TaqMan probe does not emit fluorescence due to the function of the quencher. Next, the TaqMan probe was hybridized to the genomic DNA or cDNA of the subject using a PCR primer designed to amplify the region containing the polymorphic site in the contig and the complementary region thereof, and Taq DNA polymerase. A PCR reaction is performed in this state. Since Taq DNA polymerase has 5 'nuclease activity, if the TaqMan probe actually hybridizes to the genomic DNA or cDNA of the subject, the fluorescent dye is cut out and the fluorescence is observed. Therefore, if a TaqMan probe is prepared by appropriately selecting a base complementary to the above polymorphic site, the type of the base at the polymorphic site in the genomic DNA or cDNA of the subject can be determined by whether or not it emits fluorescence (that is, both are completely fluorescent). Whether or not to hybridize).
[0219]
In one example of SNP typing using the RCA method, first, a complementary sequence to a sequence of about 20 nucleotides each including a polymorphic site on a genomic DNA or cDNA of a subject is arranged at both ends, and the two complementary sequences are arranged. A single-stranded probe (budlock probe) in which the sequences are linked by a specific sequence called a backbone is prepared. The badlock probe is designed such that a base complementary to the base at the polymorphic site is located at one end (usually at the 3 ′ end). Next, a ligated probe is used as a template with the genomic DNA or cDNA of the subject, and an amplification reaction is performed using a combination of appropriate primers and DNA polymerase.
[0220]
Here, when the base at the polymorphic site of the genomic DNA or cDNA serving as a template and the base at one end of the badlock probe are actually complementary, ligation (here circularization) and amplification of the badlock probe are performed. A reaction occurs and the base at the polymorphic site can be typed. On the other hand, if they are non-complementary, this ligation and amplification reaction does not occur. The RCA method is particularly suitable for large-scale analysis of a sample because a DNA amplification reaction can be performed at a constant temperature (usually about 65 ° C.). Further, the above-mentioned badlock probe contains a sequence that becomes an oligonucleotide A only when circularized (the above-mentioned complementary sequence arranged at both ends), and such a sequence is also included in the category of the oligonucleotide A according to the present invention. include.
[0221]
Oligonucleotide A is used as one of the PCR primers, and hybridization (annealing) between the oligonucleotide A and the DNA derived from the subject and an amplification reaction by the PCR method are attempted. That is, a method (for example, ASA (allele-specific amplification) method) of typing an SNP by whether or not both hybridize completely is also an example of the indirect method.
[0222]
Each of the SNP typing kits used in the indirect method comprises oligonucleotide A, and thus corresponds to the composition according to the present invention. That is, the composition according to the present invention includes 1) a product in which oligonucleotide A is immobilized on a carrier such as a DNA chip, and 2) SNP typing such as for TaqMan PCR, Invader, and RCA. In general, a kit in which the oligonucleotide A is not immobilized, such as a kit for use, is also included.
[0223]
As described above, by using the method using the oligonucleotide A and other SNP typing methods, various barley genus plants (particularly, the barley varieties “H. spontanum”, “Akami power”, and “Haruna”) are used. The base at the above polymorphic site or its complementary site can be determined for (Niijo)). The types of bases at the polymorphic site or its complementary site are substantially the same within the same barley cultivar, and there are differences between specific different cultivars, so that barley cultivar can be identified.
[0224]
For example, using any of the SNP typing methods according to the present invention, a polymorphic site on DNA derived from “H. spontanum”, “Akagami power” and “Haruna Nijo”, for example, contig (6) (SEQ ID NO: 6) As a result of identifying the base at the position corresponding to the 1083th nucleotide (polymorphic site) on the DNA sequence shown in (1), "H. spontanum" was used for adenine, and "Haruna Nijo" or "Haruna Nijo" was used for thymine. Red God power "is determined.
[0225]
It should be noted that varieties may be identified for barley and organisms other than those of the genus Barley in which similar SNPs are highly likely to be recognized (see examples of SNP typing subjects). In addition, if varieties are identified by combining two or more of these polymorphic sites (or their complementary sites), or if varieties are identified in combination with other gene markers, the identification accuracy is further improved. Breed identification using genetic polymorphism information, such as SNPs, can be performed by tasks that are difficult for the naked eye, such as testing the quality accuracy of barley grains and processed barley foods (such as starch) (confirming the presence or absence of different types of barley). Particularly effective.
[0226]
(G) Use of SNP as a gene marker
In addition, when the base at the polymorphic site (which may be a complementary site) differs between a gene and its allele, resulting in a different phenotype, (1) a gene or a gene involved in a desired phenotype SNPs can be used as genetic markers for the purpose of obtaining one of the alleles, (2) selecting plants having one of the genes or alleles, and the like. Hereinafter, the cases (1) and (2) will be described in detail, but for convenience, the base at the polymorphic site is any one of the genes of the “Akagami power” type, the “Haruna Nijo” type, or the “H. spontanium” type. One of them is simply referred to as a “gene”, and the one having a different base at the polymorphic site (having a different genotype) from the “gene” is referred to as an “allyl”. It is also assumed that the gene and its allele confer a more useful phenotype (ie, are useful genes).
[0227]
In the case of the above (1), generally, an oligonucleotide B that hybridizes to only one of allele or gene is used as a probe, and for example, a genomic DNA library or a cDNA library of barley is screened by a known method. The above gene (useful gene) may be obtained (isolated). Alternatively, using a gene amplification method (PCR method or the like) using such an oligonucleotide B as a primer, for example, a clone containing the gene is identified from a barley genomic DNA library or cDNA library, and the gene is obtained. do it.
[0228]
The isolated gene is introduced into a target cell (host cell) so as to be expressible by a known genetic engineering technique (gene manipulation technique), and a transformant expressing the gene is produced. Specific examples of the above-mentioned genetic engineering methods include, for example, a method utilizing infection of Agrobacterium bacteria such as a protoplast coculture method and a leaf disk method, and a polyethylene glycol method, an electroporation method, and a microinjection method. , A particle gun method, a liposome method, a direct DNA transfer method, a transfer method using an appropriate vector system, and the like. An optimum one may be selected according to the type of the target cell.
[0229]
As the target cells, prokaryotic cells such as Escherichia coli, or eukaryotic cells such as plant cells are used.Because the gene to be introduced is derived from barley, plant cells are preferably used, and wheat cells are more preferably used. Plant cells, particularly preferably cells of the genus Barley, including barley are used. When selecting a prokaryotic cell, cDNA may be selected as the gene.
[0230]
When the phenotype given by the gene and its allele is substantially the same, or when a homolog of the gene is obtained, an oligonucleotide B capable of non-selectively isolating these is used for gene acquisition. Probe or primer. The gene, its allele, and its homolog are preferably isolated from barley plants (particularly, barley varieties such as "H. spontanum", "Akagami power" or "Haruna Nijo"). Can be isolated from an organism having a partial sequence or full length gene of the oligonucleotide (more preferably, each contig or its complementary sequence), or a homolog thereof.
[0231]
The kind of the above-mentioned gene (useful gene) is not particularly limited, but a useful gene or the like of an ancestral wild-type barley (H. spontaneum) of a cultivated barley is particularly preferable. Particularly, cultivated barley cells are particularly suitable.
[0232]
On the other hand, in the case of the above (2), the base of the polymorphic site (or the complementary site) is specified according to any of the SNP typing methods already described, and based on this, any of the desired gene or allele (generally, Can be used to select plants having the "gene" which is a useful gene. In addition, such a plant selection operation includes, for example, 1) when selecting a mating parent having the above-mentioned useful gene, and 2) from a hybrid (transformant; transformed plant) group obtained in the process of backcrossing. It is mainly performed in the field of plant breeding, for example, when selecting a hybrid candidate to be further crossed to a return parent or a new variety stably retaining the useful gene.
[0233]
Hereinafter, a specific method for producing a transformant by crossing will be described. In the combination of parents (referred to as parent plants A and B) to be crossed, one (parent plant A) has the above gene (hereinafter referred to as a useful gene) and the other (parent plant B) has the useful gene Or a plant species that has a relationship with an allele of the useful gene and that can be crossed with each other.
[0234]
In other words, the parent plant A is not particularly limited as long as it is a plant having a useful gene having a partial sequence or full length of the oligonucleotide according to the present invention (more preferably, each contig or its complementary sequence). In view of the high possibility of having the useful gene, a plant of the wheat ream is more preferable, and a barley genus plant (for example, a barley variety “H. spontanum”, “Akagami power” or “Haruna Nijo”) is further preferable. Among them, ancestral wild-type barley (H. spontaneum) having a gene not found in cultivated barley is particularly preferable.
[0235]
On the other hand, the parent plant B is not particularly limited as long as it does not have the useful gene or has a useful gene allele and can be crossed with the parent plant A. From the viewpoint of easy crossing with the parent plant A), a wheat plant is more preferable, and a barley genus plant including barley is more preferable. Needless to say, the parent plants A and B are different varieties. In addition, crosses between heterologous plants belonging to the wheat ream are known among barley plants, wheat plants, rye and the like (the latest biotechnology book (1) Propagation and breeding of cereals, potatoes, and pastures 112). (See p. Agricultural Books, published in 1992.)
[0236]
Furthermore, the combination in which the parent plant A is “H. spontanum (an ancestral wild-type barley)” and the parent plant B is a cultivated barley having a “Haruna Nijo” type or a “Red God power” type polymorphic site shows that the hybridization is It is particularly preferable because it is easy and there is no possibility that the hybrid progeny will become sterile (because of cross between different varieties). It is also known that there is an ancestral wild-type plant gene that is well expressed only under the genetic background of the cultivar. Therefore, it is extremely significant to cross ancestral wild-type barley with cultivated barley.
[0237]
When the “cross” is backcrossing, the parent plants A and B may be plants having a single parent / repeating parent (returning parent) relationship in turn, or may be backcrossing in order. May be the relationship between the hybrid obtained as a result of the above and a recurrent parent that crosses the hybrid back.
[0238]
The purpose of the above "crossing" is to select and establish a practical line having the genetic background of the parent plant B and having the useful gene of the parent plant A introduced therein. In other words, it is to select and establish a practical line in which the inferior trait of the parent plant B is eliminated and only the useful characteristics of the parent plant A are introduced. Therefore, in the process of crossing, a step of selecting a hybrid having the useful gene from a hybrid plant group produced by crossing parent plants A and B (selection step) is appropriately performed.
[0239]
The above selection step utilizes that the useful gene has the oligonucleotide according to the present invention as a partial sequence or full length. For example, when the parent plant B has an allele of the useful gene, a suitable hybrid may be selected from the obtained hybrid plant group using any of the SNP typing methods described above. The selection method using such a gene marker does not require breeding the hybrid and actually confirming the phenotype. For example, since the selection using the seed of the hybrid can be performed, the advantage is that it is quick and simple. Having.
[0240]
When the parent plant B does not have the above-mentioned useful gene or its allele, besides using the above-mentioned SNP typing method, the hybrid-derived DNA or RNA and the oligonucleotide according to the present invention (the oligonucleotide for detecting SNP) (Including other than A) can also be used to confirm the introduction of a useful gene.
[0241]
The hybrids selected in the above selection step include, for example, 1) a hybrid candidate to be further crossed to a return parent in the process of backcrossing, and 2) a new variety in which the useful gene is stably retained. Correspondingly, it is desired that these hybrids have substantially inherited only the useful gene from parent plant A (but not the poor trait of parent plant A). Therefore, using a gene marker other than the SNP described in the present invention (or a SNP on another gene), a molecular map (chromosome map) capable of identifying various chromosomal portions of the parent plants A and B is prepared. It is particularly preferable to select a hybrid into which only the useful gene has been substantially introduced from the obtained hybrid group (chromosome replacement line group) based on the map information. The molecular map may be prepared from, for example, a group of doubled haploids of parent plants A and B.
[0242]
Further, the “transformant or transformed plant” refers to a hybrid that has been produced using crossing or genetic engineering techniques and has the useful gene. The category of the transformant or the transformed plant includes an individual organism; various organs such as roots, stems, leaves, and reproductive organs (including flower organs and seeds); various tissues; cells; It should also include protoplasts, spheroplasts, induced calli, regenerating individuals and their progeny, and the like.
[0243]
【Example】
Hereinafter, the present invention will be described in detail with reference to examples. It should be noted that the present invention is not limited to the description of the following examples.
[0244]
[Example 1: Extraction of mRNA from barley, preparation of cDNA library] As barley, "Akami Riki", "Haruna Nijo", and "H. spontanum (OUH602)" (wild-type barley) were used. These barley seeds were germinated and grown as described in Cell Engineering Separate Volume, Plant Cell Engineering Series 14, Plant Genome Research Protocol, pages 193 to 195 (see Shujunsha (2000)) to obtain a material for RNA extraction. For Haruna Nijo, the buds at the time of germination, the leaf blade of the young seedling, and the top three leaves of the flag leaf stage were used as materials, and for "Akagami power", the leaf blade of the vegetative growth period was used as a material. As for “H. spontanum (OUH602)”, the top three leaves in the flag leaf stage were used as materials.
[0245]
Next, total RNA was extracted from the above-mentioned materials using Sepasol RNA (manufactured by Nacalai Tesque) according to the method described in the instruction manual of the kit. Further, only mRNA was extracted from the total RNA using an mRNA purification kit (mRNA Purification Kit) (manufactured by Amersham Pharmacia Biotech) according to the method described in the instruction manual of the kit.
[0246]
The cDNA library was prepared using a λZAP-cDNA synthesis kit (Stratagene), except for the following changes (a) and (b): “Genome analysis lab manual p63-p75: Springer Verlag Tokyo ( 1998)).
[0247]
(A) In the second centrifugation step in the step of blunting cDNA ends (see Genome Analysis Lab Manual, p71), 20 μl of sodium acetate solution (3M) and 400 μl of 100% ethanol were added to the aqueous layer containing cDNA. After sufficiently mixing by vortexing, the mixture was allowed to stand at -20 ° C overnight (overnight) and then centrifuged at 4 ° C and 15,000 rpm for 60 minutes.
[0248]
(B) The reaction solution obtained in the XhoI cleavage step (see Genome Analysis Lab Manual, p. 72) was allowed to stand at −20 ° C. overnight, and then centrifuged at 40 ° C. and 15,000 rpm for 60 minutes. . Thereafter, the supernatant was discarded and the pellet was completely dried. The pellet was dissolved in 14 μl of STE (1 ×), and 3.5 μL of a column loading dye was added thereto, followed by a ligation step to a vector. .
[0249]
The titer of the prepared cDNA library was 10 ⁶ pfu or more, and the average insert (cDNA insert) size in the clone, measured by the method described in the kit using Insert Check-Ready- (manufactured by TOYOBO), was about 1. 5 kb, 6 kb or more at maximum.
[0250]
[Example 2: Analysis of cDNA insert]
Each clone cut out from the cDNA library was cultured overnight in an LB medium by a mass excitation method (see ZAP-cDNA Gigapack III (manufactured by Stratagene) Gold cloning kit instruction manual), and plasmid DNA was extracted. .
[0251]
Next, the cDNA insert fragment of the plasmid DNA was subjected to sequence analysis from both the 5 ′ end and the 3 ′ end. For the sequence analysis, an ABI PRISM ™ 3700 Genetic Analyzer (manufactured by PE Biosystems) was used. Furthermore, in order to examine the degree of duplication of each clone from the results of the sequence analysis, clustering of the clones was performed. At the same time, the inserted fragments of each clone were assembled to prepare each of the contigs (1) to (534). These inserts were subjected to multiple alignment to detect SNPs between different clones.
[0252]
As software for clustering clones, assembling inserts, and detecting SNPs, dynarust SNP explorer (dynatrust ver. 4 additional module: manufactured by Dynacom) was used. More specifically, using the blastn program built in the software, the Expected Value is set to 1e. ^{? 10} Was set and clustering of clones was performed. In addition, the read error probability of each base at the time of base calling was fixed (Pred score = 15 (default value)), Phrap was adopted as an assembly tool, and inserted fragments were assembled according to the parameter settings shown in Tables 111 and 112. .
[0253]
[Table 111]

[0254]
[Table 112]

[0255]
【The invention's effect】
As described above, the oligonucleotide according to the present invention is a part or the entire length of the oligonucleotide consisting of the DNA sequence represented by any one of SEQ ID NOs: 1 to 534, and includes the polymorphic site represented by the universal code. Among them, it is composed of a continuous DNA sequence of 7 nucleotides or more including at least one polymorphic site causing non-synonymous substitution, or a complementary sequence thereof.
[0256]
Since the above-mentioned oligonucleotide contains the above-mentioned polymorphism site resulting from the difference of barley varieties or its complementary site, it can be used, for example, for the purpose of testing single nucleotide polymorphism existing among various barley varieties. In addition, it is possible to identify a genetic variety of barley, determine which type of gene the barley has, and the like.
[0257]
Furthermore, a method of isolating a useful gene using the above-described oligonucleotide as a probe or primer and introducing the useful gene into a target cell using a genetic engineering technique, or a hybrid plant obtained by crossing a parent plant Among them, the method of selecting a gene having a useful gene by using the above-described oligonucleotide has an effect that a desired transformant can be produced.
[0258]
[Sequence list]

Claims (7)

In the DNA sequence represented by any one of SEQ ID NOs: 1 to 534, at least one of the polymorphic sites where non-synonymous substitution occurs is included among the polymorphic sites whose bases are described in the “universal code”. An oligonucleotide comprising a continuous DNA sequence of 7 nucleotides or more, or a complementary sequence thereof. A composition for testing or identifying a single nucleotide polymorphism occurring in a gene, comprising at least one of the oligonucleotides according to claim 1. In the DNA sequence represented by any one of SEQ ID NOs: 1 to 534, among the polymorphic sites in which the notation of bases is described in "universal code", a polymorphic site causing non-synonymous substitution or a complementary site thereof An assay method for assaying or identifying a single nucleotide polymorphism generated in a gene of a subject based on at least one of the methods. In the DNA sequence represented by any one of SEQ ID NOs: 1 to 534, among the polymorphic sites in which the notation of bases is described in "universal code", a polymorphic site causing non-synonymous substitution or a complementary site thereof A method for producing a transformant, comprising a step of isolating a gene containing at least one gene and a step of introducing the obtained gene into a target cell using a genetic engineering technique. In the DNA sequence represented by any one of SEQ ID NOs: 1 to 534, among the polymorphic sites in which the notation of bases is described in "universal code", a polymorphic site causing non-synonymous substitution or a complementary site thereof A step of crossing a parent plant A having a gene containing at least one gene and a parent plant B not having this gene or having an allele having a single nucleotide polymorphism in this gene to produce a hybrid plant And then selecting a plant having the gene from the hybrid plants using at least one of the oligonucleotides according to claim 1 or using the assay method according to claim 3. A method for producing a transformant, comprising: A transformant produced by the method according to claim 4. In the polynucleotide consisting of the DNA sequence represented by any one of SEQ ID NOs: 1 to 534, at least one of the polymorphic sites where non-synonymous substitutions occur among the polymorphic sites whose bases are described in the “universal code” A primer designed to amplify a region containing one or at least one of its complementary regions.